Acta histochem. 92, 207-215 (1992)

Gustav Fischer Verlag lena

Laboratory of Experimental Neurology, Department of Neurology'), Academic Medical Centre, University ofAmsterdam, The Netherlands, Insitute of Pathology'), Bezirkskrankenhaus fUr Psychiatrie, Leipzig, Germany,Department of Cytology and Histology3), Medical School, University of Nijmegen, The Netherlands, Depart-

ment of Biochemistry4), Faculty of Veterinary Medicine, University of Leipzig, Germany

Pitfalls in the histochemical demonstration of a-glucan phosphorylaseactivity in glycogen-depleted skeletal muscle fibres

By ALBERT EDUARD FRIEDRICH HUGO MEIJER I), JOSEPH BERNHARD ZIEGAN2),PETER WIRTZ3) and HEINZ DITTRICH4)

With 3 Figures

(Received May 21, 1991)

Keywords: a:-glucan phosphorylase, histochemistry, biochemistry, glycogen-depleted skeletal musclefibres.

Summary

In the present communication, an investigation is described into the reliability of histochemical methodsfor the demonstration of a:-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres. Humanskeletal muscles with glycogen-depleted fibres from patients with diseases of the neuromuscular system andfrom subjects who had suffered from malignant hyperthermia were used for the study. The location ofphosphorylase activity and glycogen was demonstrated with histochemical techniques. Biochemical techniqueswere used to assay the activity of phosphorylase and the content of glycogen.

Biochemical determinations of phosphorylase activity did frequently not reveal significant differencesbetween glycogen-depleted and non-depleted skeletal muscle fibres. In contrast, all histochemical methodsinvestigated, showed little or no phosphorylase activity in the glycogen depleted fibres, indicating that none ofthe existing histochemical methods revealed reliable staining results in these fibres. Owing to the invalidstaining results of the histochemical methods for glycogen-depleted muscle fibres, it is necessary that formetabolic studies a biochemical assay for phosphorylase activity is also to be performed.

1. Introduction

Since enzyme histochemistry is a tissue-saving specialism which bridges the deep rift

between morphology and biochemistry, the application of suitable enzyme histochemicaltechniques makes possible a better and greater understanding of the physiological andmetabolic characteristics of tissues and cells.

For that reason, nowadays histochemical methods for the demonstration of activity of 0:­

glucan phosphorylase (EC 2. 4. I. I) are frequently used for the detection and classification

of metabolic disorders in human skeletal muscles, e. g. phosphorylase deficiency in McArd­le's disease (McARDLE 1951; MOMMAERTS et al. 1959; SCHMID und MAHLER 1959),molecular heterogeneity of McArdle's disease (DAEGELEN et al. 1986; MITSUMOTO 1979),variants of late-onset phosphorylase deficiency (FElT and BROOKE 1976; KOST and VARITY1980), other phosphorylase deficiencies (ABARBANEL et al. 1987) and myopathies withdepletion of glycogen in part of fibres, including exertional myopathic alterations (MEIJER et

208 A. E. F. H. MEIJER et al.

al. 1989). Histochemical phosphorylase methods are also used for the study of human andanimal skeletal muscles with malignant hyperthermia (ZIEGAN et al. 1985; 1989).

However, the kinetic properties of phosphorylase are very complex since the enzyme issubjected to complicated allosteric mechanisms for the regulation of enzyme activity (ARA­GON et al. 1980; FISCHER et al. 1964; FUKUI et al. 1982; HASCHKE et al. 1970; HEILMEYER1970; ILLINGWORTH et al. 1964; KILLILAE et al. 1976; MEYER et al. 1970; MORGAN andPARMEGGIANI 1964). Moreover, these mechanisms are very vulnerable and the kineticproperties of the enzyme are changed in frozen sections (MEIJER, to be published). Hence,we obtained a growing realization that histochemical methods for the demonstration ofphosphorylase activity have not been properly validated, and consequently the data andconclusions arising from their employment in biological investigations may be of unreliablevalue.

In the present communication is ascertained the unreliability of histochemical phosphory­lase methods cited in the literature.

2. Material and methods

Human and animal skeletal muscle biopsies: The human specimens were from m. quadriceps femorisbiopsies of 11 patients with camitine deficiency, dystrophia myotonica, or non-specific myopathies and fromm. deltoideus and vastus lateral is of II subjects suspected of malignant hyperthermia. All muscle specimenscontained groups of glycogen depleted fibres. Specimens of II normal human skeletal muscles withoutpathological findings in the muscles were used for control studies. Specimens from m. longissimus dorsi of 2normal pigs, fresh and after storage at 4°C for 8, 24, 48, 99, and 120 h were also investigated.

The muscle specimens were rapidly frozed by immersing small blocks of tissue in isopentane cooled to-150°C with liquid nitrogen.

Histochemistry: Cryostat sections were cut at a thickness of 81-lm. Sections were stained with haematoxy­lin and eosin for morphological studies. Serial sections were used to demonstrate glycogen with the periodicacid (PAS) technique with and without a diastase (EC 3.2.1.1) pretreatment. The activity of phosphorylase awas demonstrated according to the dextran method of MEIJER (1968a) and demonstrated with other his­tochemical methods (described by LoJDA et al. [1979] and PEARSE [1972]) using exogenous glycogen asglucosyl acceptor. To test the effect of dextran on the demonstration of enzyme activity, 7 dextrans withvarying average molecular masses were used, according to the procedure described by MEIJER (l968b). Theaverage molecular masses were M,= 19,900; 40,000; 76,100; 124,000; 200,000; 500,000; and 2,000,000.These dextrans were obtained from Pharmacia (Uppsala, Sweden).

Biochemistry: Determination of glycogen and phosphorylase a was performed on pieces of skeletalmuscle of control and glycogen-depleted material. Glycogen was estimated according to the procedure ofKEPPLER and DECKER (1974). 1,4-a:-D-Glucan glucohydrolase (EC 3.2.1.3) was used for the hydrolyses ofglycogen. The auxiliary enzymes hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC1.1.1.49) were used for the spectrophotometric assay. Phosphorylase a was estimated according to Wu andRACKER (1959) with the auxiliary enzymes phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphatedehydrogenase.

Diffusion experiments: The biochemical activity of phosphorylase a was determined in a series of sectionsof normal and glycogen-depleted skeletal muscles. In a 2nd series of sections, the activity was determinedafter wetting the sections with a few drops of double-distilled water (supernatant) for 10 min and 60 min. In a3rd series, the activity was determined after wetting the sections with a few drops of a solution of dextran(average Me =40,000, and 200,000, 2 g per 25 ml double-distilled water) for 10 min and 60 min. The activityof the enzyme was also determined in the supernatant.

3. Results

3.1. Histochemistry

All investigated m. quadriceps femoris specimens obtained from patients with carnitinedeficiency, dystrophia myotonica, or non-specific myopathies showed fibres with depletionof glycogen, according to the PAS-reaction. The percentage of depleted fibres varied

Histochemical demonstration of Ct'-glucan phosphorylase activity 209

rig. I. Serial sections of a human skeletal muscle with dystrophia myotonica. The PAS technique only revealedmainly muscle fibres with a very slight quantity of glycogen (left). The same staining results were obtained withthe phosphorylase technique, without dextran in the medium. With the phosphorylase technique, and usingdextran with an average molecular mass M, = 40,000 in the medium, various glycogen depleted muscle fibresshow moderate to strong enzyme activity (right). Biochemical assay demonstrated a diminished activity ofphosphorylase a as well. x30.

greatly. In muscle specimens from these patients, the percentage of fibres with a completeglycogen depletion varied from about 10 % to about 40 %. A varying number of fibres wasonly partially glycogen depleted. The depleted fibres were usually present in groups andsometimes filled whole fascicles (Fig. 1).

With the histochemical methods studied, except the dextran method, the glycogen­depleted fibres, according to the PAS-reaction, did not reveal any phosphorylase activity.Fibres with a partial loss of glycogen frequently showed a normal activity. Provided that anincubation period of 2 h was employed, the dextran method revealed only a moderatedecrease in enzyme activity in the glycogen-depleted fibres in some muscle specimens.However, in other specimens the glycogen-depleted fibres showed little or no activity(Fig. I). A slight positive effect on the staining intensity by an increasing molecular weightof dextran could be demonstrated. In the diseased human muscles (7 cases of II), ahistochemically demonstrated reduction of phosphorylase activity, obtained by the dextranmethod, agreed with a lowered activity measured biochemically. 4 cases showed a strongdecrease in activity in many muscle fibres with the dextran method, whereas the phosphoryl­ase activity in homogenates was normal.

The PAS-reaction for the muscle fibres of the II subjects and of the pig suspected ofmalignant hyperthermia was negative. A great number of the muscle fibres revealed almostregular or irregular patterns of light stripes (Fig. 2) with the dextran technique for thedemonstration of activity of phosphorylase a. The specific staining pattern is called

14 Acta histochem .. Bd. 92. 2

210 A. E. F. H. MEIJER et aI .

..-

Fig. 2. Deltoideus muscle of a subject with malignant hyperthennia. The fibres show almost regular andirregular stripes of phosphorylase activity, demonstrated with the dextran method. The PAS-reaction forglycogen was negative. Biochemical assay demonstrated an increased activity of phosphorylase Q. x 104.

Fig. 3. Activity of phosphorylase Q in a skeletal muscle of a pig with malignant hyperthermia. Activitydemonstrated with the dextran method. In transverse sections the typical stripes could not be observed. ThePAS-reaction for glycogen was negative. x6S.

Histochemical demonstration of lX-glucan phosphorylase activity

Table I. Glycogen content and phosphorylase activity in normal and glycogen depleted skeletal muscle.

211

Biochemistry Histochemistry

Glycogen l ) Phospho- GlycogenJ) Phospho- Phospho-rylase2) rylase2), J) rylase2), J)

without withdextran dextran

Normal human m. quadriceps femoris, 2.4 ± 0.8 8.4±4.9 1+/2+ 3+ 4+n=4

Normal human m. deltoideus, 5.9±3.1 1+/2+ 2+14+ 3+/4+n=7

Normal human m. vastus 7.9±3.4 1+/2+ 2+14+ 3+/4+lateralis, n =7

Glycogen depleted human m. 0.3 14.2 -I± -I± -/2+quadriceps femoris

Human m. deltoideus with pathological 15.2±7.7 -I±myophosphorylase reaction, n = II

Human m. vastus lateralis with patho- 12.0±4.7 -I±logical myophosphorylase reaction, n = II

Normal pig death in transit 0 13.5

Normal minipigs m. longissimus 12; 14 21.3; 15.5 3+ 4+dorsi, n =2, fresh

Normal minipigs m. longissimus 5; 8 18.5; 18.6 2+ 3+dorsi, n=2, 8h at 4°C

Normal minipigs m. longissimus «I 19.4; 18.3 -I± 1+dorsi, n =2, 24 h at 4°C

Normal minipigs m. longissimus 0 18.8; 18.1 ±dorsi n=2, 48 h at 4°C

Normal minipigs m. longissimus 0 12.2; 12.5 ±dorsi, n =99 h at 4°C

I) mg glycogenlg wet tissue. ') Activity not stimulated with AMP, U/g wet tissue, J) Staining intensity isdependent on the metabolic type of the muscle fibres. - absent; + slight, I + low; 2+ moderate; 3+ strong;4+ abundant.

Table 2. Leakage [%1of phosphorylase from cryostat sections of skeletal muscle during treatment with aquabidest.

Period of treatmentaverage ± S.D., n = 5

normal skeletal muscle

without dextranwith dextran')

glycogen depleted muscle

without dextranwith dextran l )

10min

58± 950± 5

83± II69± 7

60 min

85± 979± II

96± 784± 8

I) Average mol. mass Mr = 40,000. Concentration 8 g/lOO ml aqua bidest, corresponding with the concentra­tion used in the procedure of MEIJER (1968 a).

14*

212 A. E. F. H. MEIJER et al.

"myophosphorylase reaction" (ZIEGAN et al. 1985, 1989). Frequently, a strongly decreasedactivity could be observed for all muscle fibres in the sections (Fig. 3).

3.2. Biochemistry

Areas with groups of glycogen depleted fibres, selected by the PAS-reaction, weredissected from the muscle specimens of patients with diseases of the neuromuscular system.In accordance with the histochemical findings, the biochemical assays confirmed that thefibres contained less glycogen (Table I). On the other hand, there was a very greatdiscrepancy between the histochemical demonstration of phosphorylase activity and thebiochemical values. Moreover, the staining findings suggesting that the activity of phos­phorylase a is strongly decreased as a result of the malignant hyperthermia is in conflict withthe biochemical data, which even demonstrate a significant rise in activity. From the kineticpoint of view, the rise in activity is understandable (see discussion).

The results of the storage experiments reveal a gradual decrease in the glycogen contentof the muscle fibres. In contrast to the histochemical findings, the biochemically foundactivity of the enzyme was not significantly decreased.

3.3. Diffusion experiments

Data from the leakage experiments of cryostat sections demonstrate considerable diffu­sion of phosphorylase into the medium during treatment with double-distilled water. Deple­tion of glycogen increased the leakage (Table 2). The addition of dextran, irrespective of thetype used, produced no reduction of the leakage.

4. Discussion

Most histochemical methods for the demonstration of lX-glucan phosphorylase (EC 2.4.1.1), with thesystematic name 1,4-lX-D-glucan: orthophosphate lX-D-glucosyltransferase, are based on de novo synthesis ofglycogen in tissue sections (LOJDA et al. 1979; MEIJER 1968a; PEARSE 1972). This implies that thehistochemical reaction is dependent on the presence of acceptor molecules such as glycogen or dextrancontaining preformed lX-I,4-linked polyglucose chains (FRENCH 1964). The cited methods use the syntheticability of phosphorylase according to the reaction:

[Glucoselx+ y(Glucose-l-phosphate2-)---+ [Glucoselx+y + Y(HPOi-)

If glycogen is present in the sections, it may serve the purpose, but difficulties arise when the tissue isdepleted of its glycogen.

Several investigators have sought to overcome this drawback by using "water soluble" glycogen as aglucosyl acceptor. However, exogenous glycogen cannot serve as acceptor (MEIJER 1968a). Commercialglycogen preparations have a high average molecular mass (2,5OO,OOSM, s 5,000,(00), and cannot penetrateinto the cells of the tissue sections. It is therefore not surprising that the application of these methods provedunsuccessful in demonstrating phosphorylase activity in glycogen-depleted skeletal muscle fibres. MEIJER(1968 a, b) described a technique in which dextran was used as a glucosyl acceptor, to demonstratephosphorylase activity in infarcted glycogen-depleted heart muscle fibres. The choice of dextran was based onbiochemical findings concerning its possible role as a glucosyl acceptor (SWANSON and CORI 1948; HESTRIN1949).

By using dextran as glycosyl acceptor, the staining method is much improved for glycogen depletedischaemic heart muscle fibres. It is disappointing that this improvement in staining could not be established forskeletal muscle fibres. Only in 7 of II cases of diseased skeletal muscles with glycogen-depleted fibres, thehistochemical and biochemical findings were in agreement.

The staining results obtained with muscle tissue from subjects after attacks of malignant hyperthermia arevery disappointing. Whereas the histochemical dextran method revealed no phosphorylase activity in nearly all

Histochemical demonstration of ex-glucan phosphorylase activity 213

glycogen-depleted muscle fibres, the biochemical data presented in Table I indicate that in malignanthyperthermia, the activity of phosphorylase a was actually increased. The increase in activity of phosphorylase ais in accordance with what is known about the kinetic properties of phosphorylase. The rate-limiting enzyme ofglycogen degradation exists in 2 inconvertible forms, a and b. Phosphorylase a is active in the presence andabsence of AMP, phosphorylase b is only active in the presence of AMP (CORI and GREEN 1943). In both anoxiaand ischaemia, the conversion into the a form of phosphorylase b is stimulated (CORNBLATH et al. 1963;WILLNER et al. 1980; WOLLEN BERGER et al. 1969), and it is the oxygen depletion present in malignanthyperthermia which increases the activity of phosphorylase a.

Another serious drawback is the enzyme leakage during the incubation period. The percentage of diffusedphosphorylase, however, appears to be less than that of non-bound or loosely-bound enzymes (MEIJER 1980).The reduced diffusion rate might be explained by the enzyme being partly or fully bound to the insolubleglycogen in the muscle fibres. In that case, it is understandable that sections of glycogen depleted muscle fibres,showed a somewhat increased rate of phosphorylase leakage. Our leakage findings agree with the conclusion ofTATA (1964) that the enzyme is closely associated with glycogen in rat liver under normal circumstances, but isfound in the soluble fraction after depletion of liver glycogen by fasting. Kinetic studies of skeletal musclephosphorylase and glycogen were performed by MADSEN and CORI (1958). The authors described the physico­chemical features of the binding. Findings of ANDERSON-CEDERGREN and MUSCATELLO (1963) and MEYER etal. (1970) render it likely that in skeletal muscle the enzyme is also bound to glycogen. The study of MEYER etal. (1970) strongly indicates that the enzyme-glycogen complex is not an artifact of a given isolation procedure,but that the complex actually exists in the cell.

Basically, the problem of leakage of the enzyme during the incubation period could be solved by theapplication of semipermeable membranes. The methods of FREDERIKS et al. (1987) and HACKER (1978) whichmake use of semipermeable membranes, would serve this purpose, but they have the disadvantage that, due tothe absence of a glucosyl acceptor, these techniques cannot optimally reveal the activity of phosphorylase inglycogen depleted tissue. The same conclusion can be drawn for the gel film technique of SIGEL and PETTE(1969). This staining reaction consists principally of coupling the specifically catalysed reaction to a NADPH­producing dehydrogenase reaction, which can be visualised by the phenazine methosulphate mediate reductionof nitro blue tetrazolium. The addition of glycogen into the incubation medium is pointless since the glucosylacceptor cannot penetrate into the cell structures.

In consequence for reliable metabolic classifications of diseases of the neuromuscular system it is necessaryto perform a biochemical assay. Finally it can be stated that in spite of its shortcomings the dextran method isvery useful in detecting malignant hyperthermia in skeletal muscles. The pathological myophosphorylasereaction, characterised by a weakened staining reaction, regular patterns of light stripes and negatively stainedfibres, is highly specific for the malignant hyperthermia syndrom (ZIEGAN 1988; ZIEGAN et al. 1985, 1989).

Acknowledgements

The authors would like to express appreciation to the Muscle Research Centre, Amsterdam (Prof. Dr. J.BETHLEM) and to colleagues of the Belgian-Dutch Study Group on Neuromuscular Diseases, for providinghuman muscle specimens. The study was supported by a grant of the "Prinses Beatrix Fonds".

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Author's address: Prof. Dr. A. E. F. H. MEIJER, Krakeling 14, NL - 2121 BM Bennebroek.