003 1-399818812403-0308$02.00/0 PEDIATRIC RESEARCH Vol. 24, No. 3, 1988 Copyright O 1988 International Pediatric Research Foundation, Inc. Printed in U.S.A.

Hepatic and Muscular Presentations of Carnitine Palmitoyl Transferase Deficiency: Two Distinct Entities

FRANCE DEMAUGRE, JEAN-PAUL BONNEFONT, GRANT MITCHELL, NAM NGUYEN-HOANG, ANNA PELET, MARC0 RIMOLDI, STEFAN0 DI DONATO, AND JEAN-MARIE SAUDUBRAY Dkpartement de Biochimie, INSERM U75, CHU Necker-75730 Paris Cedex 15, France [F.D.. N.N-H.]; Clinique et Unite de Recherche de Ginetique Medicale, INSERM U12, H6pital des Enfants Malades, 149 Rue de Skvres- 75743 Paris Cedex 15, France [J-P.B., G.M., A.P., J-M.S.]; and Neurometabolic Disease Laboratory, Istituto

Neurological "C. Besta, " 20133 Milan, Italy [M.R., S.D.D.]

ABSTRACT. Human carnitine palmitoyl transferase priately low ketogenesis. In this disorder, CPT deficient activity (CTP) deficiency results in two different clinical variants, has been demonstrated in liver. However, the defect is probably one with "hepatic" and one with "muscular" symptoms. not limited to the liver because this patient's fibroblasts also We studied CPT activity and long-chain fatty acid oxida- exhibited impaired long-chain fatty acid oxidation (1 3). In the tion in fibroblast cell lines from four patients, two from "muscular" presentation, the defect has been demonstrated in all each group. Overall CPT activity was deficient in patients' tissues studied, including muscle, liver, leukocytes, platelets, and fibroblasts with the hepatic presentation, as previously fibroblasts (1 1, 14). In all these studies, the methodology of CPT demonstrated in patients' fibroblasts with the muscular assays did not allow a clear discrimination between CPTl and presentation. The hepatic patients' fibroblasts displayed a CPT2 activities. Thus it was not possible to evaluate if the two CPTl deficiency which resulted in impaired long-chain different clinical presentations resulted from distinct enzymatic fatty acid oxidation. In contrast, CPTl activity and palmi- defects affecting CPT, and/or CPT2 activities. tate oxidation were normal in muscular patients' fibro- We compared CPT activities and long-chain fatty acid oxida- blasts. In these latter patients, the mutation presumably tion in four patients' fibroblasts, two with "hepatic" and the involved CPT2 activity. These data suggest that CPT defi- other two with "muscular" presentations. Our results suggest that ciency is due to at least two different mutations, resulting there are at least two varieties of CPT deficiency, each presenting in two distinct patterns of clinical and biochemical abnor- distinct biochemical abnormalities in cultured fibroblasts. malities. (Pediatr Res 24: 308-31 1, 1988)

Abbreviation CASE REPORTS Case I. The patient was fully reported in 198 1 (12). She was CPT, carnitine palmitoyl transferase referred at 8 months of age for coma with seizures related to hypoglycemia after 16 h of fasting. She had minimal hepatomeg- aly without clinical muscular symptomatology or biochemical muscular abnormalities. A 19-h fasting test resulted in a marked Long-chain fatty acids constitute a major source of energy in hypoglycemia (1.8 mmol/liter) with deficient ketogenesis (<0.2 man. Their oxidation in muscles is essential for energy homeo- mmol/liter) reversible by the administration of medium-chain stasis during prolonged exercise (1). During fasting, the oxidation triglycerides. The hepatic histology showed an accumulation of of long-chain fatty acids in liver mitochondria produces ketone triglycerides, whereas total liver carnitine was normal. Hepatic bodies, enhances gluconeogenesis, and therefore contributes to CPT activity (measured by an "isotope exchange" assay) was the maintenance of normoglycemia (2). CPT (EC 2.3.1.21) con- greatly reduced. CPT activity was not determined in other tissues trols the transport of long-chain fatty acids into mitochondria. but impaired oxidation of palmitate was demonstrated in cul- CPT activity is classically considered to be distributed on both tured fibroblasts (13). The child is now l l yr old and has no the outer (CPT1) and the inner (CPT2) surfaces of the inner clinical or biologic muscular symptoms. mitochondria1 membrane, although this assumption has been Case ZZ. The patient, a 1-yr-old boy, was referred for hypogly- recently questioned (3). CPT, activity is specifically inhibited by cemic coma. At the end of a 19-h fasting test, blood glucose fell malonyl CoA in animal and human tissues (4-6). Recent data to 1.3 mmol/liter. Blood ketones level was 0.3 mmol/liter, (3, 7-9) suggested that CPTl and CPT2 activities involve two or contrasting with blood free fatty acid concentration of 2.5 mmoll more distinct proteins. liter. Total and free blood carnitine were 55 and 45 pmol/liter. Human CPT deficiency displays two clinical presentations. Urinary organic acid excretion displayed no abnormal pattern. Inasmuch as the first description by Di Mauro and Dimauro in A liver biopsy was not accepted by the parents. The child is now 1973 (lo), muscular CPT deficiency has been recognized as an 10 yr old and no clinical or biologic symptom can be demon- important cause of episodic muscle necrosis with paroxysmal strated. myoglobinuria (1 1). A second clinical presentation associated Case ZII. The patient has been described previously (15). At with liver-CPT deficiency has been documented in a single the age of 12 yr, the boy first experienced muscular cramps and patient (12) consisting of fasting hypoglycemia with inappro- rhabdomyolysis after prolonged physical exercise. Many subse- Received November 27, 1987; accepted April 29, 1988. quent episodes of rhabdomyolysis were all induced by prolonged Correspondence and reprints to Dr. France Demaugre, Dkpartement de Bio- effort, particularly when performed during a fasting test. At the chimie, INSERM U75, CHU Necker, 75730 Paris Cedex 15, France. age of 22 yr, an identical episode resulted in acute renal failure. 30 HEPATIC AND MUSCULAR CPT DEFICIENCY 309 An ischemic forearm exercise test performed several months was determined on two different control fibroblasts homoge- after this attack resulted in a normal rise of plasma lactate. The nates. At 500 pM L-carnitine,Km for palmitoyl CoA was 28 and total carnitine content of muscle was not decreased. The CPT 23 pM. K, for carnitine was 38 and 35 pM when determined activity was markedly deficient in muscle and fibroblasts using with 100 pM palmitoyl CoA. the isotope exchange method. The assay of CPTl activity is hampered by its localization on Case ZV. The patient has been described ( 14) and was clinically the inner face of mitochondrial membrane. CPT activity was similar to case 111. The CPT defect was demonstrated in muscle measured after disruption of mitochondrial membranes with and liver without noticeable clinical hepatic symptomatology. palmitoyl carnitine as a detergent (assays B and C). Assay B was Fibroblasts failed to exhibit any CPT defect when tested with a modification of assay B from McGarry et al. (4). It measured forward and isotope exchange assays (described in the case report) L-(methy13H)carnitine release from palmitoyl L-(methy13H)car- but expressed the CPT defect with an assay similar to assay B nitine and CoA in the absence of albumin. The 2 10 d incubation (Rimoldi M, unpublished data).

MATERIALS AND METHODS Fibroblasts were cultured as previously described (13) from controls and CPT-deficient patients. Cell lines I and I1 were from patients with hepatic presentation and cell lines I11 and IV from patients with muscular presentation. The oxidation of 5 mM [I ,4-'4C]succinate, 500 pM[1-'4C]octanoate and 100 pM[1-I4C] palmitate was measured as described (13) with modifications (16). CPT activity was assayed in the 150 x g supernatant of fibroblast homogenates in which mitochondrial integrity was preserved. The homogenization was performed in 150 mM KC1 50 mM tris, pH 7.4, by sonication (MSE sonicator; medium power; 14 pM peak to peak amplitude; 3 s; 4" C). Mitochondria1 integrity was assessed by assaying citrate synthase (EC 4.1.3.7) (17) in the supernatant after a 9000 X g centrifugation for 15 min. Usually less than 20% of the total citrate synthase activity was present in the supernatant. The assay of CPTl activity only requires conditions preserving mitochondrial membrane integrity. CPTl activity is character- ized by its total inhibition by malonyl CoA (4). We tested these conditions in assay A, similar to assay 2 of McGarry et al. (4). This assay measured palmitoyl-L-(methy13H)carnitine produc- is I . - i isio 50 100 tion from L-(methy13H)carnitine and palmitoyl-CoA. The 550 MALONYLCoA pl incubation mixture contained 115 mM Tris pH 7.4; 4.5 mM reduced glutathione; 70 mM KC1; 110 pM palmitoyl CoA com- (rM) plexed with 1% fatty acid-free bovine serum albumin (wtlvol), Fig. 1. The effect of malonyl CoA on CPT activity (assay A). Fibro- and homogenate. After a 2.5-min preincubation with and with- blasts homogenates were preincubated for 2.5 min with or without out malonyl CoA, the reaction was initiated by the addition of malonyl CoA and the reaction was initiated by the addition L-~H- 540 pM L-(methy13H)carnitine. The actual concentrations of carnitine. Concentrations of palmitoyl CoA and L-(methy13H)carnitine malonyl CoA added in the preincubation mixture are indicated into the assays were 110 and 550 pM, respectively (for details see in figure 1 and Table 1. The reaction was stopped as described Materials and Methods). Results of a typical experiment are displayed. by Solberg (18). Km of CPT, for carnitine and palmitoyl CoA Similar results were obtained with another cell line.

- Table 1. CPT activities of patients with hepatic (I and ZI) and muscular (ZZZ and ZV) presentations* Patients Malonyl CoA 50 UM Controls I I1 111 IV Assay A 2.45 + 0.65 0.39 + 0.04 0.56 + 0.18 3.18 + 0.24 2.86-2.72 - (1.45-3.35) n=3 n=3 n=3 n=2 N= 12 0.23 + 0.06 0.21 + 0.05 0.26-0.30 0.30 + 0.04 0.22-0.25 + (0.15-0.31) n=3 n = 2 n=3 n=2 N= 12

Assay B

Assay C 2.30 + 0.32 2.50 + 0.45 2.02-2.28 0.60 + 0.1 1 0.67-0.63 N=3 n=3 n=2 n=3 n=2 * CPT activities were measured in duplicate by three distinct assays (A, B, C) as indicated in Materials and Methods. Assay A (palmitoylcarnitine synthesis) was performed in nondetergent conditions; assay B (carnitine release), and assay C (palmitoylcarnitine synthesis) in detergent conditions. Results (mean + 1 SD) are expressed as nmol of palmitoyl-L-(methyl3H) carnitine produced .min-'. mg NCP-I (assay A and C) and as nmol of L- (methyl 3H) carnitine released.min-'.mg NCP-' (assay B). N = number of different control cell lines. n = number of independent experiments performed in deficient strains (each experiment was performed in duplicate). 3 10 DEMAUGRE ET AL. medium contained 115 mM Tris pH 7.4; 70 mM KC1; 8 mM mal. Conversely, in cell lines I11 and IV, long-chain fatty acid CoA; 4.5 mM reduced glutathione; and homogenate. After a 2.5- oxidation was not reduced, irrespective of the concentration of min preincubation, the reaction was initiated by the addition of palmitate used in the experiments and of the presence of L- 2 mM palmitoyl-L-(methy13H) carnitine. The reaction was carnitine. The apparent Km for palmitate was 19.5 f 4.5 pM (n stopped with 0.6 M perchloric acid. After centrifugation, an = 4) for controls and 18.3 pM (n = 2) and 20.5 pM (n = 2) in aliquot of supernatant containing L-(methy13H)carnitine was cell lines I11 and IV, respectively. counted in scintillation medium. Assay C was performed as described (19). It measured palmitoyl-L-(methy13H) carnitine DISCUSSION production from L-(methy13H)carnitine and palmitoyl CoA in the absence of albumin and in the presence of palmitoyl-D- CPT deficiency with a muscular presentation has been de- carnitine in a volume of 500 p1 containing 40 p~ palmitoyl scribed in nearly 40 patients and has been recently reviewed (1 1). COA, 1 mM L-carnitine, and 1 mM palmitoyl-~-carnitine.The The clinical presentation is stereotyped. The patient is most often assays were linear with incubation time (3-8 min) and added an adolescent male and experiences acute episodes of rhabdo- protein when performed with 50-250 g protein in assays A and myolisis after prolonged exercise or occasionally after prolonged C and 50-120 g protein in assay B. Incubation time was 5 min. fasting. Spontaneous attacks are possible but muscle cramps are Protein was determined by the method of Lowry et al. (20). rare. Acute renal failure can occur during acute attacks. Serum COA,palmitoyl COA,palmitoyl-L-carnitine, octanoic acid, pal- and muscle carnitine levels are not usually decreased but lipid rnitic acid, succinic acid, and fatty acid-free bovine serum albu- accumulation is occasionally demonstrated in muscle biopsies. min were purchased from Sigma Chemical Co. (St. Louis, MO). CPT deficiency is not restricted to muscle but is also found in Palmitoyl-~-camitinewas synthetized as described (2 1). L-Car- all other tissues (e.g. liver, leukocytes, platelets, fibroblasts) stud- nitine was a gift from Sigma-Tau France. Reduced glutathione ied. The hepatic consequences of this defect are minor and fasting was purchased from Boehringer. [ 1-14C]sodium octanoate, [ 1- hypoglycemia has not been 0b~~~ed.Ketone body production 14CIpalmitic acid, [1,4-14C]succinic acid, and methyl 3H) Car- was delayed during fasting in the few patients thus studied. TO nitine were purchased from Amersham (UK). Palmitoyl-L- our knowledge, patient I represents the only reported case (12) (methyl 3H) carnitine was synthetized as described (22). with hepatic presentation of CPT deficiency. Symptomatic hy- poketotic hypoglycemia was the main clinical problem. In con- RESULTS trast to patients with long-chain fatty acid oxidation defects, such as systemic carnitine deficiency (23) or long-chain acyl CoA CPTl activity was identified by its suppressibility by malonyl dehydrogenase deficiency (24), heart and skeletal muscle were CoA (4). In control fibroblasts (Fig. l), CPT activity measured not affected in patients I and 11. Although direct assays in tissues by assay A was inhibited by malonyl CoA in a dose-dependent other than liver were not performed, the defect was probably not fashion. The maximum effect (90% inhibition) was obtained restricted to liver, because this patient's fibroblasts exhibited a with 50pM malonyl CoA. The Iso was approximately 1.2 pM, severe impairment of long-chain fatty acid oxidation (12). similar to values obtained in rat and human fetal liver (6). In These distinct clinical presentations of CPT deficiency could cell lines I and I1 (hepatic presentation), CPT activity measured result from distinct mutations affecting either CPTl or CPT2 by assay A was impaired (residual activities 15 and 25% of activities. The review of the literature failed to answer this control, respectively) (Table 1). By contrast, CPT activity meas- question, as the assays used did not discriminate between CPTl ured by this assay was normal in cell lines I11 and IV (muscular and CPT2 activities. In intact mitochondria, CPT is known to presentation). Inhibition by 50 pM malonyl CoA, V,,,, and Km exist as an overt (CPTI)and a latent (CPT2) form. The latter is for palmitoyl CoA and L-carnitine were similar to controls in exposed by agents or procedures that disrupt membrane. CPT cell lines I11 and IV (data not shown). In assays B and C, activity of fibroblasts was measured by three assays (A, B, C), in performed in the presence of detergent, cell lines I11 and IV homogenates in which mitochondrial integrity was preserved (see exhibited an impaired CPT activity (residual activity 25 to 30% Materials and Methods). CPT activity, as measured as palmitoyl of control). By contrast, in cell lines I and 11, CPT activity carnitine synthesis in nondetergent conditions (assay A), was measured by these assays was normal. suppressed (>go%) by 50 pM malonyl CoA, a specific inhibitor The consequences of impaired CPT activity on the oxidation of CPTl (4), suggesting this assay specifically measured CPTl of fatty acids were studied in intact fibroblasts. In cell lines I and activity. The assay of CPT2 required treatments that disrupted 11, the oxidation of palmitate was dramatically decreased, mitochondrial membranes. This requirement was fulfilled in whereas the oxidation of succinate and octanoate (which enters assays B (used in most published clinical cases) and C, performed the mitochondria by a CPT-independent mechanism) were nor- in detergent conditions (19). Carnitine release was measured in

Table 2. Oxidation of fatty acids and succinate injbroblastsfrom CPT-deficient patients with hepatic (cell lines I and II) and muscular (cell lines I11 and ZV) presentations* Patients Controls I I1 111 IV

* Results (mean + I SD) are expressed as nanomoles of radioactive product. h-l. lo6 cells. N = number of different control cell lines. n = number of independent experiments performed in deficient strains (each experiment was performed in duplicate). HEPATIC AND MUSCULAR CPT DEFICIENCY 31 1 assay B and palmitoyl carnitine synthesis was measured in assay ketone body production. Annu Rev Biochem 49:395-420 3. Murthy MSR, Pande SV 1987 MalonylCoA binding site and the overt carni- C. Although these assays are supposed to investigate total (I + tine-palmitoyltransferase reside on the opposite sides of the outer mitochon- 2) CPT activity, D- and L-palmitoyl carnitine used as detergents drial membrane. Proc Natl Acad Sci USA 84:378-382 in assays B and C also inhibited a fraction of CPT activity, 4. Mc Gany JD, Leatherman GF, Foster DW 1978 Carnitinepalmitoyltransferase presumably CPT, activity (7-9). As a consequence, assays B and I. The site of inhibition of hepatic fatty acid oxidation by malonylCoA. J Biol Chem 253:4128-4136 C likely measure CPTl activity only. 5. Saggerson ED, Carpenter CA 198 1 Carnitine palmitoyl transferase and carni- We studied CPT activities in four patients' fibroblasts, two of tineoctanoyltransferaseactivities in liver, kidney cortex, adipocyte, lactating them presenting the hepatic form and the other two the muscular mammary gland, skeletal muscle and heart. Relative activities, latency and form. Patients' cell lines with hepatic presentation exhibited a effect of malonyl CoA. FEBS Lett 129:229-232 6. Mc Gany JD, Mills SE, Long CS, Foster DW 1983 Observationson the affinity defective CPT, activity. Conversely, in these cells, CPT activity for carnitine and malonylCoA sensitivity of carnitinepalmitoyltransferase I was not impaired when measured in detergent conditions, sug- in animal and human tissues. Biochem J 214:21-28 gesting an intact CPT2 activity. In patients' cell lines with mus- 7. Declercq PE, Falck JR, Kuwajima M, Tuminski H, Foster DW, Mc Gany JD cular presentation, assay A failed to evidence a decreased activity, 1987 Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system 1. Use of inhibitors. J Biol Chem 2629812-9821 irrespective of the concentration of albumin (0.1 to I %) and 8. Woeltje KF, Kuwajima M, Foster DW, Mc Garry JD 1987 Characterization palmitoyl CoA (10 to 300 pM) used in the assay (data not shown), of the mitochondrial carnitine palmitoyl transferase enzyme system. J Biol contrasting with Zierg and Engel's results (25). They demon- Chem 262:9822-9827 strated an impaired CPT activity in muscle mitochondria from 9. Lund H 1987 Carnitine palmitoyltransferase: characterization of a labile deter- gent-extracted malonylCoA sensitive enzyme from rat liver mitochondria. muscular presentation patients in an assay similar to assay A Clin Chim Acta 918:67-75 performed with 0.1 % albumin and 200 pM palmitoyl CoA. This 10. Di Mauro S, Dimauro PMM 1973 Muscle carnitinepalmitoyltransferasedefi- discrepancy between our data and those of Zierg and Engel (25) ciency and myoglobinuria. Science 182:929-93 1 might be due to differences in the cellular material used (intact 11. Di Mauro S, Trevisan C 1982 Carnitine palmitoyl transferase (CPT) deficiency: a review. In: Schotland DL (ed) Disorders of the Motor Unit. John Wiley fibroblast mitochondria versus disrupted muscle mitochondria). and Sons, New York, pp 657-665 However, CPT activity of "muscular" cell lines was markedly 12. Bougneres PF, Saudubray JM, Marsac C, Bernard 0, Odibvre M, Girard J reduced when measured in detergent conditions, in agreement 198 1 Fasting hypoglycemia resulting from hepatic carnitinepalmitoyltrans- with earlier data (15, 25); indeed, muscular CPT deficiency was ferase deficiency. J Pediatr 98:742-746 13. Saudubray JM, Coude FX,Demaugre F, Johnson C, Gibson KM, Nyhan WL usually shown under such conditions. Thus the impaired CPT 1982 Oxidation of fatty acids in cultured fibroblasts: a model system of the activity measured by assays B and C could be explained by a identification and study of defects in oxidation. Pediatr Res 16:877-881 defect of CPT2 activity. These data show that the two clinical 14. Di Donato S, Castiglione A, Rimoldi M, Cornelio F, Vendemia F, Gardace G, forms of CPT deficiency are caused by different mutations. As Bertagnolio B 1981 Heterogeneity of carnitinepalmitoyltransferase defi- ciency. J Neurol Sci 50:207-215 we only studied two cases of each clinical presentation of CPT 15. Di Donato S, Cornelio F, Pacini L, Peluchetti D, Rimoldi M, Spreafico S 1978 deficiency, investigations of other patients must be performed to Muscle carnitinepalmitoyltransferasedeficiency in cultured fibroblasts. Ann establish a firm correlation between clinical and biochemical Neuro14465-467 data. 16. Mitchell G, Saudubray JM, Pelet A, Bemaugre F, Labarthe JC 1984 The effects of carnitine on palmitate oxidation in cultured fibroblasts. Clin Chim Acta The metabolic consequences of CPT deficiency also differen- 143:23-27 tiated muscular and hepatic cell lines; only the latter exhibited 17. Saggerson ED, Carpenter CA, Tselentis BS 1982 Effects of thyroidectomy and reduced long-chain fatty acid oxidation. These data might be starvation on the activity and properties of hepatic carnitinepalmitoyltrans- compared with clinical consequences of CPT defects in hepatic ferase. Biochem J 208:667-672 18. Solberg ME 1972 Different carnitinepalmitoyltransferase~in calf liver. Biochim and muscular forms. It was striking that, despite liver CPT Biophys Acta 280:422-433 deficiency in both forms (4), hypoketotic hypoglycemia only 19. Stakkestad JA, Bremer J 1983 The outer carnitinepalmitoyltransferaseand occurred in the hepatic form. These data suggest that CPTl regulation of fany acid metabolism in rat liver in different thyroid states. activity determines overall fatty acid oxidation in fibroblasts and Biochim Biophys Acta 750:244-252 20. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ 195 1 Protein measurement liver as well. The lack of muscular symptoms in the hepatic with the folin phenol reagent. J Biol Chem 193:265-275 presentation is puzzling. Along this line, we hope to perform 21. Bremer J 1968 Long-chain acylcarnitines. Biochem Prep 12:69-73 muscle biopsies in our two hepatic patients in order to measure 22. Murthy MSR, Pande SV 1984 Mechanism of carnitine acylcarnitine translo- muscular CPT activities. case-catalyzed import acylcarnitines into mitochondria. J Biol Chem 259:9082-9089 23. Rebouche CJ, Engel AG 1983 Carnitine metabolism and deficiency syndromes. REFERENCES Mayo Clin Proc 58533-540 24. Hale DE, Batshaw ML, Coates PM, Frerman FE, Goodman SI, Singh I, Stanley 1. Ahlborg C, Felig P, Hagenfeldt L 1974 Substrate turnover during prolonged CA 1985 Long-chain acylCoenzymeA dehydrogenase deficiency: an inher- exercise in man: splanchnic and leg metabolism of glucose, free fatty acids ited cause of nonketotic hypoglycemia. Pediatr Res 19:666-67 1 and amino acids. J Clin Invest 53:1080-1090 25. Zierg S, Engel AG 1985 Regulatory properties of a mutant carnitinepalmitoyl- 2. Mc Gany JD, Foster DW 1980 Regulation of hepatic fatty acid oxidation and transferase in human skeletal muscle. Eur J Biochem 149:207-214