Defects in activation and transport of fatty acids - Springer Link

J. Inher. Metab. Dis. 22 (1999) 428È441 ( SSIEM and Kluwer Academic Publishers. Printed in the Netherlands

Defects in activation and transport of fatty acids M. BRIVET1*, A. BOUTRON1, A. SLAMA1, C. COSTA1, L. THUILLIER2, F. DEMAUGRE3, D. RABIER2, J. M. SAUDUBRAY4 and J. P. BONNEFONT2,5 1 Department of Biochemistry, AP-HP Hoü pital de Biceü tre, L e Kremlin Biceü tre ; 2 Departments of Biochemistry and 4 Pediatrics, 3 INSERM U370 and 5 INSERM U393, Hoü pital Necker, Paris, France * Correspondence : L aboratoire de Biochimie 1, Hopital de Biceü tre, 94275 L e KremlinBiceü tre Cedex, France Summary : 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 carnitineÈacylcarnitine translocase. Defects in activation and uptake of fatty acids a†ect these four steps : CPT II deÐciency leads to either exerciseinduced 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 deÐciency 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 proÐle 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 conÐrmed the existence of di†erent related genetic diseases. Mutation analysis of CPT II defects has given some clues for correlation of genotype and phenotype. The Ðrst molecular analyses of hepatic CPT I and translocase deÐciencies were recently reported.

The oxidation of long-chain fatty acids in mitochondria plays an important role in energy production, especially in skeletal muscle, heart and liver. Defects in activation and transport of fatty acids a†ect the four steps of the carnitine cycle which is 428

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required for the entry of long chain acyl-CoAs into the mitochondrial matrix. All are autosomal recessive disorders. One of them, the muscular form of carnitine palmitoyltransferase deÐciency, is frequent, the others are rather rare (fewer than 30 reported patients), although many patients have not been fully reported (Saudubray et al 1999). For ten years, our group has performed in vitro fatty acid oxidation studies in lymphocytes (Brivet et al 1995) and/or Ðbroblasts (Saudubray et al 1982) in approximately 600 patients. Ninety-two cases of fatty acid oxidation disorders were found, half of them corresponding to defects of long-chain fatty acid entry into mitochondria. Among these patients, we further recognized 7 cases of primary carnitine deÐciency, 9 cases of carnitine palmitoyltransferase type I deÐciency, 20 cases of carnitine palmitoyltransferase II deÐciency (9 with the muscular form, 11 with the generalized form) and 12 cases of carnitineÈacylcarnitine translocase deÐciency. We also carried out 9 prenatal diagnosis for a fetus at risk for these two latter defects. We review here the four disorders of the carnitine cycle with emphasis on clinical hallmarks, clues to diagnosis and advances in pathophysiology and genetics.

ROLE OF THE CARNITINE CYCLE IN THE MITOCHONDRIAL OXIDATION PATHWAY (Figure 1) Fatty acids are mobilized from adipose tissue stores and transported in the circulation primarily bound to albumin. During late stages of fasting, they become the predominant substrate for energy production : they are used for hepatic ketone body synthesis and for oxidation in skeletal muscle. Fatty acids are also the preferred fuel for the heart and serve as an essential source of energy for skeletal muscle during sustained exercise. The tissue uptake of fatty acids and their transfer from the cell membrane to mitochondria remain poorly understood. Fatty acid transporters (FATP) and cytosolic fatty acid binding proteins (FABP) are probably involved in these processes (Roe and Coates 1995). Cytosolic long-chain fatty acids are activated to their corresponding acyl-CoA thioesters by a long-chain acyl-CoA synthetase on the outer mitochondrial membrane. Transport of long-chain acyl-CoAs into mitochondria then occurs by means of a cycle involving four proteins : a plasma membrane carnitine transporter (CT) to maintain the intracellular supply of carnitine ; an outer membrane carnitine palmitoyltransferase I (CPT I), which transfers acyl residues from CoA to carnitine ; a carnitineÈacylcarnitine translocase (translocase) to shuttle acylcarnitines across the inner membrane in exchange for free carnitine ; and an inner membrane carnitine palmitoyltransferase II (CPT II) that transfers acyl residues back from carnitine to CoA. Once present in the matrix, fatty acyl-CoAs enter the b-oxidation spiral, which removes electrons via FADH and NADH for ATP 2 synthesis and sequentially degrades the fatty acids to acetyl-CoA, converted to ketone bodies in the liver. Fatty acids of less than 12 carbons, such as are provided by dietary supplements of medium-chain triglycerides, are activated within the mitochondrial matrix and do J. Inher. Metab. Dis. 22 (1999)

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Figure 1 Schematic representation of the pathway of fatty acid activation and uptake. FATP, fatty acid transporter ; FABP, fatty acid binding protein ; AS, acyl-CoA synthetase ; CT, carnitine transporter ; CPT I, carnitine palmitoyltransferase I ; Translocase, carnitineÈ acylcarnitine translocase ; CPT II, carnitine palmitoyltransferase II

not require the carnitine-dependent transport process to enter the b-oxidation pathway. SYSTEMIC CARNITINE DEFICIENCY (SCD) Clinical presentation Although systemic carnitine deÐciency has long been recognized by clinicians (Chapoy et al 1980), the Ðrst demonstration of a defect in the plasma membrane transport of carnitine was made only in 1988 (Ericksson et al 1988 ; Treem et al 1988). To date, approximately 30 cases have been described, but many patients have not been reported (Saudubray et al 1999). SCD is a potentially lethal but eminently treatable inborn error of fatty acid oxidation. While cardiomyopathy and skeletal muscle weakness are the common features in all a†ected patients, attacks of hypoglycaemia are encountered, usually before 2 years of age, in children exposed to fasting (Stanley et al 1991). Sudden and unexpected deaths have been reported ; one patient born to a vegetarian mother died at 5 days (Rinaldo et al 1997). Without carnitine supplementation, cardiac failure progresses to death. J. Inher. Metab. Dis. 22 (1999)


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Diagnosis Dicarboxylic aciduria is generally mild, even in crisis urines, as reported in other defects of the carnitine cycle. Plasma carnitine concentration is very low, below 10 kmol/L, contrasting with persistent urinary excretion. The overall oxidation of radiolabelled long-chain fatty acids ([1-14C]palmitate, [9,10(n)-3H]palmitate and [9,10(n)-3H]myristate) is impaired in fresh lymphocytes and cultured Ðbroblasts. However, the defect appears in Ðbroblasts only if the culture medium is carnitine-depleted before incubation of cells with labelled fatty acids. b-Oxidation Ñuxes return to normal values when 1 mmol/L L-carnitine is added during incubation. SCD is currently diagnosed by measuring the uptake velocity of tritiated carnitine into Ðbroblasts or lymphoblasts (Tein and Xie 1996). Results are below 10% of control rates in patients and often below 40% in parents (Tein et al 1990). A reduced carnitine uptake was also shown in muscle cultures from one patient (Pons et al 1997) and in amniotic Ñuid cells of an a†ected fetus (Christodoulou et al 1996). Pathogenesis SCD results from a multisystem disorder of the high-affinity (K \ 5 kmol/L), m sodium-dependent system of carnitine transport in kidney, muscle and heart. Kinetic studies have shown that this carnitine transport does not exist in liver and brain (Bieber 1988). The failure to concentrate carnitine in cardiac and skeletal muscle leads to insufficient carnitine to support fatty acid oxidation. Failure to reabsorb carnitine in the kidney results in very low plasma carnitine levels which, in turn, diminish the hepatic uptake of carnitine by passive di†usion. Hence ketogenesis may be impaired. However, it must be noted that heart and skeletal muscle have a higher requirement for carnitine than does liver, since the K for carnitine of CPT I in cardiac and m skeletal muscle has been reported to be 5- to 10-fold higher than for the isoform of the enzyme in the liver (McGarry et al 1983). The absence of dicarboxylic aciduria is thought to result from partial peroxisomal oxidation of long-chain fatty acids, followed by complete mitochondrial oxidation of the medium-chain intermediates (Roe and Coates 1995). Carnitine supplementation restores plasma and liver carnitine levels to normal, whereas muscle carnitine levels remain less than 10% of control levels (Treem et al 1988). Nevertheless, clinical signs of myopathy and cardiomyopathy are corrected, suggesting than normal muscle carnitine level greatly exceeds that necessary to support fatty acid oxidation (Stanley et al 1991). Genetics An animal model, mouse with juvenile visceral steatosis (“ jvs Ï mouse) exhibits the clinical features of systemic carnitine deÐciency. These mice have very low levels of J. Inher. Metab. Dis. 22 (1999)

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carnitine in blood and tissues and a reduced transport of carnitine in the kidney (Horiuchi et al 1994). The locus responsible for human systemic carnitine deÐciency has recently been assigned to the long arm of chromosome 5 (Shoji et al 1998) ; this locus is syntenic to the murine “ jvs model Ï locus on chromosome 11. Since the submission of our manuscript, the Ðrst molecular analysis of systemic carnitine deÐciency was reported (Lamhonwah and Tein 1998).

CPT DEFICIENCIES After a long dispute, it is now clearly established that the mitochondrial outer membrane CPT I and the inner membrane CPT II are distinct proteins (for a review, see McGarry and Brown 1997). Some important steps in the unravelling of the CPT enzyme system were (1) the discovery that malonyl-CoA potently inhibits the outer CPT selectively (McGarry et al 1978) ; (2) the demonstration in vesicles prepared from the outer and the inner membranes that the outer CPT is detergent labile, whereas detergents enhance the activity of the inner CPT (Murthy and Pande 1987) ; (3) the recognition of clearly identiÐable di†erent diseases associated with the loss of malonyl-CoA insensitive and malonyl-CoA sensitive CPT activities (Demaugre et al 1988) ; (4) the demonstration of normal CPT I activity in muscle of patients with hepatic CPT I deÐciency (Tein et al 1989) ; (5) the characterization of distinct cDNAs encoding rat liver CPT I and CPT II (Esser et al 1993 ; Woeltje et al 1990) ; and (6) the identiÐcation of two isoforms of the outer membrane CPT I, namely the liver-type CPT I and the muscle-type CPT I (Weis et al 1994). Clinical presentation CPT I deÐciency is a rare disorder. Only liver-type CPT I deÐciency, has been recognized so far. Since the Ðrst description by Bougneres and colleagues in 1981, 13 patients have been reported (Bergman et al 1994 ; Ijlst et al 1998 ; Schaefer et al 1997). Patients usually present in infancy, with recurrent episodes of hypoketotic hypoglycaemic coma triggered by fasting or intercurrent illness, and mimicking episodes of Reye syndrome. Myopathy or cardiomyopathy has not been noted in any patients. Renal tubular acidosis was found in three cases. Five patients had a persistent neurological deÐcit, probably resulting from the initial insult. DeÐciency of CPT II occurs in two distinct phenotypes. The most frequent is the classical muscular form of CPT II deÐciency. Since the Ðrst report by DiMauro and Melis-DiMauro in 1973, more than 100 similar cases have been described. Patients with this defect (mostly males) generally present in adulthood, with recurrent episodes of muscle pain, rhabdomyolysis and paroxysmal myoglobinuria, triggered by exercise, fasting, infections, or cold exposure (Ziers 1994). It is the most common cause of hereditary myoglobinuria. A rare hepatocardiomuscular form of CPT II deÐciency has been recognized in infants (Hug et al 1991) and children (Demaugre et al 1991). The neonatal form is J. Inher. Metab. Dis. 22 (1999)


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rapidly lethal : the patient of Hug had severe hepatopathy, encephalopathy and cardiomegaly and died at age 5 days of arrhythmia with multiorgan failure. All patients died before 2 weeks of age in our own series of 5 cases. Renal dysgenesis was noted in 3 observations (North et al 1995). Patients with late-infantile onset have fasting hypoketotic hypoglycaemia, liver failure, cardiomyopathy and mild signs of muscle involvement. They are at risk of sudden death : only 2/6 patients in our series and 3/6 in the literature were still alive after 6 months of age (Demaugre et al 1991 ; Elpeleg and Gutman 1994 ; Fontaine et al 1998 ; Taroni et al 1992 ; Vianney-Saban et al 1995).

Diagnosis Increased dicarboxylic aciduria is not a feature of CPT deÐciencies as in the other carnitine cycle disorders. The plasma carnitine status is strikingly di†erent in CPT I- and CPT II-deÐcient patients. Total plasma carnitine concentration is in the highÈnormal range in CPT I-deÐcient patients, as a consequence of an unusually high renal threshold for free carnitine (Stanley et al 1992a). In the generalized form of CPT II deÐciency, patients present with a very low concentration of free plasma carnitine combined with decreased total plasma carnitine (Demaugre et al 1991). Plasma carnitine levels are usually not altered in patients with the muscular form of CPT II deÐciency. Tandem mass spectrometry enables the determination of the acylcarnitine proÐle from blood samples as small as spots collected on a Guthrie card (Millington et al 1992). A prominent peak of C species characterizes samples from patients with a 16 generalized CPT II deÐciency. Only one presumptive case of CPT I deÐciency was investigated with this method : a proÐle with high free carnitine and almost absent long-chain acyl carnitines was observed (Al Aqeel and Rashed 1998). The overall oxidation of radiolabelled long-chain fatty acids is reduced to less than 30% of control values in lymphocytes or Ðbroblasts from patients with the infantile form of both CPT defects (Saudubray et al 1982 ; Slama et al 1996). Results in lymphocytes are available within 24 h (Brivet et al 1995). However, this method does not distinguish CPT I-deÐcient and CPT II-deÐcient patients. Developments in analytical techniques have provided tools to measure the accumulation of boxidation intermediates in cells incubated with a labelled long-chain fatty acid. RadioÈhigh-pressure liquid chromatography analysis of Ðbroblasts incubated with [U-14C]palmitate will demonstrate the accumulation of large amounts of either palmitoyl-CoA in CPT I-deÐcient cells or palmitoylcarnitine in CPT II-deÐcient cells (Schaefer et al 1997). Tandem mass spectrometry can demonstrate the accumulation of palmitoylcarnitine in CPT II-deÐcient Ðbroblasts incubated with deuterated linoleate and L-carnitine (Nada et al 1995). CPT deÐciencies are currently diagnosed by measuring both CPT activities in Ðbroblasts by the same isotope exchange assay : palmitoylcarnitine synthesis is measured from palmitoyl-CoA and carnitine (Demaugre et al 1988). The CPT I activity of the outer mitochondrial membrane is measured in non-detergent conditions and J. Inher. Metab. Dis. 22 (1999)

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is identiÐed by its suppressibility by malonyl-CoA. Conversely, the CPT II activity of the inner mitochondrial membrane is measured after disruption of mitochondrial membranes in detergent conditions. Recently, the use of permeabilized cells, rather than gently sonicated cells, was proposed for the assay of CPT activities (Schaefer et al 1997). The CPT I activity measured in Ðbroblasts from CPT I-deÐcient patients ranges from 10% to 23% of control values. There appears to be no correlation between the degree of enzyme deÐciency and the severity of clinical presentation. The clinical heterogeneity of CPT II deÐciency is poorly understood. Our studies in Ðbroblasts support the hypothesis that di†erences in residual enzyme activity could give rise, at least in part, to the phenotype (i.e. muscular or generalized forms) : the residual activity of CPT II-deÐcient Ðbroblasts was lower in our infantile set of patients ( \ 15% of controls) than in the adult one (15È25% of controls). A marked decrease of long-chain fatty acid oxidation was observed in cells with CPT activity below 10% of controls, while a residual activity over 15% appears sufficient to maintain long-chain fatty acid oxidation over 50% (Bonnefont et al 1996). However, other authors have not found a signiÐcant di†erence between residual CPT II activities of muscle from adult-type and infantile-type patients (Taroni et al 1993). Prenatal diagnosis for CPT I deÐciency has never been attempted to our knowledge. Prenatal diagnosis of infantile CPT II deÐciency was established by ultrasonography, revealing enlarged echogenic kidneys in one fetus (Witt et al 1991). We have excluded CPT II deÐciency in 3 fetuses at risk for the infantile form by mutation analysis on chorionic villi (one case) and in vitro fatty acid oxidation studies coupled to enzymatic assay in amniocytes (two cases). Results were conÐrmed postnatally.

Pathogenesis Defects of CPT I and CPT II each a†ect the entry of long-chain fatty acids into the mitochondrion. CPT I deÐciency results in the inability to produce long-chain acylcarnitines from long-chain acyl-CoAs, whereas in CPT II deÐciency long-chain acylcarnitines that have been translocated across the inner mitochondrial membrane are not efficiently converted to their corresponding acyl-CoAs. The absence of dicarboxylic aciduria in both defects may result from alternative pathways for long-chain fatty acid oxidation, as in the other carnitine cycle defects. CPT I deÐciency is expressed in liver, kidney and Ðbroblasts, but not in muscle (Tein et al 1989). The high renal threshold for free carnitine observed in CPT IdeÐcient patients is thought to be due to the lack of formation of long-chain acylcarnitines, which are potent inhibitors of free carnitine transport (Stanley et al 1991). Long-chain acylcarnitines, accumulated in the mitochondrial matrix in the generalized form of CPT II deÐciency, may be transported out of mitochondria, as demonstrated by the prominent long-chain acylcarnitine species seen in plasma. Two deleterious e†ects have been attributed to these compounds : they may induce a defect in the cellular uptake of free carnitine, leading to a secondary carnitine deÐJ. Inher. Metab. Dis. 22 (1999)


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ciency (Stanley et al 1991), and they may promote cardiac arrhythmia (Roe and Coates 1995). Genetics CPT I, tightly associated with the outer mitochondrial membrane, exists as two di†erent, tissue-speciÐc, isoforms : a liver-type (L-CPT I) and a muscle-type (M-CPT I), which di†er by their kinetic properties. Human L-CPT I cDNA (Britton et al 1995) and M-CPT I cDNA (Yamazaki et al 1997) encode proteins of 773 and 772 amino acids with molecular mass of 88.1 and 88.2 kDa, respectively. L-CPT I, the sole isoform expressed in human Ðbroblasts, is also the primary form in liver and kidney ; M-CPT I is expressed intensively in skeletal muscle and adipose tissue ; both isoforms are present in heart, although this tissue expresses M-CPT I predominantly (McGarry and Brown 1997). The gene structure is partially known for the L-CPT I gene (Britton et al 1995) and completely established for the M-CPT I gene (Yamazaki et al 1997). The two chromosomal loci have been assigned to 11q13 (LCPT I) and 22q13.3 (M-CPT I) (Britton et al 1997). The Ðrst molecular analysis of hepatic CPT I deÐciency was recently reported (Ijlst et al 1998). Analysis of the patientÏs cDNA revealed a homozygous mutation changing aspartate into glycine (D454G). This amino acid is conserved through 17 di†erent acyltransferases. Heterologous expression in yeast has shown that the expressed mutant displayed only 2% of residual activity. No case of an inherited defect of muscle CPT I deÐciency has been recognized so far. CPT II is loosely associated with the inner mitochondrial membrane and insensitive to malony-CoA. The human full-length cDNA clone isolated by Finocchiarro and colleagues in 1991 predicted a protein of 658 amino acids containing a 25 amino acid NH -terminal leader sequence that is cleaved upon mitochondrial 2 import to yield a mature protein with molecular size of 71 kDa. In a wide variety of rat tissues, the CPT II mRNA was found to be identical in size, and its product immunologically indistinguishable, implying the operation of a single gene in all tissues (McGarry and Brown 1997). The CPT II gene is approximately 20 kb in size and is composed of Ðve exons ranging from 81 bp to 1305 bp, separated by four introns varying from 1.5 kb to 8 kb (Verderio et al 1995). It was localized by Gellera and colleagues (1994) to chromosome 1p32. Mutation analysis of CPT II defects has given some clues for a genotypeÈ phenotype correlation. A common mutation, S113L, associated with the muscular form was described (Taroni et al 1993). To date, more than 25 other mutations have been recognized in patients with muscular or generalized CPT II deÐciency. All mutations reported so far in the homozygous state are strongly associated with a given phenotype, either muscular (P50H, S113L, E174K) or generalized (F383Y, Y628S, R631C) (Bonnefont et al 1996 ; Taroni et al 1992, 1993 ; Verderio et al 1995 ; Wataya et al 1998). Compound heterozygotes between one adult-type mutation and one infantile-type mutation seem at risk for severe episodes : one patient (F383Y/E174K) had a severe J. Inher. Metab. Dis. 22 (1999)

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hepatic presentation (Yamamoto et al 1996) ; we reported another patient (S113L/ Y628S) with a muscular presentation at 6 years who experienced a cardiac arrest (Thuillier et al 1997). The percentages of residual CPT II activity after transfection experiments do not show a tight correlation between the phenotype and the enzymatic activity (Wataya et al 1998). Thus, additional parameters must be involved : for instance, mutations may distinctly impair the interaction between CPT II and other components of the b-oxidation machinery. TRANSLOCASE DEFICIENCY Translocase deÐciency seems not to be very rare since, to our knowledge, 23 patients have been diagnosed, but only 9 cases have been reported since the Ðrst description by Stanley and colleagues (1992b). Clinical presentation In most cases, patients experienced a life-threatening episode in the neonatal period, characterized by neonatal distress with hyperammonaemia, inconstant hypoglycaemia, heart beat disorders and early muscle involvement (skeletal muscle weakness and high plasma CK) (Chalmers et al 1997 ; Niezen-Koning et al 1995 ; Pande et al 1993 ; Stanley et al 1992b). Sustained elevations of plasma ammonia, resistant to dietary changes, have been recorded in several cases, as in severe forms of CPT II deÐciency ; they can lead to a misdiagnosis of urea cycle defect (Ogier de Baulny et al 1995). A few patients with carnitine translocase deÐciency survived the severe neonatal condition and are now doing well, without any cardiomyopathy or myopathy, at ages varying from 3 to 9 years (Huizing et al 1997 ; Morris et al 1998 ; Olpin et al 1997). Diagnosis Dicarboxylic aciduria is mild, as observed in the other defects of the carnitine cycle. An important Ðnding suggestive of either CPT II or translocase deÐciency is the severe plasma free carnitine deÐciency associated with a high level of acylcarnitines (up to 90% of total plasma carnitine). Acylcarnitines are primarily C species, as 16 demonstrated by mass tandem spectrometry. We retrospectively diagnosed a patient who died suddenly in the neonatal period by investigating acylcarnitine proÐle from a Guthrie card (Brivet et al 1996). Long-chain fatty acid oxidation is markedly reduced in lymphocytes or Ðbroblasts, but the residual value seems to be somewhat higher in surviving patients (Morris et al 1998). CarnitineÈacylcarnitine translocase activity can be measured in fresh cells (lymphocytes, Ðbroblasts or fetal cells) permeabilized by digitonin (Pande et al 1993). The translocase activity is reÑected by the rate of [14C]acetylcarnitine efflux through the mitochondrial membrane, after oxidation of [14C]pyruvate to J. Inher. Metab. Dis. 22 (1999)


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[14C]acetyl-CoA and further conversion of [14C]acetyl-CoA to [14C]acetylcarnitine by carnitine acetyltransferase. Efficiency of pyruvate oxidation is checked by monitoring acetyl-CoA conversion to citric acid cycle intermediates and carnitine acetyltransferase activity is determined by measuring [14C]acetylcarnitine formation from [14C]acetyl-CoA and carnitine. We have been able to determine the translocase activity in the Ðbroblasts from 12 patients. The translocase activity was \1% in all cell lines but one. In these cells, the residual activity was about 3% of control values. The corresponding patient is now 3 years old and doing well (Morris et al 1998). There is occasionally an overlap between translocase activity in controls and heterozygotes, but the difficulty is overcome when the results are expressed as the ratio between the values of pyruvate oxidation into either citric acid cycle intermediates or acetylcarnitine (Brivet et al 1996). We have performed a prenatal diagnosis for 6 fetuses at risk of translocase deÐciency by in vitro fatty acid oxidation studies, coupled to enzymatic assay in cultured trophoblasts (n \ 3) or amniocytes (n \ 4). Results were conÐrmed postnatally for the Ðve non-a†ected fetuses and the a†ected fetus. Two other prenatal diagnoses of translocase deÐciency have been reported (Chalmers et al 1997).

Pathogenesis The absence of dicarboxylic aciduria may result from alternative pathways for longchain fatty acid oxidation, as already suggested in the other carnitine cycle defects. DeÐciency of carnitineÈacylcarnitine translocase leads to the accumulation of long-chain acylcarnitines outside the mitochondrial matrix, with the same deleterious consequences as observed in CPT II deÐciency (secondary carnitine deÐciency, cardiac toxicity). Patients with almost no translocase activity have been reported to have signiÐcant rates of long-chain fatty acid oxidation in Ðbroblasts (Huizing et al 1998). These Ðndings raise the issue of whether a secondary pathway might be able to bypass the translocase block.

Genetics The human translocase cDNA has recently been cloned (Huizing et al 1997) : it is 1.2 kb in length and encodes a 301 amino acid protein (32.9 kDa). The translocase protein contains a 3-fold repeated sequence of about 100 amino acids, which is a characteristic feature of the proteins belonging to the mitochondrial carrier family. A model of the folding in the inner mitochondrial membrane has been proposed with the presence of six transmembrane a-helices, connected by Ðve hydrophilic loops and cytoplasmic exposure of the N- and C-terminal regions (Indiveri et al 1997). The gene was assigned to chromosome 3p21.31 (Viggiano et al 1997). It genomic organization is only partially known. Molecular defects in translocase cDNA sequence were identiÐed in 3 patients. The Ðrst recognized defect, reported in J. Inher. Metab. Dis. 22 (1999)

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a mild case, was a homozygous cytosine insertion resulting in a frameshift and elongation of the protein by 21 amino acids at the C-terminal region (Huizing et al 1997). Compound heterozygosity for two extensive deletions has been described in a severe case (Huizing et al 1998). We identiÐed a homozygous 558C ] T transition in the translocase cDNA, leading to a premature stop codon at amino acid 166. The presence of this C ] T transition was directly conÐrmed on genomic DNAs from the patient and her parents (Costa et al 1999). CONCLUSION During the past 10 years, the recognition of defects of the carnitine cycle has increased dramatically. In the future, it is likely that progress in pathophysiology will arise from knowledge of structureÈfunction relationships for the di†erent proteins involved in this pathway. Description of new defects such as defects of transport of fatty acids across the plasmalemmal membrane and muscle-type CPT I deÐciency remains an exciting challenge. ACKNOWLEDGEMENTS The authors thank Drs P. Delonlay, D. Devictor, J. Hammond, A. Lombes, L. Majewski, D. Mitanchez, A. A. A. Morris, A. Munnich, H. Ogier de Baulny, P. Reinert, C. Roe, C. Sansaricq, G. Touati, C. Vianney and B. Wilcken for referring cell lines. This work was supported in part by grants from AP-HP Recherche Clinique 95170 and 97010. REFERENCES Al Aqeel A, Rashed M (1998) Carnitine palmitoyl transferase I deÐciency (CPT I), three a†ected siblings in one family (Abstract). J Inher Metab Dis 21 (supplement 2) : 61. Bergman AJ, Donckerwolcke RA, Duran M, et al (1994) Rate-dependent distal renal tubular acidosis and carnitine palmitoyltransferase I deÐciency. Pediatr Res 36 : 582È588. Bieber LL (1988) Carnitine. Annu Rev Biochem 57 : 261È283. Bonnefont JP, Taroni F, Cavadini P, et al (1996) Molecular analysis of carnitine palmitoyltransferase II deÐciency with hepatocardiomuscular expression. Am J Hum Genet 58 : 971È 978. Bougneres PF, Saudubray JM, Marsac C, et al (1981) Fasting hypoglycermia resulting from hepatic carnitine palmitoyltransferase deÐciency. J Pediatr 98 : 742È746. Britton CH, Schultz RA, Zhang B, et al (1995) Human liver mitochondrial carnitine palmitoyltransferase I : characterization of its cDNA and chromosomal localization and partial analysis of the gene. Proc Natl Acad Sci USA 92 : 1984È1988. Britton CH, Mackey DW, Esser V, et al (1997) Fine chromosome mapping of the genes for human liver and muscle carnitine palmitoyltransferase I (CPTIA and CPT1B). Genomics 40 : 209È211. Brivet M, Slama A, Saudubray JM, et al (1995) Rapid diagnosis of long-chain and medium chain fatty acid oxidation disorders using lymphocytes. Ann Clin Biochem 32 : 154È159. Brivet M, Slama A, Millington D, et al (1996) Retrospective diagnosis of carnitine acylcarnitine translocase deÐciency by acylcarnitine analysis in the proband Guthrie card and enzymatic studies in the parents. J Inher Metab Dis 19 : 181È184.

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