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Late onset lipid storage myopathy due to multiple acyl CoA dehydrogenase deficiency triggered by valproate
Neuromuscular Disorders, Vol. 1, No. 4, pp. 247-252, 1991 Printed in Great Britain
096~8966/91 $3.00 + 0.00 Pergamon Press plc
LATE ONSET LIPID STORAGE MYOPATHY DUE TO MULTIPLE ACYL CoA D E H Y D R O G E N A S E DEFICIENCY TRIGGERED BY VALPROATE ALEXANDROSPAPADIMITRIOU*t+ +and SERENELLASERVIDEIt§ *Department of Neurology, Red Cross Hospital, Athens 11526,Greece;tCollege of Physiciansand Surgeons of Columbia University, New York, U.S.A. (Received 18 December 1990,"accepted 5 September 1991)
Abstract--Late onset Multiple Acyl CoA dehydrogenase (MAD) deficiency myopathy is a rare disorder. Only five cases have been reported. We report one case with MAD deficiency in which the clinical features appeared during valproic acid therapy. A 47-yr-old man taking valproic acid for 4 months presented nocturnal calf cramps, exercise intolerance, difficulty in climbing stairs and shortness of breath. Muscle biopsy revealed ragged red fibres and neutral lipid storage. Electron microscopy showed enlarged abnormal mitochondria with abnormal internal structure. The total and free muscle carnitine was decreased. The activities of all short, medium and long chain acyl CoA dehydrogenases were 40% of the normal. In this case a partial defect of MAD is noted, possibly triggered by valproic acid, causing the clinical manifestations of the pre-existing myopathy. After discontinuation of the drug a clinical improvement was observed while therapy with riboflavin resulted in a total relief of the symptoms. Key words: Lipid storage myopathy, Multiple Acyl CoA dehydrogenase deficiency, valproic acid.
INTRODUCTION Multiple Acyl CoA dehydrogenation (MAD) deficiencies are a group of rare diseases in which three clinical subgroups have been proposed [1]. The first is known as neonatal onset MAD and is characterized by hypoglycaemia, severe acidosis and is associated with premature birth and congenital abnormalities. The second is also fatal and is characterized by a similar clinical course associated with cardiomyopathy but with no congenital abnormalities. The third group constitutes late onset and a milder form of MAD, with clinical symptoms appearing during adult life. Biochemically, and depending on the type of organic acids excreted in the urine, the glutaric aciduria type II, ethylmalonic adipic aciduria and riboflavin responsive form are included in the MAD group [2]. Five documented cases of late onset M A D myopathy have been reported [3-7] and clinical heterogeneity has been found. In some of them [4-7] clinical improvement during riboflavin therapy has been described. There are no known Author to whom offprintrequests should be addressed. § Current address: Department of Neurology, University Cattolica del Sacro Cuore, Largo Gemelli8, Rome, Italy.
precipitated factors apart from one case in which the symptomatology developed during pregnancy [4]. We report one case with M A D deficiency in which the clinical features of the disease appeared during valproate therapy. After discontinuation of the drug a clinical improvement was observed while therapy with riboflavin resulted in a total relief of the symptoms.
CASE REPORT
The patient is a 47-yr-old man without any family history of neuromuscular disease. He has two normal children and his parents are in good health. The patient was well until the age of 18 yr when he had four major seizures and was treated with phenobarbital and phenytoin. When the patient was 43 yr old the antiepileptic drug was changed to valproate (600 mg daily) and 4 months later he began to have typical nocturnal calf cramps and exercise intolerance. After walking for relatively short distances, up to 500 m, he began having pains throughout the legs; however, these symptoms were relieved when he stopped walking. He had difficulty
247
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A. PAPADIMITRIOU and S. SERVIDEI
climbing stairs and had to take a rest every 10 Finally it was decided to place him on ribosteps and also had to sit down in order to shave. flavin, 100 mg daily, where a dramatic improveHe also felt shortness of breath and sometimes ment was shown. Before starting therapy a had difficulty in chewing and was able to eat only treadmill test was carried out to determine the soft food. Progressively the patient's condition patient's physical ability; he was able to excel on deteriorated and he was unable to perform any the bicycle for 2½ min after which he became daily activities. He was admitted to the depart- exhausted. Ten days after riboflavin treatment ment of Neurology at the Red Cross Hospital of and following a repeat treadmill test the patient Athens, where the routine laboratory tests and was able to accelerate for a full 15 rain. At the neurological examination during rest were nor- present time, the patient does not seem to have mal. CPK was 400 U 1-1 (normal < 100) any of the previous symptoms and feels well. Aldolase was 18 U 1-~ (normal < 9) and serum CPK is normal and lactic acid is in the upper lactic acid was 19.2/~g (normal 8-12/tg ml-~). normal limit. He has been on this regimen since then and is now able to perform manual work The Tensilon test was negative. and all other necessary activities. During hospitalization the valproic acid was discontinued. He was started on Cambamazepine and a slight improvement was observed. A MATERIALS AND METHODS muscle biopsy was performed for histochemical and biochemical analysis. He started also treatThree muscle biopsies (one from the left deltoid, ment with DL-carnitine (3 g daily) and was placed one from the left quadriceps and another from on a low fat diet; however, his symptoms con- the right quadriceps) in three different periods tinued during the next 5 months. In the same were obtained. The muscle was divided into four period he was admitted to the Columbia Presby- portions for light and electron microscopy, terian Medical Center, New York. Laboratory histochemical and biochemical analysis. For investigation urinalysis, ESR, CBC, T3, T4, histochemical studies muscle was frozen in liquid TSH, serum electrolytes, glucose, BUN, creatinine, nitrogen-cooled isopentane, cross-sectioned in a Ca, P, total protein, cholesterol, bilirubin, cryostat and stained with haematoxylin-eosin, alkaline phosphatase, SGOT, SGPT were within adenosine triphosphatase at pH 9.4 - 4.3 oil red normal values. The CPK was increased to 128 U 0, NADH-TR, and modified trichrome staining. 1-I (normal 1-50), LDH was 223 (normal 90- For electron microscopic analysis muscle was 200). The ECG showed a right bundle branch fixed as described by Engel and Dale [8]. block. CSF protein was 58 mg%. Motor and For biochemical analysis the muscle biopsies sensory nerve conduction velocities were normal were immediately frozen in liquid nitrogen. and needle EMG showed a myopathic pattern. Muscle mitochondria were isolated by the Repetitive nerve stimulation was normal. During method of Bookelman et al. [9]. rest period the serum lactic acid was 4.3 mM 1-~ Total and free carnitine were measured radio(normal 0.5-2.2 mM 1-1) rising to 10.8 mM 1-1 chemically [10]. The beta oxidation enzymes after having exercise for only 1 min (control after were measured spectophotometrically in both 5 min exercise 6.6 mM 1-~). Organic acids in the crude muscle homogenate and in mitochondrial urine were not detected during this period. A preparation. Palmityl-CoA DH, Butyryl-CoA second muscle biopsy was performed. DH and Octanoyl-CoA DH were estimated Progressively his symptoms began to improve according to the method of Fong and Schultz and within 2 months he was able to walk more [11]. The reaction mixture contained 66 mM K than 500 m. However, after this exercise, he phosphate (pH 7.4) 100 /~M dichlorophenol started complaining of muscle cramps and indophenol (DCPIP), 1 /~M phenazine methoweakness which were relieved only when he sulphate, 0.03% BSA, 0.06% Triton-X100 and stopped exercise. Gradually the patient was able the appropriate substrate (Acyl CoA) in a final to resume his office work but his physical ability concentration of 0.2 mM. Crotonase, hydroxyacyl CoA DH and thiolase were measured as was limited. Approximately a year later a third muscle described previously [12]. biopsy was performed for repeated biochemical Mitochondrial enzymes were measured in crude analysis. However, the patient had still showed homogenates and in mitochondria preparations no physical difference in condition. In all the according to methods described previously [13]. laboratory examinations, CPK and lactic acid Non-collagen protein was estimated by the method of Lowry et al. [14]. during rest were always high.
L a t e Onset Storage M y o p a t h y
249
Fig. 1. Excess of s a r c o p l a s m i c neutral lipid (oil red 0 staining).
Table 1. Carnitine concentrations of muscle (/~mol g-')
RESULTS
Total
Free
0.500 0.502 0.920 2.34 + 0.68
0.350 0.434 0.700 1.87 5:0.66
Muscle biopsy. The muscle fibres were normal except for a few scattered atrophic fibres in E-H staining. About half of the fibres contained dark red granules in the modified Gomori trichrone stain. By the oil red 0 staining a similar proportion of muscle fibres contained an excess of sarcoplasmic neutral lipid (Fig. 1). By electron microscopy, unusual, enlarged, abnormal mitochondria with abnormal internal structure and lamellar concentric inclusions were found (Fig. 2A and B). Although the three muscle biopsies were performed at different times the morphology was similar. Biochemistry. Total and free carnitine were decreased (20--40% of the control) in all three biopsies (Table 1). It is important to note that the second and third biopsies were performed while
I st biopsy 2nd biopsy 3rd biopsy Controls (n= 20) x 5: S.D.
the patient was on carnitine therapy. The ratio of acylcarnitine to free carnitine was higher (0.42) as compared with normal controls (0.25). The mitochondrial enzymes in crude muscle and isolated mitochondria were normal in both the second and third biopsies (Tables 2 and 3). The fl-oxidation mitochondrial enzymes showed a decreased activity (40% of the control values) of all the short, medium and long chain fatty acyl CoA DH. All the other enzymes of fl-oxidation were normal (Table 4). The same results were also found in isolated mitochondria (Table 5).
Table 2. Mitochondrial enzymes in muscle homogenate (pmol rain 'g ~)
Cytochrome oxidase NADH Cyt c reductase Succinate Cyt c reductase Citrate synthetase Succinate dehydrogenase NADH dehydrogenase
Patient (2nd biopsy)
Patient (3rd biopsy)
4.43 3.89 1.24 20.50 1.41 37.10
3.35 3.78 0.99 18.35 0.99 26.10
Controls (n = 30) x ± S.D. 2.86 3.32 0.75 11.35 1.68 35.21
+ ± ± ± ± ±
0.53 t.20 0.26 2.40 0.45 6.37
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Fig. 2. (A and B) Unusual enlarged abnormal mitochondria with abnormal internal structure and lamellar concentric inclusions (EM). (A) Magnification 15,000 X and (B) magnification 30,000 x .
Late Onset Storage Myopathy
251
Table 3. Mitochondrial enzymes in isolated mitochondria (pmol min-~mg protein-t)
Cytochrome oxidase N A D H Cyt c reductase Succinate Cyt c reductase Citrate synthetase Succinate dehydrogenase N A D H dehydrogenase
Patient (2nd biopsy)
Patient (3rd biopsy)
1.02 0.305 0.291 2.440 0.218 0.223
1.04 0.271 0.209 1.322 0.175
Controls (n = 15) x ± S.D. 1.34 0.29 0.25 2.303 0.173 0.154
± 0.37 ± 0.04 5:0.04 ± 0.5 ± 0.04 ± 0.05
Table 4./?-oxidation mitochondrial enzymes in muscle (/tmot min- ~g-~)
Butyryl CoA D H Octanoyl CoA D H Palmityl CoA DH Crotonase Hydroxyl-acyl CoA DH Thiolase (C4)
Patient (2nd biopsy)
Patient (3rd biopsy)
0.35 0.59 0.45 15.59 7.39 5.71
0.32 0.31 0.52 13.70 6.80
Controls (n = 7) x±S.D. 1.15 0.98 1.23 14.73 5.00 5.15
± 0.20 5:0.19 ± 0.24 ± 1.72 5:1.18 ± 1.27
Table 5. fl-oxidation enzymes in isolated mitochondria (,umol min-~g protein-~)
Butyryl CoA DH Octanoyl CoA D H Palmityl CoA DH Crotonase Hydroxy-acyl CoA DH Thiolase (C4)
Patient (2nd biopsy)
Patient (3rd biopsy)
9.55 12.57 11.96 135.20 71.58 153.41
6.40 10.60 12.50 112.83 85.00
DISCUSSION
Late onset MAD deficiency is a rare disorder and only five cases have been described. In 1979 a 19-yr-old woman was presented with repeated episodes of hypoglycaemia, liver dysfunction and proximal myopathy. Increased quantities of glutaric acid, ethylmalonic acid, dicarboxylic acids with a six to ten carbons chain lengths were found in the urine of the patient. In cultured fibroblasts the ability to oxidize butyrate and lysine was reduced and multiple CoA DH deficiency was suggested [3]. In 1983 a 36-yr-old woman was described who, during pregnancy, presented with liver dysfunction and organic aciduria. The patient had eight other pregnancies resulting in one stillborn infant and six floppy infants who died during neurological distress. In all these pregnancies the mother had the same symptoms, during the last trimester [4]. In 1986 there was a report of a 17-yr-old girl with progressive muscle weakness, organic aciduria, extensive lipid accumulation in muscles and a reduction of mitochondria floxidation enzymes [5]. In 1988 a 39-yr-old man was presented with painful proximal myopathy,
Controls (n = 7) x ± S.D. 28.25 23.77 28.27 124.60 54.60 126.49
5:2.70 5:2.90 ± 3.90 + 12.56 5:11.98 5:21.88
organic aciduria, lipid accumulation in muscle and low acyl CoA dehydrogenase activity [6]. The most recent case [7] is o f a 12-yr-old girl presenting with easy fatigability and muscle weakness, lipid storage myopathy and organic aciduria. In our case there are similarities to those reported in the literature: that is myopathy, lipid accumulation in muscles, abnormal mitochondria, muscle carnitine deficiency and low acyl-CoA DH activity. However, it also presents two important differences: exercise intolerance is the main clinical symptom, symptomatology started during valproic acid therapy and improved slightly after discontinuation of the drug. Valproic acid is a branched chain fatty acid and inhibits the oxidation of fatty acids at the level of fl-oxidation I15]. It is also reported that valproic acid produces organic aciduria as in cases of MAD deficiencies [16]. Many patients tolerate valproic acid therapy well but others may present liver toxicity, hyperammonemia, and a hepatocerebral syndrome similar to Reye's syndrome. It has been suggested that the toxicity may be due to an unrecognized inborn error of metabolism in fatty acid oxidation [17,18].
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We believe that valproic acid played an important role in the appearance of symptomatology in our case. The residual activities of the three acyl-CoA DH enzymes were 40% of normal and were probably sufficient to facilitate fatty acid metabolism covering the clinical expression. By inducing valproic acid, which shares the same metabolic pathway, the enzymes become insufficient and therefore symptomatology starts. The low level of muscle carnitine in our case can be explained, as in all cases of acyl CoA DH deficiency, as a secondary phenomenon. The defect of the enzyme causes accumulation of acyl CoA in the muscle mitochondria. Carnitine reduces the amount of acyl CoA causing accumulation of acyl carnitine which is excreted in the urine resulting in secondary carnitine deficiency. The higher ratio of acylcarnitine: free in our case, is in accordance with previously described cases [5,19]. The effect of riboflavin was definitive in our case and there was complete relief of all symptoms. We have found in the literature few cases of MAD deficiency in which riboflavin showed clinical or biochemical improvement [5,20,21]. However, none of these cases showed complete relief, as in our case. The mechanism of the riboflavin effect is not known. Recently normalization of short-chain acyl CoA dehydrogenase after riboflavin therapy has been reported [7]. Riboflavin is a precursor of FAD which is a prosthetic group of Acyl CoA DH, ETF DH and play an important role in stimulating the residual activity of the deficient enzyme. It has been proposed that riboflavin responsive MAD may be due to a primary alteration of riboflavin or FAD metabolism causing accelerated breakdown or reduced synthesis of flavoprotein haloenzymes [2]. Acknowledgement--The authors would like to thank Pro-
fessor S. Di Mauro for his helpful discussion regarding the manuscript, and Dr E. Bonillia for electron microscopy study.
4.
5.
6. 7.
8.
9.
10. 11. 12.
13.
14. 15.
16. 17. 18.
REFERENCES
19. 1. Goodman S I, Frerman F. Glutaric acidaemia type II (multiple acyl CoA DH deficiency). J Inherit Metab Dis 1984; 7 (Suppl l): 33-37, 2. Di Donato S, Garavaglia B, Bloisi W, Colombo I, Finocchiaro G. Biochemical and molecular aspects of fl-oxidation defects in skeletal muscle. In: Benzi G, ed. Advances in Myochemistry. J Libbey Eurotext, 1989: 151-163. 3. Dusheiko G, Kew M C, Joffe B I, et al. Recurrent
20. 21.
hypoglycemia associated with glutarie aciduria type II in an adult. N Engl J Med 1979; 301: 1405-1409. Harpey J, Charpentrie C, Goodman S, Darbois Y, Lefehvre G, Sebbah J. Multiple acyl-CoA dehydrogenase deficiency occurring in pregnancy and caused by a defect in riboflavin metabolism in the mother. J Pediatr 1983; 103: 394-398. De Visser M, Scholte H, Schutgens R, et aL Riboflavin responsive lipid-storage myopathy and glutaric aciduria type II of early adult onset. Neurology 1986; 36: 367-372. Turnbull D, Shepherd I, Ashworth B, et al. Lipid storage myopathy associated with low acyl-CoA dehydrogenase activities. Brain 1988; 111: 815-828. Di Donato S, Gellera C, Peluchetti D, et al. Normalization of short-chain Acylcoenzyme A dehydrogenase after Riboflavin treatment in a girl with multiple Acyl CoA DH deficient myopathy. Ann Neurol 1989; 25: 479-484. Engel A G, Dale A D. Autophagic glycogenosis of late onset with mitochondrial abnormalities: light and electron microscopic observations. Mayo Clin Proc 1968; 43: 233-279. Bookelman H, Trijbels J M F, Sengers R A C, Janssen A M. Measurement of cytochromes in human skeletal muscle mitochondria isolated from fresh and frozen stored muscle specimens. Biochem Med 1978; 19: 366373. McGarry D J, Foster D W. An improved and simplified radioisotopic assay for determination of free and esterified carnitine. J Lipid Res 1976; 17:277-281. Fong J, Schultz H. On the rate-determining step of fatty acid oxidation in heart. J Biol Chem 1978; 253: 69176922. Trevisan C, Reichmann H, De Vivo D, Di Mauro S. Beta oxidation enzymes in normal human muscle and in muscle from a patient with an unusual form of myopathic carnitine deficiency. Muscle Nerve 1985; 8: 672-675. Di Mauro S, Nicholson J, Hays A, et al. Benign infantile mitochondrial myopathy due to reversible cytochrome c oxidase deficiency. Ann Neurol 1983; 14: 226-234. Lowry O H, Rosebrough N J, Farr A L, Randall R J. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-271. Thurston J, Haubart R. Hepatic toxicity of valproic acid: do inborn errors of liver metabolism underlie hepatic fatalities in some children. Ann Neuro11986; 20: 400 (abstr). Hardison 1-1,Kelley R, Stanley C, Berman P. Effect of valproic acid on ammonia and organic acid metabolism. Ann Neurol 1984; 16:378 (abstr). Gerber N, Dickinson R, Harland R C, et al. Reye-like syndrome associated with valproic acid therapy. J Pediatr 1979; 95: 142-144. Hayasaka K, Takahashi I, Kobayashi Y, Imuna K, Narisawa K, Tada K. Effects of Valpoate on biogenesis and function of liver mitochondria. Neurology 1986; 36: 351-356. StumpfD, Parker D, Angelini C. Carnitine deficiency, organic acidemias and Reye's syndrome. Neurology 1985; 35: 1041-1045. Carroll J E, Shumate J B, Brooke M H, Hagherg J M. Riboflavin responsive lipid myopathy and carnitine deficiency. Neurology 1981; 31: 1557-1559. Gregersen N, Christensen F, Christensen E, Kolvraa S. Riboflavin responsive Multiple acyl-CoA dehydrogenation deficiency. Acta Paediatr Scand 1986; 75: 676681,
096~8966/91 $3.00 + 0.00 Pergamon Press plc
LATE ONSET LIPID STORAGE MYOPATHY DUE TO MULTIPLE ACYL CoA D E H Y D R O G E N A S E DEFICIENCY TRIGGERED BY VALPROATE ALEXANDROSPAPADIMITRIOU*t+ +and SERENELLASERVIDEIt§ *Department of Neurology, Red Cross Hospital, Athens 11526,Greece;tCollege of Physiciansand Surgeons of Columbia University, New York, U.S.A. (Received 18 December 1990,"accepted 5 September 1991)
Abstract--Late onset Multiple Acyl CoA dehydrogenase (MAD) deficiency myopathy is a rare disorder. Only five cases have been reported. We report one case with MAD deficiency in which the clinical features appeared during valproic acid therapy. A 47-yr-old man taking valproic acid for 4 months presented nocturnal calf cramps, exercise intolerance, difficulty in climbing stairs and shortness of breath. Muscle biopsy revealed ragged red fibres and neutral lipid storage. Electron microscopy showed enlarged abnormal mitochondria with abnormal internal structure. The total and free muscle carnitine was decreased. The activities of all short, medium and long chain acyl CoA dehydrogenases were 40% of the normal. In this case a partial defect of MAD is noted, possibly triggered by valproic acid, causing the clinical manifestations of the pre-existing myopathy. After discontinuation of the drug a clinical improvement was observed while therapy with riboflavin resulted in a total relief of the symptoms. Key words: Lipid storage myopathy, Multiple Acyl CoA dehydrogenase deficiency, valproic acid.
INTRODUCTION Multiple Acyl CoA dehydrogenation (MAD) deficiencies are a group of rare diseases in which three clinical subgroups have been proposed [1]. The first is known as neonatal onset MAD and is characterized by hypoglycaemia, severe acidosis and is associated with premature birth and congenital abnormalities. The second is also fatal and is characterized by a similar clinical course associated with cardiomyopathy but with no congenital abnormalities. The third group constitutes late onset and a milder form of MAD, with clinical symptoms appearing during adult life. Biochemically, and depending on the type of organic acids excreted in the urine, the glutaric aciduria type II, ethylmalonic adipic aciduria and riboflavin responsive form are included in the MAD group [2]. Five documented cases of late onset M A D myopathy have been reported [3-7] and clinical heterogeneity has been found. In some of them [4-7] clinical improvement during riboflavin therapy has been described. There are no known Author to whom offprintrequests should be addressed. § Current address: Department of Neurology, University Cattolica del Sacro Cuore, Largo Gemelli8, Rome, Italy.
precipitated factors apart from one case in which the symptomatology developed during pregnancy [4]. We report one case with M A D deficiency in which the clinical features of the disease appeared during valproate therapy. After discontinuation of the drug a clinical improvement was observed while therapy with riboflavin resulted in a total relief of the symptoms.
CASE REPORT
The patient is a 47-yr-old man without any family history of neuromuscular disease. He has two normal children and his parents are in good health. The patient was well until the age of 18 yr when he had four major seizures and was treated with phenobarbital and phenytoin. When the patient was 43 yr old the antiepileptic drug was changed to valproate (600 mg daily) and 4 months later he began to have typical nocturnal calf cramps and exercise intolerance. After walking for relatively short distances, up to 500 m, he began having pains throughout the legs; however, these symptoms were relieved when he stopped walking. He had difficulty
247
248
A. PAPADIMITRIOU and S. SERVIDEI
climbing stairs and had to take a rest every 10 Finally it was decided to place him on ribosteps and also had to sit down in order to shave. flavin, 100 mg daily, where a dramatic improveHe also felt shortness of breath and sometimes ment was shown. Before starting therapy a had difficulty in chewing and was able to eat only treadmill test was carried out to determine the soft food. Progressively the patient's condition patient's physical ability; he was able to excel on deteriorated and he was unable to perform any the bicycle for 2½ min after which he became daily activities. He was admitted to the depart- exhausted. Ten days after riboflavin treatment ment of Neurology at the Red Cross Hospital of and following a repeat treadmill test the patient Athens, where the routine laboratory tests and was able to accelerate for a full 15 rain. At the neurological examination during rest were nor- present time, the patient does not seem to have mal. CPK was 400 U 1-1 (normal < 100) any of the previous symptoms and feels well. Aldolase was 18 U 1-~ (normal < 9) and serum CPK is normal and lactic acid is in the upper lactic acid was 19.2/~g (normal 8-12/tg ml-~). normal limit. He has been on this regimen since then and is now able to perform manual work The Tensilon test was negative. and all other necessary activities. During hospitalization the valproic acid was discontinued. He was started on Cambamazepine and a slight improvement was observed. A MATERIALS AND METHODS muscle biopsy was performed for histochemical and biochemical analysis. He started also treatThree muscle biopsies (one from the left deltoid, ment with DL-carnitine (3 g daily) and was placed one from the left quadriceps and another from on a low fat diet; however, his symptoms con- the right quadriceps) in three different periods tinued during the next 5 months. In the same were obtained. The muscle was divided into four period he was admitted to the Columbia Presby- portions for light and electron microscopy, terian Medical Center, New York. Laboratory histochemical and biochemical analysis. For investigation urinalysis, ESR, CBC, T3, T4, histochemical studies muscle was frozen in liquid TSH, serum electrolytes, glucose, BUN, creatinine, nitrogen-cooled isopentane, cross-sectioned in a Ca, P, total protein, cholesterol, bilirubin, cryostat and stained with haematoxylin-eosin, alkaline phosphatase, SGOT, SGPT were within adenosine triphosphatase at pH 9.4 - 4.3 oil red normal values. The CPK was increased to 128 U 0, NADH-TR, and modified trichrome staining. 1-I (normal 1-50), LDH was 223 (normal 90- For electron microscopic analysis muscle was 200). The ECG showed a right bundle branch fixed as described by Engel and Dale [8]. block. CSF protein was 58 mg%. Motor and For biochemical analysis the muscle biopsies sensory nerve conduction velocities were normal were immediately frozen in liquid nitrogen. and needle EMG showed a myopathic pattern. Muscle mitochondria were isolated by the Repetitive nerve stimulation was normal. During method of Bookelman et al. [9]. rest period the serum lactic acid was 4.3 mM 1-~ Total and free carnitine were measured radio(normal 0.5-2.2 mM 1-1) rising to 10.8 mM 1-1 chemically [10]. The beta oxidation enzymes after having exercise for only 1 min (control after were measured spectophotometrically in both 5 min exercise 6.6 mM 1-~). Organic acids in the crude muscle homogenate and in mitochondrial urine were not detected during this period. A preparation. Palmityl-CoA DH, Butyryl-CoA second muscle biopsy was performed. DH and Octanoyl-CoA DH were estimated Progressively his symptoms began to improve according to the method of Fong and Schultz and within 2 months he was able to walk more [11]. The reaction mixture contained 66 mM K than 500 m. However, after this exercise, he phosphate (pH 7.4) 100 /~M dichlorophenol started complaining of muscle cramps and indophenol (DCPIP), 1 /~M phenazine methoweakness which were relieved only when he sulphate, 0.03% BSA, 0.06% Triton-X100 and stopped exercise. Gradually the patient was able the appropriate substrate (Acyl CoA) in a final to resume his office work but his physical ability concentration of 0.2 mM. Crotonase, hydroxyacyl CoA DH and thiolase were measured as was limited. Approximately a year later a third muscle described previously [12]. biopsy was performed for repeated biochemical Mitochondrial enzymes were measured in crude analysis. However, the patient had still showed homogenates and in mitochondria preparations no physical difference in condition. In all the according to methods described previously [13]. laboratory examinations, CPK and lactic acid Non-collagen protein was estimated by the method of Lowry et al. [14]. during rest were always high.
L a t e Onset Storage M y o p a t h y
249
Fig. 1. Excess of s a r c o p l a s m i c neutral lipid (oil red 0 staining).
Table 1. Carnitine concentrations of muscle (/~mol g-')
RESULTS
Total
Free
0.500 0.502 0.920 2.34 + 0.68
0.350 0.434 0.700 1.87 5:0.66
Muscle biopsy. The muscle fibres were normal except for a few scattered atrophic fibres in E-H staining. About half of the fibres contained dark red granules in the modified Gomori trichrone stain. By the oil red 0 staining a similar proportion of muscle fibres contained an excess of sarcoplasmic neutral lipid (Fig. 1). By electron microscopy, unusual, enlarged, abnormal mitochondria with abnormal internal structure and lamellar concentric inclusions were found (Fig. 2A and B). Although the three muscle biopsies were performed at different times the morphology was similar. Biochemistry. Total and free carnitine were decreased (20--40% of the control) in all three biopsies (Table 1). It is important to note that the second and third biopsies were performed while
I st biopsy 2nd biopsy 3rd biopsy Controls (n= 20) x 5: S.D.
the patient was on carnitine therapy. The ratio of acylcarnitine to free carnitine was higher (0.42) as compared with normal controls (0.25). The mitochondrial enzymes in crude muscle and isolated mitochondria were normal in both the second and third biopsies (Tables 2 and 3). The fl-oxidation mitochondrial enzymes showed a decreased activity (40% of the control values) of all the short, medium and long chain fatty acyl CoA DH. All the other enzymes of fl-oxidation were normal (Table 4). The same results were also found in isolated mitochondria (Table 5).
Table 2. Mitochondrial enzymes in muscle homogenate (pmol rain 'g ~)
Cytochrome oxidase NADH Cyt c reductase Succinate Cyt c reductase Citrate synthetase Succinate dehydrogenase NADH dehydrogenase
Patient (2nd biopsy)
Patient (3rd biopsy)
4.43 3.89 1.24 20.50 1.41 37.10
3.35 3.78 0.99 18.35 0.99 26.10
Controls (n = 30) x ± S.D. 2.86 3.32 0.75 11.35 1.68 35.21
+ ± ± ± ± ±
0.53 t.20 0.26 2.40 0.45 6.37
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Fig. 2. (A and B) Unusual enlarged abnormal mitochondria with abnormal internal structure and lamellar concentric inclusions (EM). (A) Magnification 15,000 X and (B) magnification 30,000 x .
Late Onset Storage Myopathy
251
Table 3. Mitochondrial enzymes in isolated mitochondria (pmol min-~mg protein-t)
Cytochrome oxidase N A D H Cyt c reductase Succinate Cyt c reductase Citrate synthetase Succinate dehydrogenase N A D H dehydrogenase
Patient (2nd biopsy)
Patient (3rd biopsy)
1.02 0.305 0.291 2.440 0.218 0.223
1.04 0.271 0.209 1.322 0.175
Controls (n = 15) x ± S.D. 1.34 0.29 0.25 2.303 0.173 0.154
± 0.37 ± 0.04 5:0.04 ± 0.5 ± 0.04 ± 0.05
Table 4./?-oxidation mitochondrial enzymes in muscle (/tmot min- ~g-~)
Butyryl CoA D H Octanoyl CoA D H Palmityl CoA DH Crotonase Hydroxyl-acyl CoA DH Thiolase (C4)
Patient (2nd biopsy)
Patient (3rd biopsy)
0.35 0.59 0.45 15.59 7.39 5.71
0.32 0.31 0.52 13.70 6.80
Controls (n = 7) x±S.D. 1.15 0.98 1.23 14.73 5.00 5.15
± 0.20 5:0.19 ± 0.24 ± 1.72 5:1.18 ± 1.27
Table 5. fl-oxidation enzymes in isolated mitochondria (,umol min-~g protein-~)
Butyryl CoA DH Octanoyl CoA D H Palmityl CoA DH Crotonase Hydroxy-acyl CoA DH Thiolase (C4)
Patient (2nd biopsy)
Patient (3rd biopsy)
9.55 12.57 11.96 135.20 71.58 153.41
6.40 10.60 12.50 112.83 85.00
DISCUSSION
Late onset MAD deficiency is a rare disorder and only five cases have been described. In 1979 a 19-yr-old woman was presented with repeated episodes of hypoglycaemia, liver dysfunction and proximal myopathy. Increased quantities of glutaric acid, ethylmalonic acid, dicarboxylic acids with a six to ten carbons chain lengths were found in the urine of the patient. In cultured fibroblasts the ability to oxidize butyrate and lysine was reduced and multiple CoA DH deficiency was suggested [3]. In 1983 a 36-yr-old woman was described who, during pregnancy, presented with liver dysfunction and organic aciduria. The patient had eight other pregnancies resulting in one stillborn infant and six floppy infants who died during neurological distress. In all these pregnancies the mother had the same symptoms, during the last trimester [4]. In 1986 there was a report of a 17-yr-old girl with progressive muscle weakness, organic aciduria, extensive lipid accumulation in muscles and a reduction of mitochondria floxidation enzymes [5]. In 1988 a 39-yr-old man was presented with painful proximal myopathy,
Controls (n = 7) x ± S.D. 28.25 23.77 28.27 124.60 54.60 126.49
5:2.70 5:2.90 ± 3.90 + 12.56 5:11.98 5:21.88
organic aciduria, lipid accumulation in muscle and low acyl CoA dehydrogenase activity [6]. The most recent case [7] is o f a 12-yr-old girl presenting with easy fatigability and muscle weakness, lipid storage myopathy and organic aciduria. In our case there are similarities to those reported in the literature: that is myopathy, lipid accumulation in muscles, abnormal mitochondria, muscle carnitine deficiency and low acyl-CoA DH activity. However, it also presents two important differences: exercise intolerance is the main clinical symptom, symptomatology started during valproic acid therapy and improved slightly after discontinuation of the drug. Valproic acid is a branched chain fatty acid and inhibits the oxidation of fatty acids at the level of fl-oxidation I15]. It is also reported that valproic acid produces organic aciduria as in cases of MAD deficiencies [16]. Many patients tolerate valproic acid therapy well but others may present liver toxicity, hyperammonemia, and a hepatocerebral syndrome similar to Reye's syndrome. It has been suggested that the toxicity may be due to an unrecognized inborn error of metabolism in fatty acid oxidation [17,18].
252
A. PAPADIMITRIOUand S. SERVIDEI
We believe that valproic acid played an important role in the appearance of symptomatology in our case. The residual activities of the three acyl-CoA DH enzymes were 40% of normal and were probably sufficient to facilitate fatty acid metabolism covering the clinical expression. By inducing valproic acid, which shares the same metabolic pathway, the enzymes become insufficient and therefore symptomatology starts. The low level of muscle carnitine in our case can be explained, as in all cases of acyl CoA DH deficiency, as a secondary phenomenon. The defect of the enzyme causes accumulation of acyl CoA in the muscle mitochondria. Carnitine reduces the amount of acyl CoA causing accumulation of acyl carnitine which is excreted in the urine resulting in secondary carnitine deficiency. The higher ratio of acylcarnitine: free in our case, is in accordance with previously described cases [5,19]. The effect of riboflavin was definitive in our case and there was complete relief of all symptoms. We have found in the literature few cases of MAD deficiency in which riboflavin showed clinical or biochemical improvement [5,20,21]. However, none of these cases showed complete relief, as in our case. The mechanism of the riboflavin effect is not known. Recently normalization of short-chain acyl CoA dehydrogenase after riboflavin therapy has been reported [7]. Riboflavin is a precursor of FAD which is a prosthetic group of Acyl CoA DH, ETF DH and play an important role in stimulating the residual activity of the deficient enzyme. It has been proposed that riboflavin responsive MAD may be due to a primary alteration of riboflavin or FAD metabolism causing accelerated breakdown or reduced synthesis of flavoprotein haloenzymes [2]. Acknowledgement--The authors would like to thank Pro-
fessor S. Di Mauro for his helpful discussion regarding the manuscript, and Dr E. Bonillia for electron microscopy study.
4.
5.
6. 7.
8.
9.
10. 11. 12.
13.
14. 15.
16. 17. 18.
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