Systemic triglyceride storage disease with normal carnitine: a putative defect in long-chain fatty acid metabolism

Journal of the Neurological Sciences, 1988, 85:149-159

149

Elsevier JNS 02996

Systemic triglyceride storage disease with normal carnitine: a putative defect in long-chain fatty acid metabolism Hiroshi Ibayashi 1, Hiroshi Ideguchi 1, N a o h i k o H a r a d a l, Shinji Ishimoto 2 and Ikuo G o t o 2 ~Third Department of Internal Medicine and 2Department of Neurology, Faculty of Medicine, Kyushu University. 3-1-I MaidashL Higashi-ku, Fukuoka 812 (Japan)

(Received 30 June, 1987) (Revised, received 15 January, 1988) (Accepted 20 January, 1988)

SUMMARY A 45-year-old Japanese man presented with lipid storage myopathy, fatty liver, cardiomyopathy, vacuolated leukocytes (Jordans' anomaly) and perceptive deafness. His parents were consanguineous and his younger sister was also affected. Histopathological and biochemical studies revealed an abnormal accumulation oftriglyceride in muscle, liver, leukocytes, gastrointestinal endothelial ceils and cultured skin fibroblasts. On electron microscopy, the vacuoles lacked limiting membranes and were adjacent to the mitochondria. Total and free carnitines in muscle were normal levels. Production rate of 14CO2 or acid-soluble [ laC]metabolites from [ 1-14C]palmitate in the patient's cells was decreased to about 50~o of that in control cells, whereas that from [ 1-14C]butyrate was normal. Long-chain fatty acyl esterase activities in the patient's leukocytes were normal at both pH 4.0 and pH 8.0. Despite the strong suggestion of an impaired metabolism of long-chain fatty acids, there were no evidences of abnormalities in carnitine metabolism or uptake of fatty acids into cells. The disorder is clinically different from defects in carnitine metabolism, defects in the camitineacylcarnitine translocase system or in mitochondrial fl-oxidation enzymes. Although the underlying metabolic defect has not been elucidated, this disease seems to be an autosomal-recessively inherited disorder of systemic triglyceride storage, probably due to an impaired regulation of lipolysis and triacylglycerol synthesis.

Correspondence to: Hiroshi Ideguchi, M.D., Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.

0022-510X/88/$03.50 © 1988Elsevier Science Publishers B.V. (Biomedical Division)

150 Key words: Lipid storage myopathy; Fatty liver; Jordans' anomaly; Long-chain fatty acids; Carnitine

INTRODUCTION Since the discovery of a human carnitine deficiency syndrome (Engel and Angelini 1973), data on 43 patients with myopathic or systemic carnitine deficiency have been reported (Engel 1986). It n o w appears, however, that most carnitine deficiency syndromes are secondary to an inborn error o f metabolism, and several of these errors have been identified. These include defects in enzymes o f the mitochondrial fl-oxidation (Turnbull et al. 1984; Coates et al. 1985; Hale et al. 1985), o f the degradative pathways of branched-chain amino acids (Roe et al. 1982; Chalmers et al. 1983; Roe et al. 1983; Stanley et al. 1983) and of the mitochondrial respiratory chain or oxidative phosphorylation (Sengers et al. 1983; Clark et al. 1984). On the other hand, some patients with lipid storage myopathy have a normal level of carnitine, hence their basic defects remain unclear (Chanarin et al. 1975; Jerusalem et al. 1975; Miranda et al. 1979; Angelini et al. 1980; Snyder et al. 1982; Askanas et al. 1985). We describe here a patient with lipid storage m y o p a t h y associated with fatty liver, cardiomyopathy, Jordans' anomaly and high-tone hearing loss. This disorder resembles Chanarin disease and seems to be a variant form of Chanarin disease, which lacks congenital ichthyosis. A putative defect in fatty acid metabolism in our patient is discussed.

CASE REPORT A 45-year-oldJapanese man was admitted to Kyushu University Hospital in October 1985because of lumbago and stiffness of the lower extremities. He was born of consanguineons parents after a full term uneventful pregnancy and delivery. Since childhood he had had a slight hearing impairment and easy fatigability but normal daily activities, outings and competition in sports presented no difficulty. Several years before, a cardiomegaly was diagnosed. In July 1984, he began to complain of lumbago and stiffness of the lower extremities, especially alter his finishing daily work. The symptoms tended to exacerbate by the end of week and improve with rest. He had never experienced myoglobinuria nor attacks resembling Reye's syndrome. Physical examination on admission revealed a slight weakness of anterior tibial muscles, hypertrophy of bilateral calf muscles, a slight impairment of vibration sense and perceptive deafness. There was no muscle atrophy or sensory disturbance. The liver was not palpable. Neither ichthyosis nor retinitis pigmentosa was evident. Laboratory data were as follows:hemoglobinwas 16.4g/dl, leukocytes 6500//zl with normal differential count and platelets 18.0 x 104/#1. The peripheral blood smear showed a number of vacuoles in the cytoplasm of granulocytes and monoeytes (Jordans' anomaly). Creatine kinase was 1150 U/1 (normal: 20-90) with 96.8% of MM-type and 3.2% of MB-type, lactate dehydrogenase 589 U/1 (normal: 120-250), GOT 85 U/l, GPT 134 U/l, myoglobin 180 ng/ml and creatine 1.91 mg/dl. The 75-g oral glucose tolerance test showed a diabetic pattern (106 mg/dl at fasting and 245 mg/dl after 2 h). Serum lipid analysis was normal except for a slight increase in triglyceride(232 mg/dl). Nevertheless, prolonged fasting (18 h) caused no increase of serum ketone bodies. The ischemic forearm test showed a normal elevation of pyruvate and lactate in the blood. Cardiomegaly was noted on the chest X-ray film and complete right bundle branch

151 block was present in the ECG. Ultrasonic cardiographyshowed an increased echo levelof the left ventricular endocardium and a decreased ejection fraction. These findings suggestedthe presence of cardiomyopathy. Ultrasonic tomographyof the abdomen revealed a mild hepatosplenomegalyand an increased echogenicity of the liver. EMG showed a low amplitude and short duration of M.U. potentials in quadriceps femoris, tibialis anterior and gastrocnemius, thereby indicating a myopathy. Treatment with riboflavin,pantethine or D,L-carnitine (3 g p.o. daily for 2 months) had no effect on either clinical symptoms or laboratory data.

Family study His father and his maternal grandfather were first cousins. His father had died of gastric cancer and his mother is healthy. There are seven siblings, three have died and four are alive. His 43-year-old sister has similarabnormalities;increased creatine kinase (447 U/l) and lactate dehydrogenase(363 U/l) in serum, fatty liver, Jordans' anomaly and high-tone hearing loss. While other three siblings have diabetes mellitus, his own two children are healthy. MATERIALS AND METHODS

Histopathological examinations Tissues were obtained by biopsy from muscle (left deltoid muscle), liver, stomach, rectum and skin. A portion of the muscle was frozen in isopentane and cryostat-cut sections were processed for histochemical stainings including PAS, Sudan III, Sudan black B, modified Gomori trichrome, NADH-tetrazolium reductase, ATPase (pH 9.4 and 4.2) and acid phosphatase. Cultured skin fibroblasts and cryostat-cut sections of liver, stomach and rectum were also processed for lipid staining. For electron microscopy, muscle and liver specimens and peripheral blood buffy coat were fixed with cacodylated formaldehyde-glutaraldehyde solution, post-fixed with osmium tetroxide and embedded in Epon. The thin sections were stained with uranyl acetate and lead citrate. For examination of peroxisomes, glutaraldehyde-fixed liver slices were reacted with 3,3'-diaminobenzidine (DAB) before fixation with osmium tetroxide.

Analysis of accumulated lipid Preparation of leukocytes was based on the method of Fallon et al. (1962). Fibroblasts grown in Eagle's M E M supplemented with 10~o FCS were washed with phosphate-buffered saline and harvested with trypsin. Washed packed leukocyte or fibroblast pellets containing a known number of cells were extracted with a mixture of chloroform, methanol and water (2" 1" 0.8). After centrifugation, the lower phase was collected and spotted on a silica gel thin-layer plate (Merck, 10 x 20 cm, 0.25 m m thick). Neutral lipids were separated with the following solvent systems: (1) petroleum ether/diethyl ether/acetic acid (80 : 20 : 1, 70 : 30 : 1, 90 : 10 : 1), (2) stepwise development; benzene/diethyl ether/ethanol/acetic acid (50 : 40 : 2 : 0.2), followed by hexane/diethyl ether (94:6). Spots were developed with 4 0 ~ sulfuric acid spray and charring. The standard mixture (Nu-Chek-Prep, Inc) contained cholesterol, cholesteroyl oleate, triolein, oleic acid and methyl oleate. Quantitation of lipids were carried out densitometrically. The fatty acid composition of the triglyceride fraction was determined by derivatization to fatty acid methyl esters with 14~o BF4/CH3OH , followed by separation by gas-chromatography using 10~o Silar 10C on chromosorb W H P

152 (100-120 mesh) with helium (50 ml/min) as carrier gas. The thermal program was 155-260 °C at 4 °C/min.

Assays of carnitine and carnitine palmityltransferase activity Carnitine was assayed by a highly sensitive radiometric method described by McGarry and Foster (1976). For assay of free carnitine in tissues, the reaction system contained [acetyl-l-14C]acetyl-CoA, an excess of carnitine acetyltransferase, sodium tetrathionate and tissue extracts. Total camitine in tissues was determined after alkaline hydrolysis (0.1 N KOH, 56 ° C, 1 h). Muscle carnitine palmityltransferase (CPT) was measured by the isotope exchange method of Scholte et al. (1979).

Assays of acid and alkaline long-chain fatty acyl esterase activity Acid and alkaline long-chain fatty acyl esterases were measured with p-nitrophenylpalmitate as substrate at pH 4.0 and 8.0, respectively, based on the method of Mahadevan and Tappel (1968).

Metabolic studies Washed leukocytes (3 - 5 x 10 6 in 1 ml of phosphate-buffered saline) from the patient and normal subjects were incubated at 37 °C with [ 1 - 14C]palmitate (0.1 #Ci, 1.72 nmol in each tube) or [1-~4C]butyrate (0.1 #Ci, 1.79 nmol in each tube). After specified time intervals, liberating 14CO2 was collected in 1 M hyamine hydroxide, which was used to determine the radioactivity. A similar experiment was performed using cultured skin fibroblasts. The fibroblast monolayers from the patient and normal subjects grown in 6-cm diameter culture dishes were washed and suspended with 1 ml of Eagle's MEM containing [ 1 - ~4C]palmitate (about 2 x 105 cpm per dish). After incubation at 37 °C for 2 h, the reaction was stopped by the addition of perchloric acid and the acid-soluble 14C-metabolites were determined. For measurement of total uptake of [1-1aC]palmitate into cells, the fibroblasts incubated under the sa/ne conditions as described above were washed 3 times with PBS and solubilized twice with 1 ml of 0.2~o Triton X-100, 50 mM potassium phosphate (pH 7.5). The extracts were pooled and used to determine the radioactivity.

RESULTS

Histopathological observations The peripheral blood smear showed a number of vacuoles in the cytoplasm of granulocytes and monocytes (Fig. la). In the smear of bone marrow aspirates, the same vacuoles were also observed in immature granuloid cells, plasma cells, megakaryocytes, and rarely in erythroblasts and lymphocytes. These vacuoles were stained with Sudan III or Oil red O, a finding consistent with so-called Jordans' anomaly (Jordans 1953). The left deltoid muscle biopsy demonstrated a mild to moderate variation of muscle fiber size (30-80 #m), a slight increase of internal nuclei and an increase of connective tissue without inflammatory cells. The most characteristic abnormality was

153

Fig. 1. Light micrographs of leukocytes,muscle, liver and cultured skin fibroblasts. (a) Leukoeyteswith numerous cytoplasmic vacuoles (Jordans' anomaly) (May-Grfinwald-Giemsa, ×400, bar 40#m). (b) Musclebiopsyspecimenshowinga marked increaseof sudanophilie droplets, especiallyin type 1 fibers (Sudan black B, × 400, bar 40 #m). (e) Liver biopsy specimen showing a moderate fatty degenerationin central zone oflobuli(hematoxylin-eosin, x 100,bar 160/~m).(d) Cultured skin fibroblastscontaininglipid droplets in cytoplasm(Oil red O, × 100, bar 160#m). a marked increase of sudanophilic droplets in the muscle fibers, prominently in type I fibers (Fig. lb). There were no abnormalities in other histochemical stainings, including PAS, modified G o m o r i trichrome, NADH-TR, ATPase and acid phosphatase. On electron microscopic examination, numerous vacuoles were located in rows between the myofibrils, lacked discernible limiting membranes and were adjacent to or surrounded by mitochondria (Fig. 2). Liver biopsy showed a moderate fatty metamorphosis in the central zone of the lobuli (Fig. lc). Mallory bodies were absent. On electron microscopy, numerous lipid droplets without limiting membranes were observed in the cytoplasm of the hepatocytes (Fig. 3a). In addition, some crystalloid substances were occasionally detected in some mitochondria (Fig. 3b) and rarely in the cytoplasm. Peroxisomes were normal in number and catalase activity. Lysosomes were increased in number. Lipid accumulation was demonstrated not only in blood cells, muscle and liver but also in endothelial cells of the stomach and colon and cultures of skin fibroblasts (Fig. ld). Biochemical examinations The nature and amount of accumulated lipid were studied by thin-layer chromatography of extracts from peripheral leukocytes and cultured skin fibroblasts. As shown

154

Fig. 2. Electron micrographs of muscle. (a) Transverse section of muscle demonstrating accumulation of intermyofibrillar lipid droplets, without limiting membranes. Numerous, small mitochondria are adjacent to lipid droplets ( x 21000, bar 1/~m). (b) Longitudinal section of muscle fiber showing lipid droplets in rows between myofibrils ( x 9300, bar 2 #m). in Fig. 4, there was a p r o m i n e n t increase of triglyceride in the patient's cells, which was confirmed by other solvent systems. Triglyceride content in the patient's leukocytes determined densitometrically was increased m o r e than 20 times in c o m p a r i s o n to that o f control cells. Analysis o f fatty acid c o m p o s i t i o n o f the triglyceride fraction by gas c h r o m a t o g r a p h y revealed that 86~o o f fatty acids were long-chain fatty acids o f 16 or 18 c a r b o n s ( d a t a not shown). TABLE 1 CARNITINE AND CARNITINE PALMITYLTRANSFERASE ACTIVITY

Carnitine serum (nmol/ml) muscle ~mol/g wet weight) total free CPT activity (muscle) (nmol/min/g wet weight) " Values are expressed as mean + SD.

Patient

Control

76.0

45.0 + 7.4~ (n=6)

3.31 2.85 33.3

3.46 + 0.29a (n = 5) 3.04+ 1.10"(n=5) 61.5 + 13.2" (n=7)

155

Fig. 3. Electron micrographs of liver. (a) Lipid droplets (L) lack discernible limiting membranes and are surrounded by mitochondria (M). Peroxisomes (P) are apparently normal (DAB staining; x 20 500, bar 1 #m). (b)Intra-mitochondrial crystalloid inclusion (arrows) observed in hepatocytes (x48000, bar 0.2 #m).

Camitine content and muscle CPT activity were also determined (Table 1). Serum camitine was increased. Total and free camitines in muscle (left deltoid muscle) were within normal range, suggesting that there was no increase of acylcamitines. Muscle CPT activity was slightly decreased but above the level of typical CPT deficiency. Oxidation of long-chain fatty acid in leukocytes or cultured fibroblasts was examined in vitro (Table 2). Production of 14COu from [ 1-14C]palmitate in the patient's leukocytes was decreased to about 50~o of that in control cells, which could not be restored by the addition of increasing amounts of L-camitine (0.5 raM, 1.0 mM) in the incubation medium. In experiments using cultured fibroblasts, acid-soluble 14C-metabolites in the patient's fibroblasts was markedly decreased (43~o of control), while the total uptake of [ 1-14C ]palmitate was slightly decreased (82 Yo of control). Utilization of palmitate incorporated in patient's fibroblasts was 3.3Y/o, whereas that of control fibroblasts was 6.3 ~ . The above two experiments gave a consistent result, namely the oxidation of [ 1-14C]palmitate was suppressed to about 5 0 ~ of normal. In contrast, oxidation of [ 1-14C]butyrate in the patient's leukocytes was within normal range (88 Y/o of control). Acid esterase activity in the patient's leukocytes was 20.7 nmol of p-nitrophenylpalmitate cleaved/min/mg protein (control: 22.5 + 3.7, mean + SD, n = 6). Alkaline esterase activity in the patient's leukocytes was 4.0 nmol of p-nitrophenylpalmitate cleaved/min/mg protein (control: 4.0 + 0.2, mean + SD, n = 6).

156

C hol E

FAMe

TG

FA

Chol

PL

control

Patient Standard

Fig. 4. Thin-layerchromatographyof accumulatedlipid in leukocytes.Solventis petroleum ether/diethyl ether/acetic acid (80:20:1). Control leukocytes(left); patient's leukocytes(center); and standard lipid mixture(right)containingcholesterol(Chol),cholesteroyloleate(CholE), triolein(TG), oleicacid(FA) and methyl oleate (FAMe). Note the marked increase of triglyceridein the patient's leukoeytes.

DISCUSSION We treated a patient with lipid storage myopathy, fatty liver, cardiomyopathy, Jordans' anomaly and perceptive deafness. Histochemical examination of biopsy specimens revealed an abnormal accumulation of lipid in muscle fibers (especially in type I fiber), hepatocytes, leukocytes, endothelial cells of stomach and colon, and cultured skin fibroblasts. On electron microscopy, numerous globules without limiting membranes were observed in muscle fibers, hepatocytes and leukocytes. Accumulated lipid in leukocytes and fibroblasts was identified by thin layer chromatography as triglyceride, containing a normal pattern of ordinary long-chain fatty acids. In vitro metabolic studies showed that the oxidation rate of palmitate in the patient's cells was decreased to about half of that of control cells, whereas that of butyrate was fairly normal. Although these results implied an impaired metabolism of tong-chain fatty

157 TABLE 2 OXIDATION OF [I-14C]PALMITATE BY LEUKOCYTES AND FIBROBLASTS Patient (A) Leukoeytes (pmol 14CO2/106 celis/h) Exp. 1 Exp. 2 - L-carnitine + L-¢arnitine (0.5 mM) + L-carnitine (1.0 mM) (B) Fibroblasts (kdpm/m8 prot./h) total [14C]palmitate uptake acid-soluble ~4C-metabofites utilization efficiency (%)b

8.3

Control

15.5 ± 1.1a (n = 4)

8.7 10.8 11.1

15.0 17.4 18.5

186 6.1 3.3

228 14.3 6.3

" Value is expressed as mean ± SD. b Utilization efficiency is expressed as a percentage of acid-soluble 14C-metabolites against total [14C]palmitate uptake.

acids, there were no abnormalities in carnitine content and carnitine palmityltransferase activity in muscle. Moreover, an addition of L-carnitine could not restore the decreased rate of long-chain fatty acid catabolism. Acid (lysosomal) and alkaline (microsomal) long-chain fatty acyl esterase activities were normal in the patient's leukocytes. The present disorder is accompanied by relatively mild clinical symptoms, as compared with carnitine deficiency or CPT deficiency. He denied previous attacks of myoglobinuria or attacks resembling Reye's syndrome. Clinical features of the patient differ from those of Kearns-Sayre syndrome, Refsum's disease or Wolman's disease. In 1975, Chanarin and coworkers described a patient with systemic neutral lipid storage associated with congenital ichthyosis, and showed that about 6 times as much ['4C]palmitate was incorporated in the triglyceride fraction in the patient's fibroblasts and metabolism to 14CO2 was only 25~ of the rate observed in normal fibroblasts (Chanarin et al. 1975; Slavin et al. 1975). Similar cases were also described by Miranda et al. (1979) and Angelini et al. (1980). In those cases, clinico-biochemical findings are consistent in the following points: (1) the presence of congenitai ichthyosis, (2) systemic accumulation of triglyceride (muscle, liver, leukocytes, gastrointestinal endothelial cells and cultured fibroblasts), (3) no abnormality in content of carnitine or ratio of free and acyl carnitine in tissues, and (4)a putative genetic defect in long-chain fatty acid metabolism. The present disorder resembles those diseases in several respects except for the absence of congenital ichthyosis, and therefore it is likely to be a new variant of Chanarin disease. Indeed, despite the strong suggestion of impaired fatty acid metabolism, studies in our patient showed no evidences of accelerated uptake of long-chain fatty acids into cells or abnormality in carnitine metabolism. The activity of long-chain fatty acyl esterase was also normal in the patient's leukocytes, but this does not completely exclude a possible defect of lipolysis since the non-physiological

158 monoester ofpalmitate was employed in the assay as indicated by Miranda et al. (1979). The present disorder is not likely due to defects in carnitine-acylcarnitine translocase system or in mitochondrial fl-oxidation enzymes, because these defects are generally linked to secondary carnitine deficiency and altered proportions of carnitine and carnitine esters. The precise nature of the biochemical defect in our patient has not been elucidated. However, the defect possibly may lie in a regulation of lipolysis and triacylglycerol synthesis. The decreased rate of [ 1-~4C]palmitate oxidation may be a reflection of an expanded intracellular fatty acid pool which lowers intracellular specific activity of [ 1-14C]palmitate or a reflection of an increased incorporation of [ 1-14C]pal mitate into triglyceride. The present disorder, together with Chanarin disease, is a new clinical entity of systemic triglyceride storage, distinctive from carnitine deficiency syndrome or other disorders of known etiologies causing lipid storage in tissues. Further biochemical examinations may provide some clues to clarify the underlying defect.

ACKNOWLEDGEMENTS

We wish to thank Drs. Masanori Nakagawa and Akihiro Igata for the muscle camitine and CPT assays, Drs. Sadaki Yokota and Tetsuo Kitaguchi for the electron microscopic examinations, and Dr. Takashi Hashimoto for the metabolic study of fibroblasts. We are also pleased to acknowledge the helpful suggestions of Dr. Hideo Sugita.

REFERENCES Angelini, C., M. Philippart, C. Borrone, N. Bresolin, M. Cantini and S. Lucke (1980) Multisystem triglyceride storage disorder with impaired long-chain fatty acid oxidation. Ann. Neurol., 7: 5-10. Askanas, V., W.K. Engel, H.H. Kwan, N.B. Reddy, T. Husainy, J. Carlo, T. Siddique, R.J. Schwartzman and C.J. Hanna (1985 ) Autosomal dominant syndrome of lipid neuromyopathy with normal carnitine: successful treatment with long-chain fatty-acid-free dict. Neurology, 35: 66-72. Chalmers, R. A., C. R. Roe, B. M. Tracey, T.E. Stacey, C. L. Hoppel and D. S. Millington (1983) Secondary carnitine insufficiency disorders of organic acid metabolism: modulation of acyl-CoA/CoA ratios by L-carnitine in vivo. Biochem. Soc. Trans., 11: 724-725. Chanarin, I., A. Patel, G. Slavin, E.J. Wills, T.M. Andrews and G. Stewart (1975) Neutral-lipid storage disease: a new disorder of lipid metabolism. Brit. Med. J., 1: 553-555. Clark, J.B., D.J. Hayes, J. A. Morgen-Hughes and E. Byrne (1984) Mitochondrial myopathies: disorders of the respiratory chain and oxidative phosphorylation. J. Inher. Metab. D~., 7 (Suppl. 1): 62-68. Coates, P.M., D.E. Hale, C.A. Stanley, B.E. Corkey and J.A. Cortner (1985) Genetic deficiency of medium-chain acyl coenzyme A dehydrogenase: studies in cultured skin fibroblasts and peripheral mononuclear leukocytes. Pedlar. Res., 19: 671-676. Engel, A.G. and C. Angelini (1973) Carnitine deficiency of human skeletal muscle with associated lipid storage myopathy: a new syndrome. Science, 179: 899-902. Engel, A.G. (1986)In: A.G. Engel, B.Q. Banker (Eds.), Myology, McGraw-HiU, New York, pp. 1663-1696. Fallen, H.L, E. Frei, J.D. Davidson, J. S. Trier and D. Burk (1962) Leukocyte preparations from human blood: evaluation of their morphologic and metabolic state. J. Lab. Clin. Med., 59: 779-791. Hale, D.E., M.L. Batshaw, P.M. Coates, F.E. Frerman, S.I. Goodman, I. Singh and C.A. Stanley (1985) Long-chain acyl coenzyme A dehydrogenase deficiency: an inherited cause of nonketotic hypoglycemia. Pediat. Res., 19: 666-671.

159 Jerusalem, F., H. Spiess and G. Baumgartner (1975) Lipid storage myopathy with normal carnitine levels. J. Neurol. Sci., 24: 273-282. Jordans, G. H.W. (1953) The familial occurrence of fat containing vacuoles in the leukocytes diagnosed in two brothers suffering from dystrophia musculorum progressiva (ERB). Acta Med. Scand., 145: 419-423. Mahadevan, S. and A. L. Tappel (1968) Hydrolysis of higher fatty acid esters of p-nitrophenol by rat liver and kidney lysosomes, Arch. Biochem. Biophys., 126: 945-953. McGarry, J.D. and D.W. Foster (1976) An improved and simplified radioisotopic assay for the determination of free and estedfied carnitine, J. Lipid Res., 17: 277-281. Miranda, A., S. DiMauro, A. Eastwood, A. Hays, W.G. Johnson, M. Olarte, R. Whitlock, R. Mayeux and L.P. Rowland (1979) Lipid storage myopathy, ichthyosis, and steatorrhea. Muscle Nerve, 2: 1-13. Roe, C.R. and T.P. Bohan (1982) L-Carnitine therapy in propionicacidemia. Lancet, 1:1411-1412. Roe, C.R., C.L. Hoppel, T.E. Stacey, R.A. Cbalmers, B.M. Tracey and D. S. Millington (1983) Metabolic response to carnitine in methylmalonic aciduria, Arch. Dis. Child., 58: 916-920. Scholte, J. R., F. G. I. Jennekens and J. J. B. V. Bonvy (1979) Carnitine paimityl transferase II deficiency with normal carnitine palmityl transferase I in skeletal muscle and leukocytes. J. Neurol. Sci., 40: 39-51. Sengers, R.C.A., J.C. Fischer, J.M.F. Trijbels, W. Ruitenbeek, A.M. Stadhouders, H.J. Ter Laak and H.H.J. Jaspar (1983) A mitochondrial myopathy with a defective respiratory chain and carnitine deficiency, Eur. J. Pediat., 140: 332-337. Slavin, G., E.J. Wills, J.E. Richmond, I. Chanarin, T. Andrews and G. Stewart (1975) Morphological features in a neutral lipid storage disease. J. Clin. Pathol., 28: 701-710. Snyder, T. M., B.W. Little, G. Roman-Campos and J.B. McQuillan (1982) Successful treatment of familial idiopathic lipid storage myopathy with L-carnitine and modified lipid diet. Neurology, 32:1106-1115. Stanley, C.A., D.E. Hale, D.E.H. Whiteman, P.M. Coates, M. Yudkoff, G.T. Berry and S. Segal (1983) Systemic carnitine(carn) deficiency in isovaleric acidemia (IVA). Pedlar. Res., 17: 296A. Turnbull, D. M., K. Bartlett, D. L. Stevens, K. G. M. M. Alberti, G.J. Gibson, M. A. Johnson, A. J. McCulloch and H. S.A. Sherratt (1984) Short-chain acyl-CoA dehydrogenase deficiency associated with a lipidstorage myopathy and secondary carnitine deficiency. New Engl. J. Med., 311: 1232-1236.