Muscle lactate dehydrogenase isoenzymes in hereditary myopathies

Muscle lactate dehydrogenase isoenzymes in hereditary myopathies

137 Journal of the neurological Sciences Elsevier Publishing Company, Amsterdam - Printed in The Netherlands Muscle Lactate Dehydrogenase Isoenzyme...

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137

Journal of the neurological Sciences

Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

Muscle Lactate Dehydrogenase Isoenzymes in Hereditary Myopathies A. E. H. EMERY University Department of Medical Genetics, The Royal Infirmary, Manchester (Great Britain)

(Received 17 November, 1967")

INTRODUCTION The enzyme lactate dehydrogenase (LDH) is composed of 5 isoenzymes and evidence from several sources (VESELL 1965) suggests that each is formed by the tetrameric association of two subunits referred to as M and H, the former being predominant in skeletal muscle, the latter in heart muscle. The most rapidly migrating isoenzyme (LDH-1) has the composition H4, L D H - 2 = H a M , LDH-3=H2M2, L D H - 4 = H M a and LDH-5 = M4. The M and H subunits of L D H are referred to below as L D H - M and L D H - H respectively. In normal human skeletal muscle LDH-5 is the predominant isoenzyme. DREYFUS et al. (1962) using starch-gel electrophoresis and WIEME AND HERPOL (1962) using agar-gel electrophoresis were the first to demonstrate a reduction in the proportion of LDH-5 in human dystrophic muscle. This observation has since been confirmed by others. Some investigators have reported that LDH-5 appears to be completely absent in some patients with muscular dystrophy, particularly the severe Duchenne type of muscular dystrophy (BRoDY 1964; EMERY 1964; PEARSONet aL 1965), and in at least one type of muscular dystrophy an apparent absence of LDH-5 has been found in certain muscles but not in others (PEARSON et al. 1965). However, none of these investigators adjusted the concentrations of the homogenates before electrophoresis and the apparent absence of LDH-5 might be due to the low enzyme content of severely affected muscle, because it has been shown that when tissue homogenates are diluted there is a disproportionate loss of LDH-5 (VESELLANDBRODY 1964). SHEPARD et al. (1965) have also criticised some of these investigations because isoenzyme studies were carried out on homogenates which had been stored and it is known that LDH-5 is destroyed when tissue homogenates are stored frozen (ZONDAG1963; LAURYSSENS et al. 1964). In the present investigation an attempt has been made to avoid these difficulties by adjusting the concentration of each homogenate, before electrophoresis, Present address of the author: University Department of Human Genetics, Western General Hospital, Edinburgh (Great Britain). J. neurol. Sci. (1968) 7:137-148

138

A . E . H . EMERY

so that it contained approximately the same amount of enzyme activity as normal muscle, by determining the isoenzyme content of muscle by two different techniques: vertical starch-gel electrophoresis and urea inhibition, and by only using fresh biopsy material. Muscle from patients with a variety of hereditary myopathies has been studied and an attempt made to correlate the biochemical findings in the muscle with the degree of apparent histological abnormality and clinical involvement. MATERIAL AND METHODS

Patients

Investigations have been carried out on 21 patients in whom the diagnosis of muscular dystrophy was clearly established on clinical grounds. The classification of the muscular dystrophies is based on that of WALTON AND NATTRASS (1954) with some recent modifications (EMERY AND WALTON 1967). Throughout abbreviations have been used for the various types of muscular dystrophy*. The degree of clinical involvement of a biopsied muscle was assessed according to the Medical Research Council grading (0 = no voluntary movement possible, to 5 = normal muscle power). Controls

Normal values for the proportions of LDH-5 and L D H - M and for the amount of L D H - M have been reported els :where (EMERY 1967) and are based on results obtained in various superficial limb muscles from individuals with no history of any neuromuscular disease. Tissues

Only fresh biopsy material has been used and the tissues have all been processed within half-an-hour of being excised. The majority of the specimens (usually 300-500 mg) were obtained under general anaesthesia but in a few patients local anaesthesia was used in which case care was taken not to inject any of the anaesthetic directly into the tissue being excised. After excision the tissue was divided in two, part being used for histology and part for biochemical analysis. The latter was washed in 0.9% NaC1, to remove any contaminating erythrocytes, blotted dry, weighed (wet weight) and then homogenised in about 5 vol. of water in a Potter-Elvehjem homogenizer, the tube being kept cold by surrounding it with ice. Enzyme assays, nitrogen determinations and electrophoresis were carried out on the whole suspension suitably diluted with water. In none of the specimens of muscle did the blood content, estimated by the method described by HOLZER et al. (1956), exceed 5°,/,, and in the majority of cases the blood content was less than 2%. Differences in isoenzyme patterns in different specimens of muscle cannot therefore be due to differences in the amount of blood contained in these specimens. Chemicals

NAD, p-nitro-blue tetrazolium and phenazine methosulphate were obtained from *LGMD = limb girdle type; FSHMD = facioscapulohumeral type; DMD = Duchenne type; BMD ~ Becker type; CMD = congenital type; MD = myotonic dystrophy; MC = myotonia congenita. J. neurol. Sci. (1968) 7:137-148

MUSCLE LACTATE DEHYDROGENASE ISOENZYMES IN HEREDITARY MYOPATHIES

139

Sigma Chemical Co., London. Starch was supplied by Connaught Medical Research Laboratories, Toronto, Canada. Purified LDH-1 (pig heart) and purified LDH-5 (rabbit muscle) were obtained from C.F. Boehringer Corp., London. All other chemicals were supplied by British Drug Houses Ltd., Poole, Dorset. L D H estimation LDH activity has been determined spectrophotometrically using lactate as substrate. The reaction mixture consisted of 1.5 ml of 50mM tris-HC1 buffer pH 9.0, 1.0 ml of 0.25M sodium lactate pH 9.0, 0.3 ml of 50mM NAD and 0.2 ml of suitably diluted homogenate. The details of the method used and also of the method of urea inhibition for determining the amounts and proportions of LDH-M and LDH-H, have been described previously (EMERY 1967). Enzyme activities have been expressed as/~moles of substrate converted/min at 25°C and pH 9.0. Non-collagen nitrogen estimation The amount of non-collagen nitrogen in muscle homogenates was determined by digesting the homogenate (1 : 33, w/v, dilution in water) overnight at 20°C with 9 vol. of 0.05 N NaOH (LILIENTnALet al. 1950), centrifuging the digest and then determining the amount of nitrogen in the supernatant by a micro-Kjeldahl procedure. Electrophoresis Homogenates were subjected to vertical starch-gel electrophoresis (SMITHIES 1959) at 4°C and 4.0 V/cm for approx. 18 h (EMERY et al. 1965). The starch-gels were buffered at pH 8.5 with mM EDTA-25mM boric acid-45mM tris (final concentrations) as described by BOYER et al. (1963) The concentration of each homogenate was so adjusted that the volume (30 #1) of material subjected to electrophoresis contained approximately the same amount of LDH activity (85-95 m/~ moles/min). After electrophoresis LDH activity was located by the method of DEWEY AND CONKLIN(1960) with certain modifications (BLANCO A~D ZINKnAM 1963). The proportions of the individual isoenzymes were determined with a Chromoscan recording and integrating densitometer (Joyce-Loebl Co. Ltd.). Electrophoresis was carried out on the same day that a biopsy specimen was excised and only on freshly-prepared homogenates. For each specimen of tissue the amount of enzyme activity, the amount of noncollagen N, and the percentages of the various isoenzymes on electrophoresis are the mean values of duplicate determinations carried out on the same homogenate. Histology and histochemistry Tissue for histology was immersed in liquid nitrogen for 1 min and then sectioned at 10# in a cryostat at - - 20°C and subsequently stained with haematoxylin - eosin. Sections for the histochemical demonstration of LDH activity were not fixed but were allowed to thaw out at room temperature and then incubated in the LDH stain along with the starch-gel on which an homogenate of the same muscle had been subjected to electrophoresis. In this way it is hoped that some comparability has been achieved between the electrophoretic pattern and the display of LDH activity in tissue sections of the same muscle. J. neurol. Sci. (1968) 7:137-148

140

A . E . H . EMERY

Grading on histological appearance All the material was fixed and stained in an identical manner, and all the sections were studied without any knowledge as to the identity of the subject. The slides were then arranged according to the degree of apparent histological abnormality based on a scheme published previously (EMERY 1965) : 0 = no apparent abnormality; 1 = swelling of muscle fibres and/or hyaline fibres; 2=variation in fibre size; 3 =increase in sarcolemmal nuclei and/or evidence of fibre necrosis; 4=connective tissue proliferation and/or fatty infiltration; 5 = as grade 4 but more pronounced; 6 = as grade 5 but more pronounced; 7=extreme atrophy with few, if any, remaining muscle fibres. Cultures of fibroblasts and macrophages Fibroblasts were obtained from cultures of human skin. It was not possible to obtain pure cultures of macrophages but a concentrated suspension of cells containing 70% macrophages, 20°/,, lymphocytes and 10% polymorphs was obtained by culturing whole blood for 72 h in the absence of phytohaemagglutinin. Cell suspensions were washed several times in normal saline, then homogenised in distilled water and the homogenate subjected to electrophoresis.

RESULTS

The biochemical and histological findings in the patients who have been studied are summarised in Table 1. The proportion of LDH-5 was not less than 40°/~, in any of various superficial limb muscles from 24 controls of comparable age to the patients. The proportion of LDH-5 was found to be significantly reduced in 4 out of 6 patients with L G M D , 3 out of 4 patients with F S H M D and 6 out of 8 patients with MD. The reduction in the proportion of LDH-5 is therefore not specific for any particular type of muscular dystrophy. The results also indicate that at least in certain types of muscular dystrophy, the proportion of muscle LDH-5 may be normal even when the histology of the muscle is abnormal (e.g., patients A.M., H.S., J.B., R.W., and J.W.) and in 3 instances normal isoenzyme patterns were found in clinically affected muscles [A.M. (quadriceps), H.S., and J.W.]. However, when the results from all the patients were combined there was a negative correlation (Kendall's non-parametric rank method; SIEGEL 1956) between the proportion of LDH-5 ~nd the degree of apparent histological abnormality (correlation coefficient = - - 0.27, P<0.04). In the present investigation 2 patients with Duchenne muscular dystrophy (DMD), have been studied. In 1 of these patients (G.C.) there appeared to be no LDH-5 but unfortunately in this case the enzyme content of the muscle, which was severely affected, was not determined. In the other patient (J.M.) the concentration of the homogenate was adjusted so that the volume of material subjected to electrophoresis contained the same amount of enzyme activity as normal muscle (85-95 m/zmoles/ min), and in this case LDH-5 was present though in reduced amount. In 3 severely affected muscles in which LDH-5 appeared to be absent [patients D.T. (deltoid), P.H. and B.C.] there was so little L D H present that even with a 1 in 2 (w/v) homogenate the amount of enzyme was less than half that normally subjected

d. neurol. Sci. (1968) 7:137-148

i g

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25 54 24 35

C.D. E.W. K.L. B.L.

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.

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.

.

24.0

16.3 21.8 26.3 . .

9.9

12.7

15.8

. .

. .

111.8

44.2 . . 63.0 56.4 0.0 . .

21.5

.

23.6 .

9.1 19.9 85.9 21.2

. . 41.0 13.1 146.5 439.6

Me

18.3

37.1

40.1

82.3

79.5 72.1 0.0

81.7

62.9

59.9

74.5 73.2 89.4 55.9

49.4 49.2 88.1 83.6

M

vT :

Q =

quadriceps;

total L D H , H = L D H - H , M :

Duchenne; BMD :

17.7

20.5 27.9 100.0 . .

. .

.

.

25.5 26.8 10.6 44.1

. . 50.6 50.8 11.9 16.4

H

53.6

37.5 27.8 49.1 32.4 28.1 0.0 21.9 51.5

29.6

48.5

26.5 0.0

0.0 11.3 48.2 0.0

67.6 71.1 38.5 29.0 28.8 36.9

LDH-5

5

5 5 5 5 5 3 0 2

--

5

3 1

3 4.5 4 2

5 4 5 4 2 --

Clinical grade

0

0 1 2 3 3.5 7 5 6

4

3

4 7

3 2 1.5 5

4 5 3 4.5 6 3

Histological grade

congenital; M D ~ myotonic dystrophy; M C :

14.8

21.8 19.8 16.7 22.8 14.8 44.2 27.9 17.1

23.6

17.6

26.5 44.0

29.0 40.9 20.1 41.4

16.4 12.2 20.2 24.3 23.8 28.0

LDH-3

Becker; C M D :

0.0

8.4 8.0 4.4 16.3 7.4 14.0 8.4 1.3

14.3

1.1

4.2 8.0

31.9 12.9 7.6 11.0

1.0 0.4 1.1 11.6 8.3 5.1

LDH-1

D : deltoid; BF = biceps femoris; R = rectus; T A -- tibialis anterior. LDH-M.

135.8

54.1 . . 79.3 78.2 26.3 200.0 476.5

34.2

.

.

3.1 7.3 10.1 16.7

. . 42.0 13.5 19.8 86.0

He

%

b G = gastrocnemius;

G

G G TA G G G G G

G

G

39.4 .

12.2 27.2 96.0 37.9

136.5 180.9 83.0 26.6 166.3 525.6

Te

t~moles/min / lOOmg o f non-collagen N

facioscapulohumeral; D M D :

23

29 37 41 34 28 48 39 32

4

11

G G

D Q D D

G Q G D BF R

Muscle b

Enzyme activity

a L G M D : limb girdle; F S H M D : myotonia congenita.

R.V.

MC

S.L. M.Q. R.W. M.S. J.C. B.C. M.R. J.W.

MD

A.H.

CMD

J.B.

BMD

J.M. G.C.

12 16

45 28

H.S. P.H.

DMD

16

D.T.

FSHMD

18

Age (years)

A.M.

LGMD

Type o f muscular dystrophy a

TABLE 1

SUMMARY OF THE BIOCHEMICAL AND HISTOLOGICAL FINDINGS IN PATIENTS WITH MUSCULAR DYSTROPHY

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142

A.E.H. EMERY

to electrophoresis. However, when a fresh homogenate of normal muscle was diluted to 1 in 55 (w/v), though there was an apparent reduction in LDH-5 (Fig. 1), the actual proportions remained unchanged: 1 in 11, 78.5°/~; 1 in 22, 84.1%; 1 in 33, 85.6%; 1 in 44, 82.2°/,7 ; I in 55, 85.2°4. Thus the enzyme content of a 1 in 11 homogenate of normal muscle may be reduced at least 5-fold without significantly affecting the proportion of LDH-5. It would seem therefore that the apparent absence of LDH-5 in patients D.T. (deltoid), P.H. and B.C. could only partly be attributed to the effects of dilution. In less severely-affected muscles the reduction in LDH-5 cannot be due to dilution because the concentration of each homogenate was adjusted before electrophoresis to contain the same amount of enzyme activity as normal muscle. Further, if normal muscle is suitably diluted so that the staining intensity of band 5 is comparable to that found in dystrophic muscle in which there is an apparent reduction in the proportion of LDH-5, the staining of the anodic bands is often greater in the dystrophic muscle than in the diluted normal muscle (Fig. 1). The proportion of L D H - M was not less than 75°'~, in any of various superficial limb muscles from 21 controls of comparable age to the patients. The proportion of L D H - M was found to be significantly reduced in 2 out of 5 patients with L G M D , 3 out of 4 patients with F S H M D and 2 out of 4 patients with MD. The proportion of L D H - M was also significantly reduced in 1 patient with D M D who was studied.

i

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~

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~ ~ /i

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Fig. 1. LDH isoenzyme patterns of successive dilutions (left to right: 1/I I, 1/22, 1/33, 1/'44,and 1/55) of an homogenate of normal quadriceps muscle. On the extreme right is the LDH isoenzyme pattern of muscle from patient J.M. J. neurol. Sci. (1968) 7:137-148

MUSCLE LACTATEDEHYDROGENASEISOENZYMESIN HEREDITARYMYOPATHIES 143 There was good agreement between the results of electrophoresis and urea inhibition: in each instance where the proportion of L D H - M was abnormal there was a reduction in the proportion of LDH-5. However in 4 cases the proportion of L D H - M was apparently normal but the proportion of LDH-5 was reduced. This may be because electrophoresis is more sensitive than urea inhibition in detecting slight changes in isoenzyme concentrations. In 2 cases [patients D.T. (deltoid) and P.H.] where there was apparently no LDH-5 on electrophoresis, L D H - M was not completely absent, though the proportion was significantly reduced, which suggests that the apparent absence of LDH-5 in these cases may be due to artefact. However in 1 patient (B.C.) in which there was no LDH-5, there was also no L D H - M . When the results from all the patients were combined there was a significant negative correlation (Kendall's non-parametric rank method; SIEGEL 1956) between the proportion of L D H - M and the degree of apparent histological abnormality (correlation coefficient = - - 0.48, P < 0.01). I f the amount of L D H - M in dystrophic muscle remains constant as the disease progresses and functioning muscle tissue is replaced by connective tissue, it would be expected that when referred to a unit weight of non-collagen N there would be an apparent increase in the amount of L D H - M as the amount of non-collagen N decreases. Knowing the 95% confidence limits from the combined results of 21 normal controls for the amount of LDH-M/100 mg of non-collagen N (EMERY 1967), it is possible to calculate the expected relationship between the amount of LDH-M/100 LGMD FSHMD

'L °

MC

,.

0

¢:

°"° CMD

E :1,. 2oo

2: A

a, 10

20

30

NCN(mg/E)

Fig. 2. Relationship between the amount of non-collagen N (mg/g) and LDH-M activity ~ moles/ min/100 mg non-collagen N) in dystrophic muscle. The normal 95 % confidence limits (mean ± 1.96 S.D.) for superficial limb muscles from 21 controls is indicated . . . . . . ; LGMD ( I ) ; FSHMD (Q); MD Ok); MC (A); DMD (D); CMD (©). mg of non-collagen N and the amount of non-collagen N/g (Fig. 2). There would seem to be no objection to combining the results from various superficial limb muscles in controls because it has been shown that in normal muscle though the amount of L D H - M is related to age it is not significantly different in different superficial limb muscles (EMERY1967). When the amount of LDH-M/100 mg of non-collagen N in dystrophic muscle was plotted against the amount of non-collagen N/g (Fig. 2), with the exception of a patient with M C all the results fell below the predicted mean for the controls and 10 J. neurol. Sci. (1968) 7:137-148

144

A . E . H . EMERY

o u t o f 15 (67%) were below the p r e d i c t e d 95% confidence limits (Fig. 2). In a n o t a b l e e x c e p t i o n (patient B.L.) the a m o u n t o f L D H - M was a p p a r e n t l y within n o r m a l limits y e t the muscle was severely affected with a very low non-collagen N content. A p r o m inent feature o f the muscle histology in this p a t i e n t was its cellular c o n t e n t due to extensive invasion by fibroblasts and m a c r o p h a g e s . In the present investigation histochemical studies have shown that t h o u g h L D H activity in individual d y s t r o p h i c muscle fibres is frequently reduced, there is often considerable staining in adjacent proliferating connective tissue rich in fibroblasts and m a c r o p h a g e s . This was particularly m a r k e d in p a t i e n t B.L. B o t h fibroblasts a n d m a c r o p h a g e s a p p e a r to contain a p r e p o n d e r a n c e o f L D H - 5 (Table 2). A q u e o u s h o m o g e n a t e s o f 5 d y s t r o p h i c muscles, all with a r e d u c e d p r o p o r t i o n o f L D H - 5 (patients E.W., A . H . , P.H., S.L., J.C.) and 6 n o r m a l muscles were s t o r e d at

TABLE 2 THE PROPORTIONS OF

LDH

ISOENZYMES IN AQUEOUS HOMOGENATES OF CELL SUSPENSIONS OF MACROPHAGES AND FIBROBLASTS

LDH isoenzymes

1

2

3

4

5

3.1 4.2 5.2

8.3 11.4 10.7

25.5 26.4 15.2

17.6 22.4 21.2

45.5 35.6 47.7

0.0 0.0 0.0

0.0 0.0 0.0

13.2 14.4 10.0

27.4 27.7 35.0

59.4 57.9 55.0

Macrophages

Ia lI III Fibroblasts

I~ lI III

al, II and III represent the results of 3 different cell preparations. TABLE 3 PROPORTION OF ORIGINAL ACTIVITY REMAINING IN AQUEOUS HOMOGENATES OF NORMAL AND DYSTROPHIC MUSCLE AFTER STORAGE AT 4 ° C FOR l 0 DAYS

Total L D H

LDH-M

47.5 61.5 35.0 28.2 26.9 22.3

48.5 63.5 38.0 27.0 26.7 22.4

57.6 43.2 22.0 9.0 0.0

52.0 43.1 21.5 I 1.0 0.0

Normal

I 2 3 4 5 6 Dystrophic

J.C. A.H. S.L. E.W. P.H.

J. neurol. Sci. (1968) 7:137-148

MUSCLE LACTATE DEHYDROGENASEISOENZYMES IN HEREDITARY MYOPATHIES 145

4°C under aseptic conditions. After l0 days the percentage loss in total activity and LDH-M activity was determined (Table 3). According to the Mann-Whitney nonparametric U-test for small samples (SIEGEL 1956), there was no significant difference between the 2 groups (U=9, P=0.165): the rate of disappearance of LDH-M in stored homogenates was no greater in dystrophic muscle than in normal muscle. DISCUSSION

The results of the present investigation have confirmed the findings of others (BRoDY 1964; LAURYSSENSet al 1964; EMERYet al. 1965; PEARSONet al. 1965; PEARSONAND KAR 1966) that the reduction in the proportion of LDH-5 is not specific for any particular type of muscular dystrophy. The results also indicate that at least in certain types of muscular dystrophy normal isoenzyme patterns may be found in muscles which are affected both clinically and histologically. One patient (R.V. aged 23) had myotonia congenita with severe generalised myotonia dating from early childhood but with no apparent muscle weakness. In this patient the histology and all the biochemical parameters of a gastrocnemius muscle biopsy were within normal limits. These findings are in marked contrast to the histological and biochemical findings in a gastrocnemius muscle biopsy from a patient of similar age and with a similar degree of myotonia who had myotonic dystrophy (J.C. aged 28). These findings perhaps lend support to the idea that myotonia congenita and myotonic dystrophy are different disease entities as has been suggested on genetic grounds (BELL 1947; PENROSE 1947; DE JONG 1957). In 2 patients biopsy specimens from 2 different muscles were studied; in 1 patient the proportion of LDH-5 was normal in both specimens whereas in the other patient there was a significant reduction in the proportion of LDH-5 in both specimens. It would be informative to study multiple biopsy specimens in more cases but this is unlikely to be acceptable to most patients. Some investigators have reported that LDH-5 may be completely absent in some patients with muscular dystrophy, particularly in the severe Duchenne type of muscular dystrophy (BRODY 1964; EMERY 1964; PEARSONet al. 1965) but this might be because of the low enzyme content of severely affected muscle. In the present investigation when the concentration of a homogenate of muscle from a patient with Duchenne muscular dystrophy was adjusted so that the volume of material subjected to electrophoresis contained the same amount of enzyme activity as normal muscle, LDH-5 was present though in reduced amount. VESELLAND BRODY(1964) have shown that excessive dilution of tissue homogenates may lead to a disproportionate loss of LDH-5. The results of the present study indicate, however, that if the enzyme content of a dystrophic muscle were reduced to at least a fifth of the enzyme content of normal muscle, which would be found only in very severely affected muscles (HooFT et aI. 1966), this would not be sufficient to account for a reduction in the proportion of LDH-5 in the dystrophic muscle. The suggestion (SHEPARDe t al. 1965) that the reduction or apparent absence of LDH-5 in dystrophic muscle may be an artefact due to destruction of the isoenzyme by freezing homogenates (ZONDAG 1963; LAURYSSENSet al. 1964), is irrelevant in the J. neurol. Sci. (1968) 7:137-148

146

A.E.H. EMERY

present investigation where a reduction in the proportion of LDH-5 has been demonstrated in fresh biopsy specimens of dystrophic muscle. The reduction in the proportion of M subunits of L D H in dystrophic muscle has been confirmed in the present investigation using the technique of urea inhibition (EMERY 1967). The explanation for the change in isoenzyme pattern in dystrophic muscle remains obscure though many investigators favour the idea that the abnormal pattern may reflect a change in the metabolism of dystrophic muscle away from anaerobic to a more aerobic metabolism (BRODY 1964; LATNER 1964; LAURYSSENSet al. 1964; DAWSON AND KAPLAN 1965). There is certainly evidence that the rate of anaerobic glycosis is significantly reduced irt dystrophic muscle (DREYFUS et al. 1956). However, KATZ AND KALOW (1966) have recently suggested that the change in isoenzyme pattern in dystrophic muscle may not be due to decreased synthesis of M subunits of LDH but to increased destruction by proteolytic enzymes in the diseased muscle. Some preliminary findings in the present investigation make this seem rather unlikely because when muscle homogenates were stored the rate of disappearance of LDH-M was no greater in the case of dystrophic muscle than in normal muscle. However, in order to answer this question completely, further and more detailed investigations would have to be carried out. As the disease progresses dystrophic muscle is gradually invaded by fibroblasts and macrophages which are known to contain significant amounts of L D H activity (BRAUNSTEIN AND GALL 1962; WULFF 1963) and the present study has shown that both types of cells contain a preponderance of LDH-5. These cells would add little to the non-collagen N content of diseased muscle but might contribute significantly to the total amount of L D H - M activity. This would explain the findings in one patient (B.L.) where the muscle was severely affected with a very low non-collagen N content yet had a normal amount of L D H - M activity. A prominent feature of the histological picture in this case was extensive invasion by fibroblasts and macrophages. The situation is comparable to that of the enzyme 5'-nucleotidase. Assays on muscle homogenates have shown that this enzyme is virtually absent in normal muscle but greatly increased in dystrophic muscle (PENNINGTON 1962) where histochemical studies have demonstrated that it is mainly confined to the proliferating connective tissue (GOLARZ e t al. 1961). It is possible that the increase in such enzymes as glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in dystrophic muscle (HEYCK e t al. 1963) may also be due to invasion of the tissues by macrophages since these cells have been shown to contain significant amounts of NADP-linked enzymes (RumNSTEIN e t al. 1962; RUBINSTEINAND SMITH 1962). For these reasons the interpretation of biochemical findings in the later stages of the dystrophic process is difficult. The most valuable approach to the problem would therefore seem to be the biochemical investigation of minimally affected muscles. ACKNOWLEDGEMENTS

Grateful thanks are due to Dr. A. H. Gowenlock for much helpful advice and to Drs. C. Mawdsley and M. Rawson for help in the clinical aspects of this work which J. neurol. Sci. (1968) 7:137-148

MUSCLE LACTATEDEHYDROGENASEISOENZYMESIN HEREDITARYMYOPATHIES 147 was made possible by a research grant from the Muscular Dystrophy Group of Great Britain. Miss V. Hodson and Miss C. Mold gave valuable technical assistance.

SUMMARY

Muscle lactate dehydrogenase (LDH) isoenzymes have been studied in fresh biopsy specimens of superficial limb muscles from patients with various types of muscular dystrophy and the results compared with the findings in normal skeletal muscle. Using the technique of vertical starch-gel electrophoresis a significant reduction in the proportion of LDH-5 was found in 16 out of 23 (70%) of the muscle specimens from patients with muscular dystrophy. The alteration in isoenzyme pattern was not specific for any particular type of muscular dystrophy. When the results from all the patients were combined there was a significant negative correlation between the proportion of LDH-5 and the degree of histological abnormality. In a few cases, however, the proportion of LDH-5 was normal even though the muscle was affected clinically and the histology was abnormal. In the severe Duchenne type of muscular dystrophy LDH-5 was demonstrable when the concentration of the muscle homogenate was adjusted before electrophoresis to contain the same amount of enzyme activity as normal muscle. The reduction in the proportion of M subunits of L D H (referred to as LDH-M) in dystrophic muscle has been confirmed using the technique of urea inhibition. The rate of disappearance of L D H - M in stored homogenates was no greater in dystrophic muscle than in normal muscle, it therefore seems unlikely that there is selective destruction of L D H - M in dystrophic muscle. Interpretation of biochemical findings in the later stages of the disease is difficult because the muscle is invaded by fibroblasts and macrophages which, it has been shown, contain a preponderance of LDH-5.

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