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Myopathy due to glycogen storage disease
EXPERIMENTAL
AND
MOLECULAR
Myopathy
PATHOLOGY
38, 405-420 (1983)
Due to Glycogen
Storage
Disease:
Pathological and Biochemical Studies in Relation to Glycogenosome Formation TERUO IWAMASA,’ NOBUYUKI NINOMIYA,~ Departments Kumamoto
SEIJI FUKUDA,~ SHINICHI TOKUMITSU,~ ICHIRO MATSUDA,* AND MITSUHIRO OSAME~
of IPathology and IPediatrics, Kumamoto University Medical 860, Japan, and ‘Department of Internal Medicine, Kagoshima School, Ushikimachi, Kagoshima 890, Japan Received
December
School, l-1, Honjo, University Medical
16, 1982
Ten cases of myopathy caused by glycogen storage diseases of type II, III, and V, and phosphorylase b kinase deficiency are reported. So-called “abnormal lysosomes” or glycogenosomes which contain abundant glycogen were found in cases of type II, and in some numbers, in cases of type III, and in one case of phosphorylase b kinase deficiency which revealed a moderate decrease in debranching enzyme (amylo-1,6-glucosidase) activity. In these cases of type III and phosphorylase b kinase deficiency, the glycogenosomes are formed through deposition of abnormal glycogen (limit dextrin structure glycogen).
INTRODUCTION The biochemical details of glycogen storage disease have been established by many investigators (Cori and Cori, 1952; Anderson, 1956; Hers, 1963; Baudhuin ef al., 1964; Tarui et al., 1965; Hug el al., 1966; Garancis, 1968; Brown and Brown, 1968; Huijing and Fernandes, 1969; Van Hoof et al., 1972; Morishita et al., 1973; Koster et al., 1976; Murray et al., 1978; Howell, 1978; Corbeer et al., 1981). Based on biochemical studies and the clinical symptomatology, some attempts at classification of the disease have emerged. In each variety of the disease, glycogen accumulates abundantly in particular tissues as a result of enzyme deficiency. Among the glycogen storage diseases, type II (Pompe disease), type III (Forbes disease or Cori disease), type IV (Anderson disease), type V (McArdle disease), type VII (Tarui disease), and muscle-type phosphorylase b kinase deficiency, involve the skeletal muscle and in some cases heart muscle. Most have abundant deposits of glycogen in the skeletal muscle fibers, and clinically show atrophy. The principal histological finding is vacuolation, and the fiber sizes tend to become more variable than normal. The vacuolation ranges from a few small, centrally located vacuoles to clear large vacuoles replacing most of the sarcous substances and vacuoles in the sarcolemmal space. In such vacuolated areas, electron microscopy reveals large amounts of accumulated glycogen. In the type II disease, there are large numbers of sequestered membranesurrounded packets of glycogen (glycogenosomes) as well as dispersed glycogen in the sarcoplasm. Hers (1963) and Hug et al. (1966) ascribed the membranesurrounded glycogen accumulations (glycogenosomes) to an enzymatic defect (acid maltase deficiency) in the lysosomes. Electron microscopic studies by Baudhuin et al. (1964), Cardiff (1966), and Smith et al. (1967) have demonstrated similar glycogenosomes. The identification of such glycogenosomes or “abnormal lysosomes” in the affected muscle fibers is thus of important diagnostic value in the type II disease. However, in glycogen storage myopathies other than the type II disease, glycogenosomes have recently been observed by Osame et al. (1978), 40.5 0014-4800/83 $3.00 Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Dannon et al. (1981), and Iwamasa et al. (1982a). In this report, ten cases of glycogen storage disease with skeletal muscle involvement are described, paying special attention to the formation of glycogenosomes. MATERIALS
AND METHODS
Patients Type II glycogen storage disease: cases l-3. Type III glycogen storage disease: cases 4-7. Type V glycogen storage disease: case 8. Muscle type phosphorylase b kinase deficiency: cases 9 and 10. Case 1. A 2-year-old female infant whose main symptoms were cardiomegaly, hepatomegaly, and muscle weakness of the extremities. She was floppy at birth and began to walk at the age of 1.5 years. Biopsied muscle (M. bioceps brachii) revealed a marked decrease in acid maltase and neutral maltase activities. Case 2. A 22-year-old man whose symptoms were muscle weakness and atrophy in the extremities. Cardiomegaly and hepatomegaly have not been noted to date. He was floppy at birth and walked at the age of 1 year and 2 months. The onset of his disease was at 5 years of age. Biopsied muscle (deltoid muscle) revealed a marked decrease in acid maltase activity and an increase in glycogen content. Case 3. A male adult, aged 45, whose duration of illness was almost 5 years. Muscle weakness and atrophy of the extremities, more prominent in the proximal area, formed his main symptoms. No cardiomegaly or hepatomegaly has been noted to date. M. biceps femoris was biopsied and subjected to measurements of acid maltase activity, glycogen content, and other glycogenolytic enzyme activities. A prominent increase in glycogen content and a decrease in acid maltase activity were noted. The levels of other glycogenolytic enzyme activities were within the normal ranges. Case 4. A 39-year-old female who had slowly progressive muscle weakness and atrophy in the distal extremity muscles. Furthermore, the sternomastoid and thigh adductor muscle were similarly affected. No cardiomegaly or hepatomegaly was noted, and the patients liver function tests were normal. Her younger brother (aged 32) had very similar clinical symptoms. The femoral quadriceps was biopsied, and the debranching enzyme activity as measured with phosphorylase limit dextrin (PLD) as substrate and [14C-Iglucose incorporation into glycogen revealed extremely decreased values. Other glycogenolytic enzyme activities were within the normal ranges. Case 5. A 32-year-old female whose clinical symptoms closely resembled those of case 4, presenting distal extremity muscle weakness and atrophy. Liver function tests were normal. The serum creatinine phosphokinase (CPK) activity was slightly elevated at 42 W/liter (normal, below 25 II-J/liter). Biopsied muscle M. quadriceps femoris revealed a marked decrease in debranching enzyme activity. Case 6. A 45-year-old male whose symptoms were also very similar to cases 4 and 5, showing distal muscle weakness and atrophy. No cardiomegaly, but mild hepatomegaly have been noted to date. Case 7. A 40-year-old male whose main symptoms were muscle weakness of the extremities and hepatomegaly. The hepatomegaly had been present since the age of 3 years. Liver biopsy revealed hepatocytes of foamy appearance which contained large amounts of glycogen on PAS staining. The biopsied liver and M. biceps femoris showed a marked decrease in debranching enzyme activity.
GLYCOGEN
407
MYOPATHY
Case 8. A 50-year-old male who had noted leg muscle stiffness after running a 25-m race as a primary high school student. Since 40 years of age, muscle weakness of the upper extremities had slowly progressed and facial muscle stiffness after laughing was also noted. The ischemic exercise test showed no elevation of serum lactic acid level. Liver function tests were normal. The serum CPK activity was elevated at 94 III/liter. The phosphorylase activity in the biopsied M. triceps brachii was extremely decreased. Case 9. A 1.7-year-old female infant. She was floppy at birth and has not walked to date. Muscle hypotonia was noted in the extremities. It was slightly dominant distally, although the proximal muscles were also markedly involved. The biopsied M. quadriceps revealed a marked decrease in phosphorylase b kinase activity and a moderate decrease in debranching enzyme activity. Other glycogenolytic enzyme activities were within the normal ranges. The glycogen content was elevated more than normal. Case 10. A young female, aged 17. Muscle weakness and stiffness in the extremities during work of short duration, slightly dominant in the distal muscles, had been noted since the age of 5 years. No cardiomegaly or hepatomegaly was observed. Liver function tests were within the normal ranges. However, a slight elevation of serum CPK activity was observed. The biopsied M. biceps femoris revealed a marked decrease in phosphorylase b kinase activity. Other glycogenolytic enzyme activities yielded normal values. The glycogen content was elevated more than normal. The details of these ten cases are summarized in Table I.
TABLE I Summarized Data of the Cases Case
5w of disease
Age WeW
Sex
Main symptoms Muscle weakness of extremities. Cardiomegaly. Hepatomegaly. Muscle weakness and atrophy of extremities.
1
II
2
F
2
II
M
II
22 (Disease onset was at 5 years) 45
III
39
F
III
32
F
III
45
M
III
40
M
V Phosphorylase b kinase deficiency Phosphorylase b kinase deficiency
50 1.7
M F
Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Hepatomegaly. Muscle weakness and stiffness. Muscle hypotonia.
17
F
Muscle weakness and stiffness.
8 9 10
M
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ET AL.
Morphological Examinations Small pieces (1.0 x 1.0 x 0.5 cm) of biopsied muscle were frozen using liquid nitrogen. Fresh frozen sections were cut at a thickness of 6 km. Enzyme histochemical studies for phosphorylase (Takeuchi and Kuriaki, 1955), acid phosphatase (Barka and Anderson, 1962), ATPase with preincubation at pH 10.3,4.6, and 4.2 (Dubowitz and Brooke, 1973), and succinate dehydrogenase (Lojda et al., 1979) were performed as usual. For routine hematoxylin-eosin, PAS, and Gomori’s trichrome staining, other small pieces of tissue fixed in 10% formalin were used. For electron microscopic observation, the tissues were fixed in 2.5% glutaraldehyde for 2 hr, and postfixed in 2% osmium tetraoxide for 1 hr by the usual method, followed by embedding in Epon 812. Ultrathin sections were then cut, and after staining with lead nitrate, were observed under a Hitachi 12A electron microscope. Biochemical Examinations The amount of glycogen was determined by the anthrone method. Protein was measured by the method of Lowry et al. The activities of phosphorylase (Sutherland and Wosilait, 1956), phosphorylase b kinase (Krebs, 1966), debranching enzyme (Hers et al., 1967), and acid and neutral maltase (Hers, 1964; Salafsky and Nadler, 1973) were measured using muscle extract. To obtain the muscle extract, biopsied muscle was homogenated with 10 vol of ice-cold distilled water using a Waring blender. The resultant homogenate was centrifuged at 10,000 rpm for 30 min at 2°C. The supernatant was employed as the muscle extract. Assays of the acid and neutral maltase activities were made with 4-methylumbelliferyl (Yglucoside and maltose as substrates. The debranching enzyme activity was measured using phosphorylase limit dextrin, prepared in our laboratory, as substrate and the [i4C-Iglucose incorporation into glycogen according to Hers’ method (1964). Extraction of glycogen was performed as described previously (Iwamasa et al., 1982a). Using this glycogen, the iodine spectrum of the glycogen was determined
Fig. 1. Fibers of biopsied M. biceps brachii (case 1) showing marked vacuolation. H & E staining. x 365.
GLYCOGEN
MYOPATHY
FIG. 2. Biopsied M. biceps femoris (case 3) showing severe vacuolation. H & E staining.
X
365.
as follows. Ten microliters of 0.01 N iodine solution containing 0.02 N KI was added to 2 ml of the glycogen solution (0.1 mg/ml), and the spectrum was measured with a Hitachi 100-60 spectrophotometer at 24°C. RESULTS Morphological Findings Figures 1 and 2 show transverse sections of biopsied muscles from cases of type II glycogen storage disease. Figure 1 is from the infantile form (case 1) and
FIG. 3. Some portions of the deltoid muscle from case 3 show no vacuolation and no marked abnormality. However, in this material, the decrease of acid maltase activity was similar to that in other vacuolated portion in this case. H & E staining. X 400.
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ET AL.
Fig. 2 is from the adult form of the disease (case 3). The light microscopic findings for these forms are very similar showing vacuole formation. The vacuoles ranged from 2-5 to 60 km in diameter. The type II fibers were slightly more affected than the type I fibers. The size of the fibers was more variable than normal. Some central nuclei were found. However, necrosis and myophagy were very rare. In some portions, only variety of the fiber size was found, and vacuolation was very rare (Fig. 3). Biopsied muscle sections from generalized type III glycogen storage disease (case 7) (Fig. 4) also revealed vacuole formation, and the fiber size was more variable than normal. The vacuoles were slightly larger and more frequent than those in the type II disease. Sometimes a few centrally located nuclei were observed. Vacuoles which stained very strongly on PAS staining were observed in the sarcoplasm and subsarcolemmal area. Furthermore, rimmed vacuoles were also found elsewhere in the type III disease (Fig. 5 and 6). However, rimmed vacuoles occurred more frequently in the muscle type of type III glycogen storage disease than in the generalized type of the type III disease. Slight endomysial fibrosis was present, and rare necrosis was also found. Enzyme histochemical acid phosphatase was strongly demonstrated in the rimmed vacuoles. The type V glycogen storage disease (McArdle disease) and muscle type phosphorylase b kinase deficiency showed very similar formation of subsarcolemmal blebs (Fig. 7) which were strongly stained on PAS staining. In the biopsied muscle, bleb formation was observed at a frequency of 25-60% in the fibers. Figure 8 shows the enzyme histochemical reaction of ATPase (pH 10.3) in case 9. The type I fibers were slightly more affected than the type II tibers. However, the histochemical reaction of phosphorylase without AMP in the reaction medium revealed a similar negative reaction between the type I and type II fibers (Fig. 9).
FIG. 4. Biopsied M. biceps femoris (case 7) stained by H L E staining. Marked vacuolation of the fibers is observed. Variety in the size of fibers is also seen. The cases 4-6 showed very similar features. x 365.
GLYCOGEN
MYOPATHY
FIG. 5. Rimmed vacuole (arrow) having a distinct hematoxylinophilic E staining. x 680.
lining is found (case 7). H &
By adding AMP to the reaction medium, all of the fibers (type I and II fibers) showed a strong positive reaction (Fig. 10). There was also considerable variation in the size of the fibers when compared with that of normal muscle. Central nuclei were not frequently found. The connective tissue was not increased in these cases. Case 9 revealed very small numbers of rimmed vacuoles.
FIG. 6. Rimmed vacuoles (arrows) also have PAS-positive substance in them (case 7). PAS staining. x700.
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ET AL.
7 e ;9r
FIG. 7. Subsarcolemmal blebs of phosphorylase b kinase deficiency, indicated by arrows (case 9, M. quadriceps). Cases 8 and 10 also showed similar bleb formation. H & E staining. x 700.
Electron
Microscopic
Observations
The electron microscopic findings for muscle of type II, III, and V, and phosphorylase b kinase deficiency were very similar to those reported by Cardiff (1966), Hug et al. (1966), Smith et al. (1967) Garancis (1968), Engel et al. (1973), Howell (1978), and DiMauro and Hartlage (1978). In the type II disease, membrane-surrounded accumulations of glycogen (glycogenosomes) termed “abnormal lysosomes” were characteristically observed frequently (Fig. 11). How-
FIG. 8. ATPase reaction of case 9 at pH 10.3. The dark stained type I fibers are slightly more affected than the type II fibers. The arrows indicate vacuolation of the fibers. x 700.
GLYCOGEN
MYOPATHY
413
FIG. 9. Phosphorylase reaction without AMP (case 9). Almost all of the fibers show a negative reaction. The type I and II fibers show similar negative results. x 360.
ever, in the type III disease, glycogenosomes were demonstrated not so rarely (Fig. 12). The glycogenosome formation observed under the electron microscope was in fibers where rimmed vacuoles were seen light microscopically. In muscle fibers of phosphorylase b kinase deficiency, large amounts of glycogen accumulation were noted in subsarcolemmal areas which appeared as subsarcolemmal blebs under the light microscope. Furthermore, the fibers showed increased glycogen in such phosphorylase b kinase deficiency. Case 9, which exhibited an extreme decrease in phosphorylase b kinase activity and also a moderate decrease in debranching enzyme activity, revealed formation of glycogenosomes in considerable numbers (Fig. 13).
FIG. 10. Phosphorylase reaction with AMP using sections consecutive with Fig. 9. A marked positive reaction is demonstrated on all the fibers. Case 10 also revealed a similar reaction. The results of Figs. 9 and 10 indicate that the phosphorylase in these cases is in the b form. x 360.
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ET AL.
FIG. 11. Electron micrograph of case 2 (type II glycogen storage disease). Glycogenosomes or “abnormal lysosomes” (arrows) are present and the muscle fibrils are markedly disrupted. x 19,100.
Biochemical Data
The results are summarized in Table II. Activity measurements of phosphorylase b kinase in cases 9 and 10 are shown in Figs. 14A and B. The extracted glycogen from type III disease (case 7) demonstrated a lower P-amylolysis rate than that of normal controls. Furthermore, the extracted glycogen from case 9 also revealed a lower rate of @amylolysis. An iodine spectrum of extracted glycogen from type III disease (case 7) is given in Fig. 15. The glycogen content in each type of disease was strongly increased. In particular, in the type II and III disease, it showed extremely high levels. The acid maltase activity in each type II disease was markedly decreased. In the infantile type II disease (case l), not only the acid maltase but also the neutral maltase
GLYCOGEN
MYOPATHY
FIG. 12. Electron micrograph of case 6 (type III glycogen storage disease). Glycogenosomes (arrows) are present. Cases 4, 5, and 7 also revealed glycogenosome formation. x 10,600.
activities were decreased, although this was not the case in the adult type of the disease (case 3). DISCUSSION Glycogenosomes, membrane-surrounded glycogen or “abnormal lysosomes,” were found not only in type II glycogen storage disease (Pompe disease), but also in cases of type III disease (Forbes or Cori disease) and in one case of phosphorylase b kinase deficiency which revealed a moderate decrease in debranching enzyme activity. In these cases other than the type II disease, glycogenosomes
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ET AL.
FIG. 13. Glycogenosome in case 9 (arrow).
x
100,000.
were apparent in considerable numbers in the muscle fibers. Glycogenosome formation is therefore not considered as a specific finding of type II glycogen storage disease. As reported by us previously (Iwamasa et al., 1980a, b), glycogenosome is formed from abnormal or different structural glycogen without a decrease in acid maltase activity in newborn rat hepatocytes. Furthermore, in ascites hepatoma AH 13 cells, glycogenosome formation is frequently observed, and the acid maltase activity is not decreased in the tumor cells. Based on these results, the glycogen storage disease of type III and phosphorylase b kinase deficiency (case 9) presented here are considered to form glycogenosomes from limit dextrin-like storage glycogen different from normal glycogen. However, as reported previously (Iwamasa et al., 1982b), in the hepatocytes of generalized type III glycogen storage disease, glycogenosome formation is barely observed. It is thought that in hepatocytes of the type III disease, the limit dextrin-like structure of glycogen is not always dominant because of the influence of feeding. Determination of the glycogenosomes morphologically is considered to depend on the balance between
0.85 0.99 0.78 23.40 23.33 26.41 20.33 28.0 27.67 29.38 27.3 k 10.5
1 2 3 4 5 6
0.93 43.98 45.0 48.0 50.10 50.05 52.33 51.14 53.80 51.00 50.0 2 10.3
pH 7.0
a 4-MUG, 4-methylumbelliferyl a-glucoside. b PLD, phosphorylase limit dextrin.
8 9 10 Controls
1
pH 4.0
Case
o-Glucosidase (CMUG as substrate) (pkf/mg protein/mitt)
2.58 2.66 2.60 0.013 0.30 0.01 0.003 2.80 2.50 2.43 2.61 -c 0.5
Debranching enzyme (PLDb as substrate) Wfhng protein/min)
6993.1 + 300
121.1 134.1 101.3 98.5
[i4C]Glucose incorporation (cpm/mg glucose/g protein/hr) a 45.0 48.6 47.7 30.0 49.6 48.3 47.5 ud ud 0.80 47.7 2 13.2
76.0 75.3 19.4 70.1 70.5 75.2 73.4 ud 73.1 70.6 78.0 -t 21.1
Phosphorylase ( pkf/gplmin) Total
TABLE II Results of Biochemical Examinations
51.2 58.6 35.7 24.0 60.1 58.3 71.3 28.6 26.7 30.5 5.0-10.0
Glycogen content (m&t wet wt)
28.5 f 1.0
18.6
17.3
a-Amylolysis (%I
418
IWAMASA
ET AL.
FIG. 14A. Phosphorylase b kinase activity measurements. 0, Control; 0, case 9; Ir, case 10. Activation of phosphorylase b to a by the muscle extract was measured using 0.125 M Tris-0.125 M sodium @glycerophosphate buffer, pH 6.8. The reaction medium contained 0.2 ml of buffer, 0.2 ml of phosphorylase b (Sigma; 28 units/mg protein), 0.1 ml of 0.018 M ATP-0.06 M MgCI,, and 0.1 ml of muscle extract in neutral cysteine. The phosphorylase a activity was measured every 10 min by the method of Sutherland and Wosilait. FIG. 14B. Activation of phosphorylase b in the muscle extract to phosphorylase a by active phosphorylase b kinase (Sigma). 0, Control; 0, case 9; +, case 10. The active phosphorylase b kinase (0.1 ml, 30 units/mg protein) was added to a mixture containing 0.2 ml of 0.125 M Tris-0.125 M sodium P-glycerophosphate buffer, pH 6.8, 0.1 ml of 0.018 M ATP-0.06 M MgCl,, and 0.2 ml of the muscle extract in 0.015 M neutral cysteine.
the turnover rate of acid maltase and autophagic activity for limit dextrin-like glycogen into lysosomes. In the case of the type II disease, as reported previously (Iwamasa er al., 1979), the glycogen extracted shows an extremely large S value compared to normal glycogen by ultracentrifuge analysis. The glycogenosome formation in type II disease is therefore thought to involve extremely large glycogen. Apart from the type II disease, in the cases of type III disease and in one case of phosphorylase OD 0.500 t
0.300
c
P 0.10 I-
I
400
5cm
\
600
nm
FIG. 15. Iodine spectrum of extracted glycogen (case 7). The measurements were carried out at 24°C. The procedures were as described in the text. The peak of the spectrum in case 7 is different from that of normal control glycogen. C, Control; P, case 7.
GLYCOGEN
MYOPATHY
419
b kinase deficiency, glycogenosomes were also formed. Nevertheless, the acid maltase activity was not decreased. It is considered that the glycogen autophagied into lysosomes is digested, gradually decreases in numbers, and that the glycogenosomes are also decreased in numbers. The glycogenosomes are found in areas where rimmed vacuoles are observed light microscopically. The type III disease (case 4) presented here had very similar morphological findings to inclusion body myositis (Osame et al., 1978). The affected muscles were of the distal extremities and light microscopically, rimmed vacuoles were observed. The patient was an adult. However, in distal myopathy, many unclassified disorders other than type III glycogen storage disease are considered to exist. Detailed examinations of the metabolical disorders in myopathy must be made. In phosphorylase b kinase deficiency, not all of the fibers show bleb formation. However, the histochemical phosphorylase reaction without AMP in the incubation medium demonstrates a negative reaction in all fibers. The enzyme activity is considered to be decreased in almost all fibers. In one case (case 9) of phosphorylase b kinase deficiency, not only was the phosphorylase b kinase, but also the debranching enzyme activity decreased. However, it remains obscure as to why the debranching enzyme activity should be decreased. Furthermore, in type II glycogen storage disease, the enzyme activity becomes markedly decreased. The reason why some portions of the muscle do not reveal marked morphological abnormality remains unknown. Further examinations are clearly needed. REFERENCES D. H. (1956). Familial cirrhosis of the liver with storage of abnormal glycogen. Lab. Invest. 5, 11-20. BARKA, T. and ANDERSON, P. J. (1962). Histochemical method for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10, 741-753. BAUDHUIN, P., HERS, H. G., and LOEB, H. (1964). An electron microscopic and biochemical study of type II glycogenosis. Lab. Invest. 13, 1139-l 152. BROWN, B. I., and BROWN, D. H. (1968). The glycogen storage diseases: Type I, III, IV, V, VII and unclassified glycogenoses. In “Carbohydrate Metabolism and its Disorders” (W. J. Whelan, ed.), Vol. 2, p. 123. Academic Press, New York. CARDIFF, R. D. (1%6). A histochemical and electron microscopic study of skeletal muscle in a case of Pompe’s disease (glycogenosis II). Pediatrics 37, 249-259. CORBEEL, L., HUE, L., LEDERER, B., DEBARSY, T., VAN DEN BERGUE, G., DEVLIEGER, H., JAEKEN, J., BRACKE, O., and EECKELS, R., (1981). Clinical and biochemical findings before and after portacaval shunt in a girl with type Ib glycogen storage disease. Pediatr. Res. 15, 58-61. Corn, G. T., and Corn, C. E (1952). Glucose-6-phosphatase of the liver in glycogen storage disease. J. Eiol. Chem. 199, 661-667. DANNON, M. J., OH, S. J., DIMAURO, S., MANALIGOD, J. R., EASTWOOD, A., NAIDU, S., and Schliselfeld, L. H. (1981). Lysosomal glycogen storage disease with normal acid maltase. Neurology 31, 51-57. DIMAURO, S., and HARTLAGE, P. L. (1978). Fatal infantile form of muscle phosphorylase deficiency. Neurology 28, 1124-l 129. DUBOWITZ, V. and BROOKE, M. H. (1973). “Muscle Biopsy: A Modem Approach,” p. 32. Saunders, London. ENGEL, A. G., GOMEZ, M. R., SEYBOLD, M. E., and LAMBERT, E. H. (1973). The spectrum and diagnosis of acid maltase deficiency. Neurology 23, 95-106. GARANCIS, J. C. (1968). Type II glycogenosis: biochemical and electron microscopic study. Amer. J. Med. 44, 289-300. HERS, H. G. (1963). Alpha-glucosidase deficiency in generalized glycogen storage disease (Pompe’s disease). Biochem. J. 86, 1l-16.
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H. G. (1964). Glycogen storage disease. In “Advances in Metabolic Disorders” (R. Levine and R. Lift, eds.), Vol. 1, p. 1. Academic Press, New York. HERS, H. G., VERHUE, W., and VAN HOOF, F. (1967). The determination of amylo-1,6-glucosidase. Eur. J. Biochem. 2, 257-264. HOWELL, R. R. (1978). The glycogen storage disease. In “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds.), p. 137. McGraw-Hill, New York. HUG, G., GARANCIS, J. C., SCHUBERT,W. K., and KAPLAN, S. (1966). Glycogen storage disease, Type II, III, VIII and IX. Amer. J. Dis. Child. 111, 457-474. HUIJING, F., and FERNANDES, J. (1969). X-Chromosomal inheritance of liver glycogenosis with phosphorylase kinase deficiency. Amer. J. Human Genet. 21, 275-284. IWAMASA, T., TSURU, T., HAMADA, T., and TAKEUCHI, T. (1979). Physicochemical properties of glycogen extracted from liver of type II glycogenosis. Acta Histochem. Cytochem. 12, 197-205. IWAMASA, T., TSURU, T., HAMADA, T., and TAKEUCHI, T. (1980a). Physicochemical and ultrastructural studies on glycogenosomes in newborn rat hepatocytes. Pathol. Res. Pratt. 167, 363-373. IWAMASA, T., TSURU, T., SASAKI, M., HAMADA, T., and TAKEUCHI, T. (1980b). Ultrastructural and biochemical studies of glycogenosome formation in ascites hepatoma AH 13 cells. Pathol. Res. Pratt. 168, 173-184. IWAMASA, T., HAMADA, T., FUKUDA, S., NINOMIYA, N., and TAKEUCHI, T. (1982a). Ultrastructural and physiocochemical studies on glycogen macromolecules from ascites hepatoma AH 13 cells: A comparison with normal adult rat muscle, liver and fetal liver glycogen. Acta Pathol. Japan. 32, 461-471. IWAMASA, T., NINOMIYA, N., FUKUDA, S., HAMADA, T., HIRASHIMA, M., and OSAME, M. (1982b). Glycogen storage disease: Studies related to the mechanism of glycogenosome formation. Pathol Res. Pratt., in press. KOSTER, J. F., SLEE, R. G., VAN DER KLEI-VAN MOORSEL, J. M., REITRA, P. J. G. M., and LICAS, C. J. (1976). Physico-chemical and immunological properties of acid a-glucosidase from various human tissues in relation to glycogenosis type II (Pompe’s disease). C/in. Chim. Acta 68, 49-58. KREBS, E. G. (1966). Phosphorylase b kinase from rabbit muscle. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 8, p. 543. Academic Press, New York. LOJDA, Z., GOSSRAU,R., and SCHIEBLER,T. H. (1979). “Enzyme-histochemistry: A laboratory manual,” p. 258. Springer-Verlag, Berlin. MORISHITA, Y., NISHIYAMA, K., YAMAMURA, H., KODAMA, S., NEGISHI, H., MATSUO. M.. MATSUO, H., and NISHIZUKA, T. (1973). Glycogen phosphorylase kinase deficiency: A survey of enzyme in phosphorylase activating system. Biochem. Biophys. Res. Commun. 54, 833-841. MURRAY, A. K., BROWN, B. I.. and BROWN, D. H. (1979). The molecular heterogeneity of purified human liver lysosomal a-glucosidase (acid u-glucosidase). Arch. Biochem. Biophys. 185, 51 l-524. OSAME, M., IWAMASA, T., KAWABUCHI, M., FUKUNAGA, H., IGATA, A., and TAKEUCHI, T. (1978). Familial muscle type amylo-1,6-glucosidase deficiency. In “Proceedings, IVth International Congress, Neuromuscular Diseases, Montreal.” SALAFSKY, I. R., and NADLER, H. L. (1973). Deficiency of acid alpha glucosidase in the urine of patients with Pompe’s disease. J. Pediatr. 82, 294-297. SMITH, J., ZELLWEGER, H., and AFIFI, A. K. (1967). Muscle form glycogenosis, type II (Pompe). Neurology 17, 537-549. SUTHERLAND, E. W. and WOSILAIT, W. D. (1956). The relationship of epinephrine and glucagon to liver phosphorylase: Liver phosphorylase, preparation and properties. J. Biol. Chem. 218, 459468. TAKEUCHI, T., and KURIAKI, H. (1955). Histochemical detection of phosphorylase in animal tissues. J. Histochem. Cytochem. 3, 153-160. TARUI, S., OKIJNO, G., IKURA, Y., TANAKA, T., SUDA, M., and NISHIKAWA, M. (1965). Phosphofructokinase deficiency in skeletal muscle: A new type of glycogenosis. Biochem. Biophys. Res. Commun. HERS,
19, 517-523.
VAN HOOF. F., HUE, L., DEBARSY, T., JACQUEMIN, P., DEVOS, I?, and HERS, H. G. (1972). Glycogen storage disease. Biochemistry 54, 745-752.
AND
MOLECULAR
Myopathy
PATHOLOGY
38, 405-420 (1983)
Due to Glycogen
Storage
Disease:
Pathological and Biochemical Studies in Relation to Glycogenosome Formation TERUO IWAMASA,’ NOBUYUKI NINOMIYA,~ Departments Kumamoto
SEIJI FUKUDA,~ SHINICHI TOKUMITSU,~ ICHIRO MATSUDA,* AND MITSUHIRO OSAME~
of IPathology and IPediatrics, Kumamoto University Medical 860, Japan, and ‘Department of Internal Medicine, Kagoshima School, Ushikimachi, Kagoshima 890, Japan Received
December
School, l-1, Honjo, University Medical
16, 1982
Ten cases of myopathy caused by glycogen storage diseases of type II, III, and V, and phosphorylase b kinase deficiency are reported. So-called “abnormal lysosomes” or glycogenosomes which contain abundant glycogen were found in cases of type II, and in some numbers, in cases of type III, and in one case of phosphorylase b kinase deficiency which revealed a moderate decrease in debranching enzyme (amylo-1,6-glucosidase) activity. In these cases of type III and phosphorylase b kinase deficiency, the glycogenosomes are formed through deposition of abnormal glycogen (limit dextrin structure glycogen).
INTRODUCTION The biochemical details of glycogen storage disease have been established by many investigators (Cori and Cori, 1952; Anderson, 1956; Hers, 1963; Baudhuin ef al., 1964; Tarui et al., 1965; Hug el al., 1966; Garancis, 1968; Brown and Brown, 1968; Huijing and Fernandes, 1969; Van Hoof et al., 1972; Morishita et al., 1973; Koster et al., 1976; Murray et al., 1978; Howell, 1978; Corbeer et al., 1981). Based on biochemical studies and the clinical symptomatology, some attempts at classification of the disease have emerged. In each variety of the disease, glycogen accumulates abundantly in particular tissues as a result of enzyme deficiency. Among the glycogen storage diseases, type II (Pompe disease), type III (Forbes disease or Cori disease), type IV (Anderson disease), type V (McArdle disease), type VII (Tarui disease), and muscle-type phosphorylase b kinase deficiency, involve the skeletal muscle and in some cases heart muscle. Most have abundant deposits of glycogen in the skeletal muscle fibers, and clinically show atrophy. The principal histological finding is vacuolation, and the fiber sizes tend to become more variable than normal. The vacuolation ranges from a few small, centrally located vacuoles to clear large vacuoles replacing most of the sarcous substances and vacuoles in the sarcolemmal space. In such vacuolated areas, electron microscopy reveals large amounts of accumulated glycogen. In the type II disease, there are large numbers of sequestered membranesurrounded packets of glycogen (glycogenosomes) as well as dispersed glycogen in the sarcoplasm. Hers (1963) and Hug et al. (1966) ascribed the membranesurrounded glycogen accumulations (glycogenosomes) to an enzymatic defect (acid maltase deficiency) in the lysosomes. Electron microscopic studies by Baudhuin et al. (1964), Cardiff (1966), and Smith et al. (1967) have demonstrated similar glycogenosomes. The identification of such glycogenosomes or “abnormal lysosomes” in the affected muscle fibers is thus of important diagnostic value in the type II disease. However, in glycogen storage myopathies other than the type II disease, glycogenosomes have recently been observed by Osame et al. (1978), 40.5 0014-4800/83 $3.00 Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ET AL.
Dannon et al. (1981), and Iwamasa et al. (1982a). In this report, ten cases of glycogen storage disease with skeletal muscle involvement are described, paying special attention to the formation of glycogenosomes. MATERIALS
AND METHODS
Patients Type II glycogen storage disease: cases l-3. Type III glycogen storage disease: cases 4-7. Type V glycogen storage disease: case 8. Muscle type phosphorylase b kinase deficiency: cases 9 and 10. Case 1. A 2-year-old female infant whose main symptoms were cardiomegaly, hepatomegaly, and muscle weakness of the extremities. She was floppy at birth and began to walk at the age of 1.5 years. Biopsied muscle (M. bioceps brachii) revealed a marked decrease in acid maltase and neutral maltase activities. Case 2. A 22-year-old man whose symptoms were muscle weakness and atrophy in the extremities. Cardiomegaly and hepatomegaly have not been noted to date. He was floppy at birth and walked at the age of 1 year and 2 months. The onset of his disease was at 5 years of age. Biopsied muscle (deltoid muscle) revealed a marked decrease in acid maltase activity and an increase in glycogen content. Case 3. A male adult, aged 45, whose duration of illness was almost 5 years. Muscle weakness and atrophy of the extremities, more prominent in the proximal area, formed his main symptoms. No cardiomegaly or hepatomegaly has been noted to date. M. biceps femoris was biopsied and subjected to measurements of acid maltase activity, glycogen content, and other glycogenolytic enzyme activities. A prominent increase in glycogen content and a decrease in acid maltase activity were noted. The levels of other glycogenolytic enzyme activities were within the normal ranges. Case 4. A 39-year-old female who had slowly progressive muscle weakness and atrophy in the distal extremity muscles. Furthermore, the sternomastoid and thigh adductor muscle were similarly affected. No cardiomegaly or hepatomegaly was noted, and the patients liver function tests were normal. Her younger brother (aged 32) had very similar clinical symptoms. The femoral quadriceps was biopsied, and the debranching enzyme activity as measured with phosphorylase limit dextrin (PLD) as substrate and [14C-Iglucose incorporation into glycogen revealed extremely decreased values. Other glycogenolytic enzyme activities were within the normal ranges. Case 5. A 32-year-old female whose clinical symptoms closely resembled those of case 4, presenting distal extremity muscle weakness and atrophy. Liver function tests were normal. The serum creatinine phosphokinase (CPK) activity was slightly elevated at 42 W/liter (normal, below 25 II-J/liter). Biopsied muscle M. quadriceps femoris revealed a marked decrease in debranching enzyme activity. Case 6. A 45-year-old male whose symptoms were also very similar to cases 4 and 5, showing distal muscle weakness and atrophy. No cardiomegaly, but mild hepatomegaly have been noted to date. Case 7. A 40-year-old male whose main symptoms were muscle weakness of the extremities and hepatomegaly. The hepatomegaly had been present since the age of 3 years. Liver biopsy revealed hepatocytes of foamy appearance which contained large amounts of glycogen on PAS staining. The biopsied liver and M. biceps femoris showed a marked decrease in debranching enzyme activity.
GLYCOGEN
407
MYOPATHY
Case 8. A 50-year-old male who had noted leg muscle stiffness after running a 25-m race as a primary high school student. Since 40 years of age, muscle weakness of the upper extremities had slowly progressed and facial muscle stiffness after laughing was also noted. The ischemic exercise test showed no elevation of serum lactic acid level. Liver function tests were normal. The serum CPK activity was elevated at 94 III/liter. The phosphorylase activity in the biopsied M. triceps brachii was extremely decreased. Case 9. A 1.7-year-old female infant. She was floppy at birth and has not walked to date. Muscle hypotonia was noted in the extremities. It was slightly dominant distally, although the proximal muscles were also markedly involved. The biopsied M. quadriceps revealed a marked decrease in phosphorylase b kinase activity and a moderate decrease in debranching enzyme activity. Other glycogenolytic enzyme activities were within the normal ranges. The glycogen content was elevated more than normal. Case 10. A young female, aged 17. Muscle weakness and stiffness in the extremities during work of short duration, slightly dominant in the distal muscles, had been noted since the age of 5 years. No cardiomegaly or hepatomegaly was observed. Liver function tests were within the normal ranges. However, a slight elevation of serum CPK activity was observed. The biopsied M. biceps femoris revealed a marked decrease in phosphorylase b kinase activity. Other glycogenolytic enzyme activities yielded normal values. The glycogen content was elevated more than normal. The details of these ten cases are summarized in Table I.
TABLE I Summarized Data of the Cases Case
5w of disease
Age WeW
Sex
Main symptoms Muscle weakness of extremities. Cardiomegaly. Hepatomegaly. Muscle weakness and atrophy of extremities.
1
II
2
F
2
II
M
II
22 (Disease onset was at 5 years) 45
III
39
F
III
32
F
III
45
M
III
40
M
V Phosphorylase b kinase deficiency Phosphorylase b kinase deficiency
50 1.7
M F
Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Muscle weakness and atrophy of extremities. Hepatomegaly. Muscle weakness and stiffness. Muscle hypotonia.
17
F
Muscle weakness and stiffness.
8 9 10
M
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ET AL.
Morphological Examinations Small pieces (1.0 x 1.0 x 0.5 cm) of biopsied muscle were frozen using liquid nitrogen. Fresh frozen sections were cut at a thickness of 6 km. Enzyme histochemical studies for phosphorylase (Takeuchi and Kuriaki, 1955), acid phosphatase (Barka and Anderson, 1962), ATPase with preincubation at pH 10.3,4.6, and 4.2 (Dubowitz and Brooke, 1973), and succinate dehydrogenase (Lojda et al., 1979) were performed as usual. For routine hematoxylin-eosin, PAS, and Gomori’s trichrome staining, other small pieces of tissue fixed in 10% formalin were used. For electron microscopic observation, the tissues were fixed in 2.5% glutaraldehyde for 2 hr, and postfixed in 2% osmium tetraoxide for 1 hr by the usual method, followed by embedding in Epon 812. Ultrathin sections were then cut, and after staining with lead nitrate, were observed under a Hitachi 12A electron microscope. Biochemical Examinations The amount of glycogen was determined by the anthrone method. Protein was measured by the method of Lowry et al. The activities of phosphorylase (Sutherland and Wosilait, 1956), phosphorylase b kinase (Krebs, 1966), debranching enzyme (Hers et al., 1967), and acid and neutral maltase (Hers, 1964; Salafsky and Nadler, 1973) were measured using muscle extract. To obtain the muscle extract, biopsied muscle was homogenated with 10 vol of ice-cold distilled water using a Waring blender. The resultant homogenate was centrifuged at 10,000 rpm for 30 min at 2°C. The supernatant was employed as the muscle extract. Assays of the acid and neutral maltase activities were made with 4-methylumbelliferyl (Yglucoside and maltose as substrates. The debranching enzyme activity was measured using phosphorylase limit dextrin, prepared in our laboratory, as substrate and the [i4C-Iglucose incorporation into glycogen according to Hers’ method (1964). Extraction of glycogen was performed as described previously (Iwamasa et al., 1982a). Using this glycogen, the iodine spectrum of the glycogen was determined
Fig. 1. Fibers of biopsied M. biceps brachii (case 1) showing marked vacuolation. H & E staining. x 365.
GLYCOGEN
MYOPATHY
FIG. 2. Biopsied M. biceps femoris (case 3) showing severe vacuolation. H & E staining.
X
365.
as follows. Ten microliters of 0.01 N iodine solution containing 0.02 N KI was added to 2 ml of the glycogen solution (0.1 mg/ml), and the spectrum was measured with a Hitachi 100-60 spectrophotometer at 24°C. RESULTS Morphological Findings Figures 1 and 2 show transverse sections of biopsied muscles from cases of type II glycogen storage disease. Figure 1 is from the infantile form (case 1) and
FIG. 3. Some portions of the deltoid muscle from case 3 show no vacuolation and no marked abnormality. However, in this material, the decrease of acid maltase activity was similar to that in other vacuolated portion in this case. H & E staining. X 400.
410
IWAMASA
ET AL.
Fig. 2 is from the adult form of the disease (case 3). The light microscopic findings for these forms are very similar showing vacuole formation. The vacuoles ranged from 2-5 to 60 km in diameter. The type II fibers were slightly more affected than the type I fibers. The size of the fibers was more variable than normal. Some central nuclei were found. However, necrosis and myophagy were very rare. In some portions, only variety of the fiber size was found, and vacuolation was very rare (Fig. 3). Biopsied muscle sections from generalized type III glycogen storage disease (case 7) (Fig. 4) also revealed vacuole formation, and the fiber size was more variable than normal. The vacuoles were slightly larger and more frequent than those in the type II disease. Sometimes a few centrally located nuclei were observed. Vacuoles which stained very strongly on PAS staining were observed in the sarcoplasm and subsarcolemmal area. Furthermore, rimmed vacuoles were also found elsewhere in the type III disease (Fig. 5 and 6). However, rimmed vacuoles occurred more frequently in the muscle type of type III glycogen storage disease than in the generalized type of the type III disease. Slight endomysial fibrosis was present, and rare necrosis was also found. Enzyme histochemical acid phosphatase was strongly demonstrated in the rimmed vacuoles. The type V glycogen storage disease (McArdle disease) and muscle type phosphorylase b kinase deficiency showed very similar formation of subsarcolemmal blebs (Fig. 7) which were strongly stained on PAS staining. In the biopsied muscle, bleb formation was observed at a frequency of 25-60% in the fibers. Figure 8 shows the enzyme histochemical reaction of ATPase (pH 10.3) in case 9. The type I fibers were slightly more affected than the type II tibers. However, the histochemical reaction of phosphorylase without AMP in the reaction medium revealed a similar negative reaction between the type I and type II fibers (Fig. 9).
FIG. 4. Biopsied M. biceps femoris (case 7) stained by H L E staining. Marked vacuolation of the fibers is observed. Variety in the size of fibers is also seen. The cases 4-6 showed very similar features. x 365.
GLYCOGEN
MYOPATHY
FIG. 5. Rimmed vacuole (arrow) having a distinct hematoxylinophilic E staining. x 680.
lining is found (case 7). H &
By adding AMP to the reaction medium, all of the fibers (type I and II fibers) showed a strong positive reaction (Fig. 10). There was also considerable variation in the size of the fibers when compared with that of normal muscle. Central nuclei were not frequently found. The connective tissue was not increased in these cases. Case 9 revealed very small numbers of rimmed vacuoles.
FIG. 6. Rimmed vacuoles (arrows) also have PAS-positive substance in them (case 7). PAS staining. x700.
412
IWAMASA
ET AL.
7 e ;9r
FIG. 7. Subsarcolemmal blebs of phosphorylase b kinase deficiency, indicated by arrows (case 9, M. quadriceps). Cases 8 and 10 also showed similar bleb formation. H & E staining. x 700.
Electron
Microscopic
Observations
The electron microscopic findings for muscle of type II, III, and V, and phosphorylase b kinase deficiency were very similar to those reported by Cardiff (1966), Hug et al. (1966), Smith et al. (1967) Garancis (1968), Engel et al. (1973), Howell (1978), and DiMauro and Hartlage (1978). In the type II disease, membrane-surrounded accumulations of glycogen (glycogenosomes) termed “abnormal lysosomes” were characteristically observed frequently (Fig. 11). How-
FIG. 8. ATPase reaction of case 9 at pH 10.3. The dark stained type I fibers are slightly more affected than the type II fibers. The arrows indicate vacuolation of the fibers. x 700.
GLYCOGEN
MYOPATHY
413
FIG. 9. Phosphorylase reaction without AMP (case 9). Almost all of the fibers show a negative reaction. The type I and II fibers show similar negative results. x 360.
ever, in the type III disease, glycogenosomes were demonstrated not so rarely (Fig. 12). The glycogenosome formation observed under the electron microscope was in fibers where rimmed vacuoles were seen light microscopically. In muscle fibers of phosphorylase b kinase deficiency, large amounts of glycogen accumulation were noted in subsarcolemmal areas which appeared as subsarcolemmal blebs under the light microscope. Furthermore, the fibers showed increased glycogen in such phosphorylase b kinase deficiency. Case 9, which exhibited an extreme decrease in phosphorylase b kinase activity and also a moderate decrease in debranching enzyme activity, revealed formation of glycogenosomes in considerable numbers (Fig. 13).
FIG. 10. Phosphorylase reaction with AMP using sections consecutive with Fig. 9. A marked positive reaction is demonstrated on all the fibers. Case 10 also revealed a similar reaction. The results of Figs. 9 and 10 indicate that the phosphorylase in these cases is in the b form. x 360.
414
IWAMASA
ET AL.
FIG. 11. Electron micrograph of case 2 (type II glycogen storage disease). Glycogenosomes or “abnormal lysosomes” (arrows) are present and the muscle fibrils are markedly disrupted. x 19,100.
Biochemical Data
The results are summarized in Table II. Activity measurements of phosphorylase b kinase in cases 9 and 10 are shown in Figs. 14A and B. The extracted glycogen from type III disease (case 7) demonstrated a lower P-amylolysis rate than that of normal controls. Furthermore, the extracted glycogen from case 9 also revealed a lower rate of @amylolysis. An iodine spectrum of extracted glycogen from type III disease (case 7) is given in Fig. 15. The glycogen content in each type of disease was strongly increased. In particular, in the type II and III disease, it showed extremely high levels. The acid maltase activity in each type II disease was markedly decreased. In the infantile type II disease (case l), not only the acid maltase but also the neutral maltase
GLYCOGEN
MYOPATHY
FIG. 12. Electron micrograph of case 6 (type III glycogen storage disease). Glycogenosomes (arrows) are present. Cases 4, 5, and 7 also revealed glycogenosome formation. x 10,600.
activities were decreased, although this was not the case in the adult type of the disease (case 3). DISCUSSION Glycogenosomes, membrane-surrounded glycogen or “abnormal lysosomes,” were found not only in type II glycogen storage disease (Pompe disease), but also in cases of type III disease (Forbes or Cori disease) and in one case of phosphorylase b kinase deficiency which revealed a moderate decrease in debranching enzyme activity. In these cases other than the type II disease, glycogenosomes
416
IWAMASA
ET AL.
FIG. 13. Glycogenosome in case 9 (arrow).
x
100,000.
were apparent in considerable numbers in the muscle fibers. Glycogenosome formation is therefore not considered as a specific finding of type II glycogen storage disease. As reported by us previously (Iwamasa et al., 1980a, b), glycogenosome is formed from abnormal or different structural glycogen without a decrease in acid maltase activity in newborn rat hepatocytes. Furthermore, in ascites hepatoma AH 13 cells, glycogenosome formation is frequently observed, and the acid maltase activity is not decreased in the tumor cells. Based on these results, the glycogen storage disease of type III and phosphorylase b kinase deficiency (case 9) presented here are considered to form glycogenosomes from limit dextrin-like storage glycogen different from normal glycogen. However, as reported previously (Iwamasa et al., 1982b), in the hepatocytes of generalized type III glycogen storage disease, glycogenosome formation is barely observed. It is thought that in hepatocytes of the type III disease, the limit dextrin-like structure of glycogen is not always dominant because of the influence of feeding. Determination of the glycogenosomes morphologically is considered to depend on the balance between
0.85 0.99 0.78 23.40 23.33 26.41 20.33 28.0 27.67 29.38 27.3 k 10.5
1 2 3 4 5 6
0.93 43.98 45.0 48.0 50.10 50.05 52.33 51.14 53.80 51.00 50.0 2 10.3
pH 7.0
a 4-MUG, 4-methylumbelliferyl a-glucoside. b PLD, phosphorylase limit dextrin.
8 9 10 Controls
1
pH 4.0
Case
o-Glucosidase (CMUG as substrate) (pkf/mg protein/mitt)
2.58 2.66 2.60 0.013 0.30 0.01 0.003 2.80 2.50 2.43 2.61 -c 0.5
Debranching enzyme (PLDb as substrate) Wfhng protein/min)
6993.1 + 300
121.1 134.1 101.3 98.5
[i4C]Glucose incorporation (cpm/mg glucose/g protein/hr) a 45.0 48.6 47.7 30.0 49.6 48.3 47.5 ud ud 0.80 47.7 2 13.2
76.0 75.3 19.4 70.1 70.5 75.2 73.4 ud 73.1 70.6 78.0 -t 21.1
Phosphorylase ( pkf/gplmin) Total
TABLE II Results of Biochemical Examinations
51.2 58.6 35.7 24.0 60.1 58.3 71.3 28.6 26.7 30.5 5.0-10.0
Glycogen content (m&t wet wt)
28.5 f 1.0
18.6
17.3
a-Amylolysis (%I
418
IWAMASA
ET AL.
FIG. 14A. Phosphorylase b kinase activity measurements. 0, Control; 0, case 9; Ir, case 10. Activation of phosphorylase b to a by the muscle extract was measured using 0.125 M Tris-0.125 M sodium @glycerophosphate buffer, pH 6.8. The reaction medium contained 0.2 ml of buffer, 0.2 ml of phosphorylase b (Sigma; 28 units/mg protein), 0.1 ml of 0.018 M ATP-0.06 M MgCI,, and 0.1 ml of muscle extract in neutral cysteine. The phosphorylase a activity was measured every 10 min by the method of Sutherland and Wosilait. FIG. 14B. Activation of phosphorylase b in the muscle extract to phosphorylase a by active phosphorylase b kinase (Sigma). 0, Control; 0, case 9; +, case 10. The active phosphorylase b kinase (0.1 ml, 30 units/mg protein) was added to a mixture containing 0.2 ml of 0.125 M Tris-0.125 M sodium P-glycerophosphate buffer, pH 6.8, 0.1 ml of 0.018 M ATP-0.06 M MgCl,, and 0.2 ml of the muscle extract in 0.015 M neutral cysteine.
the turnover rate of acid maltase and autophagic activity for limit dextrin-like glycogen into lysosomes. In the case of the type II disease, as reported previously (Iwamasa er al., 1979), the glycogen extracted shows an extremely large S value compared to normal glycogen by ultracentrifuge analysis. The glycogenosome formation in type II disease is therefore thought to involve extremely large glycogen. Apart from the type II disease, in the cases of type III disease and in one case of phosphorylase OD 0.500 t
0.300
c
P 0.10 I-
I
400
5cm
\
600
nm
FIG. 15. Iodine spectrum of extracted glycogen (case 7). The measurements were carried out at 24°C. The procedures were as described in the text. The peak of the spectrum in case 7 is different from that of normal control glycogen. C, Control; P, case 7.
GLYCOGEN
MYOPATHY
419
b kinase deficiency, glycogenosomes were also formed. Nevertheless, the acid maltase activity was not decreased. It is considered that the glycogen autophagied into lysosomes is digested, gradually decreases in numbers, and that the glycogenosomes are also decreased in numbers. The glycogenosomes are found in areas where rimmed vacuoles are observed light microscopically. The type III disease (case 4) presented here had very similar morphological findings to inclusion body myositis (Osame et al., 1978). The affected muscles were of the distal extremities and light microscopically, rimmed vacuoles were observed. The patient was an adult. However, in distal myopathy, many unclassified disorders other than type III glycogen storage disease are considered to exist. Detailed examinations of the metabolical disorders in myopathy must be made. In phosphorylase b kinase deficiency, not all of the fibers show bleb formation. However, the histochemical phosphorylase reaction without AMP in the incubation medium demonstrates a negative reaction in all fibers. The enzyme activity is considered to be decreased in almost all fibers. In one case (case 9) of phosphorylase b kinase deficiency, not only was the phosphorylase b kinase, but also the debranching enzyme activity decreased. However, it remains obscure as to why the debranching enzyme activity should be decreased. Furthermore, in type II glycogen storage disease, the enzyme activity becomes markedly decreased. The reason why some portions of the muscle do not reveal marked morphological abnormality remains unknown. Further examinations are clearly needed. REFERENCES D. H. (1956). Familial cirrhosis of the liver with storage of abnormal glycogen. Lab. Invest. 5, 11-20. BARKA, T. and ANDERSON, P. J. (1962). Histochemical method for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10, 741-753. BAUDHUIN, P., HERS, H. G., and LOEB, H. (1964). An electron microscopic and biochemical study of type II glycogenosis. Lab. Invest. 13, 1139-l 152. BROWN, B. I., and BROWN, D. H. (1968). The glycogen storage diseases: Type I, III, IV, V, VII and unclassified glycogenoses. In “Carbohydrate Metabolism and its Disorders” (W. J. Whelan, ed.), Vol. 2, p. 123. Academic Press, New York. CARDIFF, R. D. (1%6). A histochemical and electron microscopic study of skeletal muscle in a case of Pompe’s disease (glycogenosis II). Pediatrics 37, 249-259. CORBEEL, L., HUE, L., LEDERER, B., DEBARSY, T., VAN DEN BERGUE, G., DEVLIEGER, H., JAEKEN, J., BRACKE, O., and EECKELS, R., (1981). Clinical and biochemical findings before and after portacaval shunt in a girl with type Ib glycogen storage disease. Pediatr. Res. 15, 58-61. Corn, G. T., and Corn, C. E (1952). Glucose-6-phosphatase of the liver in glycogen storage disease. J. Eiol. Chem. 199, 661-667. DANNON, M. J., OH, S. J., DIMAURO, S., MANALIGOD, J. R., EASTWOOD, A., NAIDU, S., and Schliselfeld, L. H. (1981). Lysosomal glycogen storage disease with normal acid maltase. Neurology 31, 51-57. DIMAURO, S., and HARTLAGE, P. L. (1978). Fatal infantile form of muscle phosphorylase deficiency. Neurology 28, 1124-l 129. DUBOWITZ, V. and BROOKE, M. H. (1973). “Muscle Biopsy: A Modem Approach,” p. 32. Saunders, London. ENGEL, A. G., GOMEZ, M. R., SEYBOLD, M. E., and LAMBERT, E. H. (1973). The spectrum and diagnosis of acid maltase deficiency. Neurology 23, 95-106. GARANCIS, J. C. (1968). Type II glycogenosis: biochemical and electron microscopic study. Amer. J. Med. 44, 289-300. HERS, H. G. (1963). Alpha-glucosidase deficiency in generalized glycogen storage disease (Pompe’s disease). Biochem. J. 86, 1l-16.
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