Journal of the Neurological Sciences, 100 (1990) 57-62
Elsevier
57
JNS 03423
Histochemical features of ragged-red fibres in diseased skeletal muscles A.E.F.H. Meijer Laboratory of Experimental Neurology, Department of Neurology. Academic Medical Centre, Universityof Amsterdam. Amsterdam (The Netherlands)
(Received 20 February, 1990) (Revised, received 27 June, 1990) (Accepted 29 June, 1990) Key words: Skeletal muscle; Ragged-red fibres; Enzyme histochemistry; Structural protein-enzyme association
Summary In the present communication, the activity of 24 oxidoreductases, transferases, isomerases and hydrolases was examined histochemically in ragged-red fibres of human skeletal muscle specimens. The biopsy material was obtained from patients with neuromuscular diseases caused by an abnormally metabolic functioning of the muscle mitochondria. The granular accumulations of the ragged-red fibres were characterized by an impressive activity of all mitochondrial and most non-mitochondrial enzymes examined, whether participating in the aerobic or in the anaerobic pathways. With the exception of mitochondrial Mg2+-stimulated ATPase, acid phosphatase and AMP-aminohydrolase, there was no activity of the other hydrolytic enzymes studied in these regions. The strong activity ofmitochondrial ATPase points to the presence of loosely coupled and/or uncoupled mitochondria. Ragged-red fibres that exhibited a diffuse and corpuscular activity of acid phosphatase, were always undergoing necrotic changes. Adsorption studies with diluted enzyme solutions demonstrated that the granular accumulations display a specific, moderate affinity for glycolytic enzymes.
Introduction
Materials and Methods
In a previous study (Meijer 1988), the enzyme histochemical features of tubular aggregates were characterized. Resuits obtained with adsorption studies pointed to a strong adsorption of some enzymes to the tubular aggregates in vivo. This phenomenon mainly determined the histochemical characteristics of these structures. The aim of the present investigation was to characterize ragged-red fibres (Fig. 1) by similar means. It is surprising that enzyme histochemical studies of ragged-red fibres are often incomplete, since these studies concentrated only on some mitochondrial enzymes and a few hydrolases. Moreover, the methods used for the demonstration of non-structurally bound, partially and weakly structurally bound enzymes were equivocal owing to diffusion artefacts (Meijer 1980). In the present study the activity and location of a number of oxidoreductases, transferases and isomerases, involved in the major aerobic and anaerobic pathways of energy metabolism, and of several hydrolases, were investigated. The most reliable conventional methods and semipermeable membrane techniques were employed.
The present study was carried out on human skeletal muscle specimens. The biopsy material was obtained from 24 patients with various metabolic disorders of the muscle mitochondria (Busch et al. 1981). The muscle biopsies contained mitochondria with a loosely coupled state of the oxidative phosphorylation (12cases), deficiency
Correspondence to: Prof. Dr. A.E.F.H. Meijer, Krakeling 14, 2121 BM Bennebroek, The Netherlands.
Fig. 1. Transverse section of human quadriceps femoris mucle with a mitochondrial defect in the energy metabolism, showing ragged-red fibres. Improved trichrome technique according to Engel and Cunningham (1963). x 140.
0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
58 of NADH :ubiquinone oxidoreductase (EC 1.6.5.3) (5 cases), deficiency of NADH : ubiquinone oxidoreductase, as well as a low level of carnitine in the skeletal muscle (1 case), significant decrease in activity of carnitine palmitoyl transferase II (EC 2.3.1.21) (2 cases), deficiency of cytochrome c oxidase (EC 1.9.3.1) (3 cases) and disturbed supply of Krebs cycle intermediates (anaplerotic reactions) by a decreased activity of pyruvate dehydrogenase (EC 1.2.2.2.) (1 case). The material comprised 17 specimens of left quadriceps femoris, 2 from left vastus lateralis and 5 from right gluteus medius muscle. All specimens exhibited ragged-red fibres. After excision, specimens were rapidly frozen by immersing the tissue blocks in isopentane, cooled to about - 150 °C with liquid nitrogen. The specimens were stored at - 96 °C. Longitudinal and transversal serial sections, 7/~m thick, were used. For the morphological study, sections were stained with haematoxylin-eosin and with the modified trichrome procedure (Engel and Cunningham 1963). The PAS reaction with and without pretreatment with diastase (EC 3.2.1.1.), the oil red O and Sudan black 10B stains were used to demonstrate glycogen and lipids, respectively. Sections were subjected to the histochemical techniques listed in Table 1.
For adsorption studies, sections were treated with 0.25 ~o HNO 3 for 5 min, following the procedure of Gomori (1950), in order to destroy enzyme activity. After subsequent thorough washing with double-distilled water, sections appeared entirely devoid of enzyme activity. The sections were then treated with diluted enzyme solutions in physiological saline for 24h at 25°C. The solutions, containing about 2 IU of enzyme per ml of solution, were prepared from products of Sigma (St. Louis, U.S.A.). Adsorption properties of ATP : D-fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11), D-glucose6-phosphate ketol-isomerase (GPI; EC 5.3.1.9), alphaD-glucose-l,6-phosphomutase (PGM; EC 5.4.2.2), D-glyceraldehyde-3-phosphate : NAD ÷ oxidoreductase (GADH; EC 1.2.1.12), 6-phospho-D-gluconate : NADP + 2-oxidoreductase (PGDH; EC 1.1.1.44), D-glucose6-phosphate:NADP ÷ 1-oxidoreductase (GPDH; EC 1.1.1.49), isocitrate:NAD +-oxidoreductase (I-NAD + ; EC 1.1.1.41), isocitrate:NADP + oxidoreductase (INADP + ; EC 1.1.1.42), malate : NAD + oxidoreductase (M-NAD + ; EC 1.1.1.37), malate : NADP + oxidoreductase (M-NADP + ; EC 1.1.1.40) and lactate:NAD + oxidoreductase (LDH; EC 1.1.1.27) were investigated. After treatment with the enzyme solutions, the sections
TABLE 1 ENZYME HISTOCHEMICAL TECHNIQUES USED Enzymes (systematic name and abbreviation)
EC number
References
(S)-Lactate:NAD ÷ oxidoreductase (LDH) (S)-Malate : NAD ÷ oxidoreductase (M-NAD * ) (S)-Malate : N A D P ÷ oxidoreductase (oxaloacetate decarboxylating) (M-NADP +) lsocitrate : N A D * oxidoreductase (decarboxylating) (I-NAD ÷ ) Isocitrate : NADP ÷ oxidoreductase (decarboxylating) (I-NADP + ) 6-Phospho-D-gluconate : N A D P ÷ 2-oxidoreductase (decarboxylating) (PGDH) D-Glucose-6-phosphate : NADP + l-oxidoreductase (GPDH) sn-Glycerol-3-phosphate : menadione 2-oxidoreductase (GPOX) D-Glyceraldehyde-3-phosphate : NAD ÷ oxidoreductase (phosphorylating) (GADH) Succinate: PMS oxidoreductase (SDH) N A D P H : nitro-BT oxidoreductase (NADPH) NADH : nitro-BT oxidoreductase (NADH) Ferrocytochrome c:oxygen oxidoreductase (CO) Donor : hydrogen peroxide oxidoreductase (PO) 1,4-~-D-Glucan: orthophosphate a-D-glucosyltransferase (GP) ATP : D-fructose-6-phosphate 1-phosphotransferase (PFK) Orthophosphoric-monoester phosphohydrolase (alkaline optimum) (ALP) Orthophosphoric-monoester phosphohydrolase (acid optimum) (AP) 5' Ribonucleotide phosphohydrolase AMP aminohydrolase (AMP-DA) ATP phosphohydrolase (H ÷-transporting, Mg 2+ activated) (ATP-Mg) Myosin ATP phosphohydrolase (actin-translocating) (ATP-Ca) D-Glucose-6-phosphate ketol-isomerase (GPI) c~-D-Glucose 1,6-phosphomutase (PGM) (phosphoglucomutase former, EC 2.7.5.1)
1.1.1.27 1.1.1.37 " 1.1.1.40 1.1.1.41 1.1.1.42 1.1.1.44 1.1.1.49 1.1.99.5 1.2.1.12 1.3.99.1 1.6.99.2 1.6.99.3 1.9.3.1 1.11.1.7 2.4.1.1 2.7.1.11 3.1.3.1 3.1.3.2 3.1.3.5 3.5,4.6 3.6,1.34 3.6.1.32 5.3,1.9 5.4.2.2
Meijer (1973) Barka and Anderson (1963) Meijer and De Vries (1975) Barka and Anderson (1963) Meijer and De Vries (1975) Meijer and De Vries (1974) Meijer and De Vries (1974) Lojda et al. (1979) De Vries et al. (1980) Nachlas et al. (1957) a Burstone (1962) Burstone (1962) Burstone (1962) b Christie and Stoward (1978) Meijer (1968) Meijer and Stegehuis (1980) Pearse (1968) c Meijer (1972) Pearse (1968) Fishbein et al. (1978) Meijer and Vloedman (1980) Meijer (1970) De Vries and Meijer (1976) De Vries and Meijer (1976)
" Phenazine methosulphate was added to the incubation medium at a final concentration of 50 pg/l. b p-Aminodiphenylamine and p-methoxy-p-aminodiphenylamine were used as substrates. The reactions were performed with and without cytochrome c added to the incubation medium at a final concentration of 0.5 mg/ml. c Naphthyl-AS-MX-phosphate was used as substrate and fast red violet LB as coupling substance.
59 were washed thoroughly with double-distilled water and incubated with the corresponding enzyme histochemical techniques (Meijer 1988),
Results The present study demonstrates that ragged-red fibres, in contrast to tubular aggregates, do not exhibit uniform histochemical properties. The only consistent finding was a considerable activity of mitochondrial enzymes in the granular aggregates. The increase of mitochondrial enzyme activity was the result of a marked increase in the number of mitochondria in the aggregates. In the most pronounced form, the heavily granular appearance of the ragged-red fibres extended from the subsarcolemmal periphery to the centre of the fibre. Accordingly, the entire fibre showed a strongly increased mitochondrial enzyme activity. The extramitochondrial ground cytoplasm of the peripheral region of the ragged-red fibres contained a slight to strong activity of non-structurally bound enzymes. Therefore, in general, the increased activity of mitochondrial and non-mitochondrial enzymes was distributed quite irregularly over the fibre periphery, often in a reciprocal mode. We have found, for example, that the peripheral zone of the ragged-red fibres contained ballooned areas, devoid of mitochondria and as a result devoid of mitochondrial enzyme activity. Such areas were surrounded by accumulations of mitochondria characterized by an intensely stained rim of mitochondrial enzyme activity. On the other hand, the activity of the glycolytic enzymes studied was strongly increased in these ballooned areas and moderately in the rim. Moreover longitudinal sections clearly establish that enzyme activities are not homogeneously distributed over the ragged-red fibres (Fig. 2).
Fig. 2. Longitudinalsectionof human giuteus medius muscle. Abundant activity of succinate dehydrogenase in the granular material that is inhomogeneouslydistributed in the ragged-red fibres, x 240.
Fig. 3. Activity of myosin adenosine triphosphatase in human gluteus medius musclewith mitochondriain a looselycoupledstate. The raggedred fibre reveals only a few myofibrils.The sarcoplasm of the muscle is unstained, x 360. The number of myofibrils is greatly reduced in the granular zones of ragged-red fibres. It is therefore understandable that these zones did not reveal myosin Ca 2÷-stimulated ATPase activity (Fig. 3). A marked deposition of lipids and glycogen was noted in the granular regions of some raggedred fibres. Other ragged-red fibres did not reveal these substances in the granular accumulations. Corpuscular and diffuse acid phosphatase activities were present in only a few of the muscle specimens containing ragged-red fibres whose content consisted entirely of mitochondrial granules. The results of the adsorption studies, all of which were performed with diluted enzyme solutions, demonstrated that fibres without pathological alterations stained only very slightly. On the other hand, the granular zones stained moderately to strong with solutions of the glycolytic enzymes PGM, PFK, LDH, GPI and G A D H examined
Fig. 4. Transverse section of human quadriceps femoris muscle with a mitochondrialdefectin the energymetabolism, showingactivityofphosphofructokinase. Apparently the ragged-red fibres belong to the aerobic type I fibres, x 140.
60
Fig. 5. Serial section of Fig. 4, showing activity of lactate dehydrogenase. Apparently the same ragged-red fibres belong to the anaerobic type II fibres. The discrepancy is caused by loosing constancy of activity ratios of enzymes in the pathological fibres (Meijer et al. 1977). x 140.
(Figs. 4 and 5). The granular zones did not stain with diluted solutions of the enzymes GPDH, PGDH, I-NAD ÷, I-NADP +, M-NAD ÷ and M-NADP+.
Discussion
The term "ragged-red" fibre was introduced by Engel (1971), denoting the presence of granular accumulations mainly in the subsarcolemmal areas of these fibres, which exhibit a bright red to reddish blue colour with the Gomori trichrome stain. Numerous publications on ragged-red fibres have subsequently appeared. Light microscopic and electron microscopic studies have demonstrated that the granular accumulations consist of mitochondria. The microscopically enlarged mitochondria often exhibit an unusual shape and frequently they contain densely packed crystalline and para-crystalline inclusions. The general opinion is that ragged-red fibres are virtually restricted to aerobic type 1 fibres (Olson et al. 1972). However, this is not true: in a myopathy with loosely coupled mitochondria, Meijer and van Wijngaarden (1983) found ragged-red fibres predominantly in anaerobic type II fibres. Furthermore ragged-red fibres frequently do not reveal a distinct aerobic or anaerobic metabolism due to a loss of constant proportion groups (Meijer et al. 1977; Figs. 4 and 5). From the staining results described above it follows that the variation in staining properties of the ragged-red fibres is caused by the structural, metabolic and adsorption aspects of these fibres. The structural aspects are determined by the location of the mitochondria and of the ground cytoplasm in the peripheral zone of the ragged-red fibres.
Concerning the metabolic aspects most remarkable is the strong activity of mitochondrial Mg2÷ -stimulated ATPase in the granular accumulations, indicating the presence of loosely coupled and/or uncoupled mitochondria (HOlsmann et al. 1970; Meijer and Vloedman 1980; Meijer 1981, 1983; Meijer and van Wijngaarden 1983; Meijer etal. 1985). The disfunction of the oxidative phosphorylation can be primary as for example in exercise-induced myopathy (Van den Hoven et al. 1986; Meijer et al. 1989) or secondary as for example caused by shortness of anaplerotic enzyme reactions (Meijer et al. 1987). Corpuscular and especially diffuse activity of the lysosomal enzyme acid phosphatase was present in the peripheral zone of the ragged-red fibres which were affected by active necrotic processes (Tappel et al. 1962; Z alkin et al. 1962; Emanuelli et al. 1969). Results of different biochemical investigations demonstrated that in vivo, diffusion of enzymes from their original site and adsorption at other sites can take place (Martin and Jacoby 1949; Gomori 1950; Yokoyama et al. 1951 and Goebel and Puchtler 1954). In other studies it has been shown that some glycolytic enzymes of skeletal muscle can be bound to structural proteins (Arnold and Pette 1968; Arnold etal. 1969, 1971; Green etal. 1965; Rose and Warms 1967). The adsorption findings presented in this communication are in accordance with the association properties of a number of enzymes as reported by Clarke and Masters (1973, 1974, 1975). These authors demonstrated strong adsorption capacities for PFK, LDH, GPI and GADH. From these findings it follows that adsorption phenomena partially determine the histochemical features of granular accumulations of ragged-red fibres. Since, at present, specific adsorption properties have been demonstrated in both tubular aggregates and granular accumulations of ragged-red fibres, it is very probable that specific adsorption phenomena will be a fairly general occurrence. Consequently these phenomena should be taken into account to explain enzyme histochemicai features of tissue structures. Acknowledgements The author would like to express appreciation to collegues of the Belgian-Dutch Study Group on Neuromuscular Diseases, for providing human muscle specimens.
References Arnold, H. and D. Pette (1968) Binding of glycolytic enzymes to structure proteins of the muscle, Eur. J. Biochem., 6: 163-171. Arnold, H., J. Nolte and D. Pette (1969) Quantitative and histochemical studies on the desorption and readsorption of aldolase in crossstriated muscle, J. Histochem. Cytochem., 17: 314-320. Arnold, H., R. Henning and D. Pette (1971) Quantitative comparison of the binding of various glycolytic enzymes to F-actin and the interaction of aldolase with G-actin, Eur. J. Biochem., 22: 121-126.
61 Barka, M.D. and P.J. Anderson (1963) Histochemistry, Theory, Practice and Bibliography, Harper and Row, New York. Burstone, M.S. (1962) Enzyme Histochemistry, Academic Press, New York. Busch, H.F.M,, F.G.I. Jennekens and H.R. Scholte (1981) Mitochondria and muscular diseases, Mefar, Beetsterzwaag, The Netherlands. Christie, K.N. and P.J. Stoward (1978) Endogenous peroxidase in mast cells localized with a semipermeable membrane technique, Histochem. J., 10: 425-433. Clarke, F.M. and C.J. Masters (1973) Multi-enzyme aggregates: new evidence for an association of glycolytic components, Biochim. Biophys. Acta, 327: 223-226. Clarke, F.M. and C.J. Masters (1974) On the association of glycolytic components in skeletal muscle extracts, Biochim. Biophys. Acta, 358: 193-207. Clarke, F.M. and C.J. Masters (1975) On the association of glycolytic enzymes with structural proteins of skeletal muscle, Biochim. Biophys. Acta, 381: 37-46. Emanuelli, G., G. Satta and G. Perpignano (1969) Lysosomal damage and its possible relationship with mitochondrial lesion in rat kidney under obstructive jaundice, Clin. Chim. Acta, 25: 167-172. Engel, W.K. (1971) "Ragged-red fibres" in opthalmoplegia syndromes and their differential diagnosis. In: Kakulas, B.A. (Ed.), Muscle Diseases, I.C.S. No. 237, Excerpta Medica, Amsterdam, p. 28. Engel, W.K. and G.G. Cunningham (1963) Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections, Neurology (Minn.), 13: 919-923. Fishbein, W.W., V.W. Armbrustmacher and J.L. Griffin (1978) Myoadenylate deaminase deficiency: a new disease of muscle, Science, 299: 545-548. Goebel, A. and H. Puchtler (1954) Untersuchungen zur Methodik des histochemischen Nachweises der alkalischen Phosphatase, Zbl. Allgem. Pathol., 92: 159-171. Gomori, G. (1950) Sources of error in enzymatic histochemistry, J. Lab. Clin. Med., 35: 802-809. Green, D.E., E. Murer, H.O. Hultin, S.H. Richardson, B. Salmon, G.P. Brierley and H. Baum (1965) Association of integrated metabolic pathways with membranes, Arch. Biochem. Biophys., 112: 635-647. Hoven, van den, R., H.J. Breukink, Th. Wensing, A.E.F.H. Meijer and A.J. Tigges (1986) Loosely coupled skeletal muscle mitochondria in exertional rhabdomyolysis, Equine Vet. J., 18: 418-421. Htilsmann, W.C., A.E.F.H. Meijer, J. Bethlem and G.K. van Wijngaarden (1970) Different mitochondrial species in human skeletal muscle. In: Walton, J.N., N. Canal and G. Scarlato (Eds.), Muscle Diseases, I.C.S. No. 199, Excerpta Medica, Amsterdam, pp. 319-322. Lojda, Z., R. Gossrau and T.H. Schiebler (1979) Enzyme Histochemistry, Springer-Verlag, Berlin, Heidelberg, New York. Martin, B.F. and F. Jacoby (1949) Diffusion phenomenon complicating the histochemical reaction for alkaline phosphatase. J. Anat., 83: 351-363. Meijer, A.E.F.H. (1968) Improved histochemical method for the demonstration of the activity of ~t-glucan phosphorylase. 1. The use of glucosyl acceptor dextran, Histochemie, 12: 244-252. Meijer, A.E.F.H. (1970) Histochemical method for the demonstration of myosin adenosine triphosphatase in muscle tissue, Histochemie, 22: 51-58. Meijer, A.E.F.H. (1972) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 1. Acid phosphatase, Histochemie, 30: 31-39. Meijer, A.E.F.H. (1973) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 3. Lactate dehydrogenase, Histochemie, 35: 165-172. Meijer, A.E.F.H. (1980) Semipermeable membrane techniques in quantitative enzyme histoehemistry. In: Trends in Enzyme Histochemistry
and Cytochemistry, Ciba Foundation Symposium, 73. Excerpta Medica, Amsterdam, pp. 103-120. Meijer, A.E.F.H. (1981) The histochemical detection of loosely coupled mitochondria in biopsies of skeletal muscles. In: Busch, H.F.M., F.G.I. Jennekens and H.R. Scholte (Eds.), Mitochondria and Muscular Disease, Mefar, Beetsterzwaag, The Netherlands, pp. 71-76. Meijer, A.E.F.H. (1983) The histochemical demonstration of loosely coupled mitochondria in human skeletal muscle, Histochem. J., 15: 331-335. Meijer, A.E.F.H. (1988) Histochemical features of tubular aggregates in diseased human skeletal muscle fibres, J. Neurol. Sci., 86: 73-82. Meijer, A.E.F.H. and G.P. de Vries (1974) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 4. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (decarboxylating), Histochemistry, 40: 349-359. Meijer, A.E.F.H. and G.P. de Vries (1975) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 5. Isocitrate:NADP ÷ oxidoreductase (decarboxylating) and malate : NADP ÷ oxidoreductase (decarboxylating), Histochemistry, 43: 225-236. Meijer, A.E.F.H., E.A. Elias and A.H.T. Vloedman (1977) The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle. 3. Human skeletal muscles with inherited or acquired disease of the neuromuscular system, Histochemistry, 53: 97-105. Meijer, A.E.F.H. and A.H.T. Vloedman (1980) The histochemical characterization of the coupling state of skeletal muscle mitochondria, Histochemistry, 69: 217-232. Meijer, A.E.F.H. and F. Stegehuis (1980) Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles, Histochemistry, 66: 75-81. Meijer, A.E.F.H. and G.K. van Wijngaarden (1983) Follow-up study of a myopathy with loosely coupled mitochondria, Pathol. Res. Pract., 178: 73-77. Meijer, A.E.F.H., H.R. Scholte and H.F.M. Busch (1985) Zur histochemischen Kennzeichnung und der mOglichen Bedeutung von Skelettmuskelmitochondrien mit einem Defekt der oxydativen Phosphorylierung, Wiss. Z. Friedrich-Schiller-Univ. Jena, Naturwiss. R, 34: 417-424. Meijer, A.E.F.H., H.R. Scholte and H.F.M. Busch (1987) Die histochemische Mg2 ÷ -ATPase-Technik zum Nachweis von Atmungskettendefekten und inad/iquater Versorgung mit Krebszyklus-Intermedi~produkten in Skelettmuskelmitochondrien, Acta Histochem., Suppl. Band, 34: 135-140. Meijer, A.E.F.H., R. van den Hoven, Th. Wensing and H.J. Breukink (1989) Histochemische ,~nderungen in Skeletmuskeln von Rhabdomyolyse-empfindlichen Trabrennpferden nach Grenzbelastung. I. Friihzeitige myopathologische ,~nderungen, Acta Histochem., 87: 1-11. Naehlas, M., K.C. Tsou, E. de Souza, C.-S. Cheng and A.M. Seligman (1957) The cytoehemical demonstration of succinate dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole, J. Histochem. Cytochem., 5: 420-436. Olson, W., W.K. Engel, G.O. Walsh and R. Einaugler (1972) Oculocraniosomatic neuromuscular disease with ragged-red fibres. Histochemical and ultrastructural changes in limb muscles of a group of patients with idiopathic progressive external ophtalmoplegia, Arch. Neurol., 26: 193-211. Pearse, A.G.E. (1968) Histochemistry; Theoretical and Applied, Vol. 1. Churchill, London. Rose, I.A. and J.V.B. Warms (1967) Mitochondrial hexokinase, release, and location, J. Biol. Chem., 242: 1635-1645.
62 Tappel, A.L., H. Zalkin, K.A. Caldwell, I.D. Desai and S. Shibko (1962) Increased lysosomal enzymes in genetic muscular dystrophy, Arch. Biochem. Biophys., 96: 340-346. De Vries, G.P. and A.E.F.H. Meijer (1976) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 6. D-6-phosphate isomerase and phosphoglucomutase, Histochemistry, 50: 1-8. De Vries, G.P., A.J. Tigges and A.E.F.H. Meijer (1980) The histochemical demonstration of glyceraldehyde phosphate dehydrogenase activity
with a semipermeable membrane technique, Histochem. J., 12: 119-122. Yokoyama, H.O., R.E. Stowell and R.M. Mathews (1951) Evaluation of histochemical alkaline phosphatase technics, Anat. Rec., 109: 139-160. Zalkin, H., A.L. Tappel, K.A. Caldwell, S. Shibko, I.D. Desai and T.A. Holliday (1962) Increased lysosomal enzymes. In: Muscular Dystrophy of Vitamin E-deficient rabbits, J. Biol. Chem., 237: 2678-2862.
Elsevier
57
JNS 03423
Histochemical features of ragged-red fibres in diseased skeletal muscles A.E.F.H. Meijer Laboratory of Experimental Neurology, Department of Neurology. Academic Medical Centre, Universityof Amsterdam. Amsterdam (The Netherlands)
(Received 20 February, 1990) (Revised, received 27 June, 1990) (Accepted 29 June, 1990) Key words: Skeletal muscle; Ragged-red fibres; Enzyme histochemistry; Structural protein-enzyme association
Summary In the present communication, the activity of 24 oxidoreductases, transferases, isomerases and hydrolases was examined histochemically in ragged-red fibres of human skeletal muscle specimens. The biopsy material was obtained from patients with neuromuscular diseases caused by an abnormally metabolic functioning of the muscle mitochondria. The granular accumulations of the ragged-red fibres were characterized by an impressive activity of all mitochondrial and most non-mitochondrial enzymes examined, whether participating in the aerobic or in the anaerobic pathways. With the exception of mitochondrial Mg2+-stimulated ATPase, acid phosphatase and AMP-aminohydrolase, there was no activity of the other hydrolytic enzymes studied in these regions. The strong activity ofmitochondrial ATPase points to the presence of loosely coupled and/or uncoupled mitochondria. Ragged-red fibres that exhibited a diffuse and corpuscular activity of acid phosphatase, were always undergoing necrotic changes. Adsorption studies with diluted enzyme solutions demonstrated that the granular accumulations display a specific, moderate affinity for glycolytic enzymes.
Introduction
Materials and Methods
In a previous study (Meijer 1988), the enzyme histochemical features of tubular aggregates were characterized. Resuits obtained with adsorption studies pointed to a strong adsorption of some enzymes to the tubular aggregates in vivo. This phenomenon mainly determined the histochemical characteristics of these structures. The aim of the present investigation was to characterize ragged-red fibres (Fig. 1) by similar means. It is surprising that enzyme histochemical studies of ragged-red fibres are often incomplete, since these studies concentrated only on some mitochondrial enzymes and a few hydrolases. Moreover, the methods used for the demonstration of non-structurally bound, partially and weakly structurally bound enzymes were equivocal owing to diffusion artefacts (Meijer 1980). In the present study the activity and location of a number of oxidoreductases, transferases and isomerases, involved in the major aerobic and anaerobic pathways of energy metabolism, and of several hydrolases, were investigated. The most reliable conventional methods and semipermeable membrane techniques were employed.
The present study was carried out on human skeletal muscle specimens. The biopsy material was obtained from 24 patients with various metabolic disorders of the muscle mitochondria (Busch et al. 1981). The muscle biopsies contained mitochondria with a loosely coupled state of the oxidative phosphorylation (12cases), deficiency
Correspondence to: Prof. Dr. A.E.F.H. Meijer, Krakeling 14, 2121 BM Bennebroek, The Netherlands.
Fig. 1. Transverse section of human quadriceps femoris mucle with a mitochondrial defect in the energy metabolism, showing ragged-red fibres. Improved trichrome technique according to Engel and Cunningham (1963). x 140.
0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
58 of NADH :ubiquinone oxidoreductase (EC 1.6.5.3) (5 cases), deficiency of NADH : ubiquinone oxidoreductase, as well as a low level of carnitine in the skeletal muscle (1 case), significant decrease in activity of carnitine palmitoyl transferase II (EC 2.3.1.21) (2 cases), deficiency of cytochrome c oxidase (EC 1.9.3.1) (3 cases) and disturbed supply of Krebs cycle intermediates (anaplerotic reactions) by a decreased activity of pyruvate dehydrogenase (EC 1.2.2.2.) (1 case). The material comprised 17 specimens of left quadriceps femoris, 2 from left vastus lateralis and 5 from right gluteus medius muscle. All specimens exhibited ragged-red fibres. After excision, specimens were rapidly frozen by immersing the tissue blocks in isopentane, cooled to about - 150 °C with liquid nitrogen. The specimens were stored at - 96 °C. Longitudinal and transversal serial sections, 7/~m thick, were used. For the morphological study, sections were stained with haematoxylin-eosin and with the modified trichrome procedure (Engel and Cunningham 1963). The PAS reaction with and without pretreatment with diastase (EC 3.2.1.1.), the oil red O and Sudan black 10B stains were used to demonstrate glycogen and lipids, respectively. Sections were subjected to the histochemical techniques listed in Table 1.
For adsorption studies, sections were treated with 0.25 ~o HNO 3 for 5 min, following the procedure of Gomori (1950), in order to destroy enzyme activity. After subsequent thorough washing with double-distilled water, sections appeared entirely devoid of enzyme activity. The sections were then treated with diluted enzyme solutions in physiological saline for 24h at 25°C. The solutions, containing about 2 IU of enzyme per ml of solution, were prepared from products of Sigma (St. Louis, U.S.A.). Adsorption properties of ATP : D-fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11), D-glucose6-phosphate ketol-isomerase (GPI; EC 5.3.1.9), alphaD-glucose-l,6-phosphomutase (PGM; EC 5.4.2.2), D-glyceraldehyde-3-phosphate : NAD ÷ oxidoreductase (GADH; EC 1.2.1.12), 6-phospho-D-gluconate : NADP + 2-oxidoreductase (PGDH; EC 1.1.1.44), D-glucose6-phosphate:NADP ÷ 1-oxidoreductase (GPDH; EC 1.1.1.49), isocitrate:NAD +-oxidoreductase (I-NAD + ; EC 1.1.1.41), isocitrate:NADP + oxidoreductase (INADP + ; EC 1.1.1.42), malate : NAD + oxidoreductase (M-NAD + ; EC 1.1.1.37), malate : NADP + oxidoreductase (M-NADP + ; EC 1.1.1.40) and lactate:NAD + oxidoreductase (LDH; EC 1.1.1.27) were investigated. After treatment with the enzyme solutions, the sections
TABLE 1 ENZYME HISTOCHEMICAL TECHNIQUES USED Enzymes (systematic name and abbreviation)
EC number
References
(S)-Lactate:NAD ÷ oxidoreductase (LDH) (S)-Malate : NAD ÷ oxidoreductase (M-NAD * ) (S)-Malate : N A D P ÷ oxidoreductase (oxaloacetate decarboxylating) (M-NADP +) lsocitrate : N A D * oxidoreductase (decarboxylating) (I-NAD ÷ ) Isocitrate : NADP ÷ oxidoreductase (decarboxylating) (I-NADP + ) 6-Phospho-D-gluconate : N A D P ÷ 2-oxidoreductase (decarboxylating) (PGDH) D-Glucose-6-phosphate : NADP + l-oxidoreductase (GPDH) sn-Glycerol-3-phosphate : menadione 2-oxidoreductase (GPOX) D-Glyceraldehyde-3-phosphate : NAD ÷ oxidoreductase (phosphorylating) (GADH) Succinate: PMS oxidoreductase (SDH) N A D P H : nitro-BT oxidoreductase (NADPH) NADH : nitro-BT oxidoreductase (NADH) Ferrocytochrome c:oxygen oxidoreductase (CO) Donor : hydrogen peroxide oxidoreductase (PO) 1,4-~-D-Glucan: orthophosphate a-D-glucosyltransferase (GP) ATP : D-fructose-6-phosphate 1-phosphotransferase (PFK) Orthophosphoric-monoester phosphohydrolase (alkaline optimum) (ALP) Orthophosphoric-monoester phosphohydrolase (acid optimum) (AP) 5' Ribonucleotide phosphohydrolase AMP aminohydrolase (AMP-DA) ATP phosphohydrolase (H ÷-transporting, Mg 2+ activated) (ATP-Mg) Myosin ATP phosphohydrolase (actin-translocating) (ATP-Ca) D-Glucose-6-phosphate ketol-isomerase (GPI) c~-D-Glucose 1,6-phosphomutase (PGM) (phosphoglucomutase former, EC 2.7.5.1)
1.1.1.27 1.1.1.37 " 1.1.1.40 1.1.1.41 1.1.1.42 1.1.1.44 1.1.1.49 1.1.99.5 1.2.1.12 1.3.99.1 1.6.99.2 1.6.99.3 1.9.3.1 1.11.1.7 2.4.1.1 2.7.1.11 3.1.3.1 3.1.3.2 3.1.3.5 3.5,4.6 3.6,1.34 3.6.1.32 5.3,1.9 5.4.2.2
Meijer (1973) Barka and Anderson (1963) Meijer and De Vries (1975) Barka and Anderson (1963) Meijer and De Vries (1975) Meijer and De Vries (1974) Meijer and De Vries (1974) Lojda et al. (1979) De Vries et al. (1980) Nachlas et al. (1957) a Burstone (1962) Burstone (1962) Burstone (1962) b Christie and Stoward (1978) Meijer (1968) Meijer and Stegehuis (1980) Pearse (1968) c Meijer (1972) Pearse (1968) Fishbein et al. (1978) Meijer and Vloedman (1980) Meijer (1970) De Vries and Meijer (1976) De Vries and Meijer (1976)
" Phenazine methosulphate was added to the incubation medium at a final concentration of 50 pg/l. b p-Aminodiphenylamine and p-methoxy-p-aminodiphenylamine were used as substrates. The reactions were performed with and without cytochrome c added to the incubation medium at a final concentration of 0.5 mg/ml. c Naphthyl-AS-MX-phosphate was used as substrate and fast red violet LB as coupling substance.
59 were washed thoroughly with double-distilled water and incubated with the corresponding enzyme histochemical techniques (Meijer 1988),
Results The present study demonstrates that ragged-red fibres, in contrast to tubular aggregates, do not exhibit uniform histochemical properties. The only consistent finding was a considerable activity of mitochondrial enzymes in the granular aggregates. The increase of mitochondrial enzyme activity was the result of a marked increase in the number of mitochondria in the aggregates. In the most pronounced form, the heavily granular appearance of the ragged-red fibres extended from the subsarcolemmal periphery to the centre of the fibre. Accordingly, the entire fibre showed a strongly increased mitochondrial enzyme activity. The extramitochondrial ground cytoplasm of the peripheral region of the ragged-red fibres contained a slight to strong activity of non-structurally bound enzymes. Therefore, in general, the increased activity of mitochondrial and non-mitochondrial enzymes was distributed quite irregularly over the fibre periphery, often in a reciprocal mode. We have found, for example, that the peripheral zone of the ragged-red fibres contained ballooned areas, devoid of mitochondria and as a result devoid of mitochondrial enzyme activity. Such areas were surrounded by accumulations of mitochondria characterized by an intensely stained rim of mitochondrial enzyme activity. On the other hand, the activity of the glycolytic enzymes studied was strongly increased in these ballooned areas and moderately in the rim. Moreover longitudinal sections clearly establish that enzyme activities are not homogeneously distributed over the ragged-red fibres (Fig. 2).
Fig. 2. Longitudinalsectionof human giuteus medius muscle. Abundant activity of succinate dehydrogenase in the granular material that is inhomogeneouslydistributed in the ragged-red fibres, x 240.
Fig. 3. Activity of myosin adenosine triphosphatase in human gluteus medius musclewith mitochondriain a looselycoupledstate. The raggedred fibre reveals only a few myofibrils.The sarcoplasm of the muscle is unstained, x 360. The number of myofibrils is greatly reduced in the granular zones of ragged-red fibres. It is therefore understandable that these zones did not reveal myosin Ca 2÷-stimulated ATPase activity (Fig. 3). A marked deposition of lipids and glycogen was noted in the granular regions of some raggedred fibres. Other ragged-red fibres did not reveal these substances in the granular accumulations. Corpuscular and diffuse acid phosphatase activities were present in only a few of the muscle specimens containing ragged-red fibres whose content consisted entirely of mitochondrial granules. The results of the adsorption studies, all of which were performed with diluted enzyme solutions, demonstrated that fibres without pathological alterations stained only very slightly. On the other hand, the granular zones stained moderately to strong with solutions of the glycolytic enzymes PGM, PFK, LDH, GPI and G A D H examined
Fig. 4. Transverse section of human quadriceps femoris muscle with a mitochondrialdefectin the energymetabolism, showingactivityofphosphofructokinase. Apparently the ragged-red fibres belong to the aerobic type I fibres, x 140.
60
Fig. 5. Serial section of Fig. 4, showing activity of lactate dehydrogenase. Apparently the same ragged-red fibres belong to the anaerobic type II fibres. The discrepancy is caused by loosing constancy of activity ratios of enzymes in the pathological fibres (Meijer et al. 1977). x 140.
(Figs. 4 and 5). The granular zones did not stain with diluted solutions of the enzymes GPDH, PGDH, I-NAD ÷, I-NADP +, M-NAD ÷ and M-NADP+.
Discussion
The term "ragged-red" fibre was introduced by Engel (1971), denoting the presence of granular accumulations mainly in the subsarcolemmal areas of these fibres, which exhibit a bright red to reddish blue colour with the Gomori trichrome stain. Numerous publications on ragged-red fibres have subsequently appeared. Light microscopic and electron microscopic studies have demonstrated that the granular accumulations consist of mitochondria. The microscopically enlarged mitochondria often exhibit an unusual shape and frequently they contain densely packed crystalline and para-crystalline inclusions. The general opinion is that ragged-red fibres are virtually restricted to aerobic type 1 fibres (Olson et al. 1972). However, this is not true: in a myopathy with loosely coupled mitochondria, Meijer and van Wijngaarden (1983) found ragged-red fibres predominantly in anaerobic type II fibres. Furthermore ragged-red fibres frequently do not reveal a distinct aerobic or anaerobic metabolism due to a loss of constant proportion groups (Meijer et al. 1977; Figs. 4 and 5). From the staining results described above it follows that the variation in staining properties of the ragged-red fibres is caused by the structural, metabolic and adsorption aspects of these fibres. The structural aspects are determined by the location of the mitochondria and of the ground cytoplasm in the peripheral zone of the ragged-red fibres.
Concerning the metabolic aspects most remarkable is the strong activity of mitochondrial Mg2÷ -stimulated ATPase in the granular accumulations, indicating the presence of loosely coupled and/or uncoupled mitochondria (HOlsmann et al. 1970; Meijer and Vloedman 1980; Meijer 1981, 1983; Meijer and van Wijngaarden 1983; Meijer etal. 1985). The disfunction of the oxidative phosphorylation can be primary as for example in exercise-induced myopathy (Van den Hoven et al. 1986; Meijer et al. 1989) or secondary as for example caused by shortness of anaplerotic enzyme reactions (Meijer et al. 1987). Corpuscular and especially diffuse activity of the lysosomal enzyme acid phosphatase was present in the peripheral zone of the ragged-red fibres which were affected by active necrotic processes (Tappel et al. 1962; Z alkin et al. 1962; Emanuelli et al. 1969). Results of different biochemical investigations demonstrated that in vivo, diffusion of enzymes from their original site and adsorption at other sites can take place (Martin and Jacoby 1949; Gomori 1950; Yokoyama et al. 1951 and Goebel and Puchtler 1954). In other studies it has been shown that some glycolytic enzymes of skeletal muscle can be bound to structural proteins (Arnold and Pette 1968; Arnold etal. 1969, 1971; Green etal. 1965; Rose and Warms 1967). The adsorption findings presented in this communication are in accordance with the association properties of a number of enzymes as reported by Clarke and Masters (1973, 1974, 1975). These authors demonstrated strong adsorption capacities for PFK, LDH, GPI and GADH. From these findings it follows that adsorption phenomena partially determine the histochemical features of granular accumulations of ragged-red fibres. Since, at present, specific adsorption properties have been demonstrated in both tubular aggregates and granular accumulations of ragged-red fibres, it is very probable that specific adsorption phenomena will be a fairly general occurrence. Consequently these phenomena should be taken into account to explain enzyme histochemicai features of tissue structures. Acknowledgements The author would like to express appreciation to collegues of the Belgian-Dutch Study Group on Neuromuscular Diseases, for providing human muscle specimens.
References Arnold, H. and D. Pette (1968) Binding of glycolytic enzymes to structure proteins of the muscle, Eur. J. Biochem., 6: 163-171. Arnold, H., J. Nolte and D. Pette (1969) Quantitative and histochemical studies on the desorption and readsorption of aldolase in crossstriated muscle, J. Histochem. Cytochem., 17: 314-320. Arnold, H., R. Henning and D. Pette (1971) Quantitative comparison of the binding of various glycolytic enzymes to F-actin and the interaction of aldolase with G-actin, Eur. J. Biochem., 22: 121-126.
61 Barka, M.D. and P.J. Anderson (1963) Histochemistry, Theory, Practice and Bibliography, Harper and Row, New York. Burstone, M.S. (1962) Enzyme Histochemistry, Academic Press, New York. Busch, H.F.M,, F.G.I. Jennekens and H.R. Scholte (1981) Mitochondria and muscular diseases, Mefar, Beetsterzwaag, The Netherlands. Christie, K.N. and P.J. Stoward (1978) Endogenous peroxidase in mast cells localized with a semipermeable membrane technique, Histochem. J., 10: 425-433. Clarke, F.M. and C.J. Masters (1973) Multi-enzyme aggregates: new evidence for an association of glycolytic components, Biochim. Biophys. Acta, 327: 223-226. Clarke, F.M. and C.J. Masters (1974) On the association of glycolytic components in skeletal muscle extracts, Biochim. Biophys. Acta, 358: 193-207. Clarke, F.M. and C.J. Masters (1975) On the association of glycolytic enzymes with structural proteins of skeletal muscle, Biochim. Biophys. Acta, 381: 37-46. Emanuelli, G., G. Satta and G. Perpignano (1969) Lysosomal damage and its possible relationship with mitochondrial lesion in rat kidney under obstructive jaundice, Clin. Chim. Acta, 25: 167-172. Engel, W.K. (1971) "Ragged-red fibres" in opthalmoplegia syndromes and their differential diagnosis. In: Kakulas, B.A. (Ed.), Muscle Diseases, I.C.S. No. 237, Excerpta Medica, Amsterdam, p. 28. Engel, W.K. and G.G. Cunningham (1963) Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections, Neurology (Minn.), 13: 919-923. Fishbein, W.W., V.W. Armbrustmacher and J.L. Griffin (1978) Myoadenylate deaminase deficiency: a new disease of muscle, Science, 299: 545-548. Goebel, A. and H. Puchtler (1954) Untersuchungen zur Methodik des histochemischen Nachweises der alkalischen Phosphatase, Zbl. Allgem. Pathol., 92: 159-171. Gomori, G. (1950) Sources of error in enzymatic histochemistry, J. Lab. Clin. Med., 35: 802-809. Green, D.E., E. Murer, H.O. Hultin, S.H. Richardson, B. Salmon, G.P. Brierley and H. Baum (1965) Association of integrated metabolic pathways with membranes, Arch. Biochem. Biophys., 112: 635-647. Hoven, van den, R., H.J. Breukink, Th. Wensing, A.E.F.H. Meijer and A.J. Tigges (1986) Loosely coupled skeletal muscle mitochondria in exertional rhabdomyolysis, Equine Vet. J., 18: 418-421. Htilsmann, W.C., A.E.F.H. Meijer, J. Bethlem and G.K. van Wijngaarden (1970) Different mitochondrial species in human skeletal muscle. In: Walton, J.N., N. Canal and G. Scarlato (Eds.), Muscle Diseases, I.C.S. No. 199, Excerpta Medica, Amsterdam, pp. 319-322. Lojda, Z., R. Gossrau and T.H. Schiebler (1979) Enzyme Histochemistry, Springer-Verlag, Berlin, Heidelberg, New York. Martin, B.F. and F. Jacoby (1949) Diffusion phenomenon complicating the histochemical reaction for alkaline phosphatase. J. Anat., 83: 351-363. Meijer, A.E.F.H. (1968) Improved histochemical method for the demonstration of the activity of ~t-glucan phosphorylase. 1. The use of glucosyl acceptor dextran, Histochemie, 12: 244-252. Meijer, A.E.F.H. (1970) Histochemical method for the demonstration of myosin adenosine triphosphatase in muscle tissue, Histochemie, 22: 51-58. Meijer, A.E.F.H. (1972) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 1. Acid phosphatase, Histochemie, 30: 31-39. Meijer, A.E.F.H. (1973) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 3. Lactate dehydrogenase, Histochemie, 35: 165-172. Meijer, A.E.F.H. (1980) Semipermeable membrane techniques in quantitative enzyme histoehemistry. In: Trends in Enzyme Histochemistry
and Cytochemistry, Ciba Foundation Symposium, 73. Excerpta Medica, Amsterdam, pp. 103-120. Meijer, A.E.F.H. (1981) The histochemical detection of loosely coupled mitochondria in biopsies of skeletal muscles. In: Busch, H.F.M., F.G.I. Jennekens and H.R. Scholte (Eds.), Mitochondria and Muscular Disease, Mefar, Beetsterzwaag, The Netherlands, pp. 71-76. Meijer, A.E.F.H. (1983) The histochemical demonstration of loosely coupled mitochondria in human skeletal muscle, Histochem. J., 15: 331-335. Meijer, A.E.F.H. (1988) Histochemical features of tubular aggregates in diseased human skeletal muscle fibres, J. Neurol. Sci., 86: 73-82. Meijer, A.E.F.H. and G.P. de Vries (1974) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 4. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (decarboxylating), Histochemistry, 40: 349-359. Meijer, A.E.F.H. and G.P. de Vries (1975) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 5. Isocitrate:NADP ÷ oxidoreductase (decarboxylating) and malate : NADP ÷ oxidoreductase (decarboxylating), Histochemistry, 43: 225-236. Meijer, A.E.F.H., E.A. Elias and A.H.T. Vloedman (1977) The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle. 3. Human skeletal muscles with inherited or acquired disease of the neuromuscular system, Histochemistry, 53: 97-105. Meijer, A.E.F.H. and A.H.T. Vloedman (1980) The histochemical characterization of the coupling state of skeletal muscle mitochondria, Histochemistry, 69: 217-232. Meijer, A.E.F.H. and F. Stegehuis (1980) Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles, Histochemistry, 66: 75-81. Meijer, A.E.F.H. and G.K. van Wijngaarden (1983) Follow-up study of a myopathy with loosely coupled mitochondria, Pathol. Res. Pract., 178: 73-77. Meijer, A.E.F.H., H.R. Scholte and H.F.M. Busch (1985) Zur histochemischen Kennzeichnung und der mOglichen Bedeutung von Skelettmuskelmitochondrien mit einem Defekt der oxydativen Phosphorylierung, Wiss. Z. Friedrich-Schiller-Univ. Jena, Naturwiss. R, 34: 417-424. Meijer, A.E.F.H., H.R. Scholte and H.F.M. Busch (1987) Die histochemische Mg2 ÷ -ATPase-Technik zum Nachweis von Atmungskettendefekten und inad/iquater Versorgung mit Krebszyklus-Intermedi~produkten in Skelettmuskelmitochondrien, Acta Histochem., Suppl. Band, 34: 135-140. Meijer, A.E.F.H., R. van den Hoven, Th. Wensing and H.J. Breukink (1989) Histochemische ,~nderungen in Skeletmuskeln von Rhabdomyolyse-empfindlichen Trabrennpferden nach Grenzbelastung. I. Friihzeitige myopathologische ,~nderungen, Acta Histochem., 87: 1-11. Naehlas, M., K.C. Tsou, E. de Souza, C.-S. Cheng and A.M. Seligman (1957) The cytoehemical demonstration of succinate dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole, J. Histochem. Cytochem., 5: 420-436. Olson, W., W.K. Engel, G.O. Walsh and R. Einaugler (1972) Oculocraniosomatic neuromuscular disease with ragged-red fibres. Histochemical and ultrastructural changes in limb muscles of a group of patients with idiopathic progressive external ophtalmoplegia, Arch. Neurol., 26: 193-211. Pearse, A.G.E. (1968) Histochemistry; Theoretical and Applied, Vol. 1. Churchill, London. Rose, I.A. and J.V.B. Warms (1967) Mitochondrial hexokinase, release, and location, J. Biol. Chem., 242: 1635-1645.
62 Tappel, A.L., H. Zalkin, K.A. Caldwell, I.D. Desai and S. Shibko (1962) Increased lysosomal enzymes in genetic muscular dystrophy, Arch. Biochem. Biophys., 96: 340-346. De Vries, G.P. and A.E.F.H. Meijer (1976) Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. 6. D-6-phosphate isomerase and phosphoglucomutase, Histochemistry, 50: 1-8. De Vries, G.P., A.J. Tigges and A.E.F.H. Meijer (1980) The histochemical demonstration of glyceraldehyde phosphate dehydrogenase activity
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