Download Glutathione depletion during experimental damage to rat skeletal

Clinical Science (1991)80,559-564
559
Glutathione depletion during experimental damage to rat
skeletal muscle and its relevance to Duchenne muscular
dystrophy
M. J. JACKSON, M. H. BROOKE*t, K. KAISER*
AND
R. H. T. EDWARDS
Muscle Research Centre, Department of Medicine, University of Liverpool, Liverpool, U.K. and *Departmentof Neurology,
Washington University School of Medicine, St Louis, Missouri, U.S.A.
(Received 13 August/l9 December 1990; accepted 4 January 1991)
SUMMARY
1. The release of glutathione has been studied in
comparison with the release of creatine kinase from
isolated rat soleus muscles subjected to certain forms of
experimental damage.
2. Excessive electrically stimulated contractile activity
or treatment of muscles with the mitochondrial inhibitor,
2,4-dinitrophenol, induced a substantial release of both
creatine kinase and glutathione and a reduction in the
total glutathione content of the muscle. The time course
of this release and depletion indicates that the efflux of the
two molecules is not directly related and that a reduction
in muscle glutathione content does not occur before
cytosolic enzyme release.
3. 2,4-Dinitrophenol-stimulated release of creatine
kinase was significantly reduced by the omission of
external calcium from the incubation media, but glutathione release and depletion was relatively unaffected by
this. Deliberate elevation of the muscle intracellular
calcium content with the calcium ionophore, A23 187,
induced a substantial loss of creatine kinase, but had no
significant effect on the release of glutathione.
4. Muscle biopsies from patients with Duchenne
muscular dystrophy were found to have an elevated
content of glutathione and an equivalent protein-thiol
content compared with control subjects.
5. We conclude that, although release of glutathione
from skeletal muscle occurs after excessive contractile
activity or inhibition of mitochondrial metabolism, this is
not a key step in the damaging processes leading to
cytosolic enzyme release, neither is it relevant to the onTPresent address: Department of Neurology, Mackenzie
Health Science Centre, University of Alberta, Edmonton,
Alberta, Canada T6G 2B7.
Correspondence:Dr M. J. Jackson, Muscle Research Centre,
Department of Medicine, University of Liverpool, PO Box 147,
Liverpool L69 3BX, U.K.
going damage to skeletal muscle which occurs in patients
with Duchenne muscular dystrophy.
Key words: calcium, damage, free radicals, glutathione,
skeletal muscle.
Abbreviations: DNP, 2,4-dinitrophenol.
INTRODUCTION
Skeletal muscle damage occurs in a number of di€ferent
physiological and pathological situations, such as after
unaccustomed or excessive exercise or in patients
suffering from the inherited muscular dystrophies. The
mechanisms by which damage to skeletal muscle occurs
have been studied in a number of different situations and
two major theories have been presented. An accumulation of intracellular calcium with a consequent activation
of calcium-sensitive degenerative pathways has been
proposed to be important by some workers [l-51, while
others have claimed that oxidizing free-radical species
play a key role in the processes of damage [6-91. Both
these processes have been suggested to be abnormal in
muscle from patients with Duchenne and other muscular
dystrophies [lo-151.
Glutathione is the most abundant intracellular nonprotein thiol compound. It has a number of important
cellular functions, mainly related to the thiol group of the
cysteine residue [16]. In particular, it plays an essential
role as a co-substrate for the enzyme glutathione peroxidase, which appears to act to detoxfy lipid peroxidase in
the cell, thereby reducing the potential damaging effects
of oxygen free-radical production. Many cells appear to
have an active transport system to remove oxidized
glutathione from the cell [ 171 and an increased release of
glutathione from cells may therefore reflect an insult to
the cell by oxidizing free-radical species. An increase in
oxidized glutathione in the blood has been reported to
560
M. J. Jackson et al.
occur after exercise [18] and has been attributed to
increased oxidizing free-radical activity in this situation,
although release of muscle glutathione may presumably
also occur as a result of the changes in membrane
permeability which lead to leakage of cytosolic proteins
after damage to muscle cells (191.
In order to clarify the role of glutathione in skeletal
muscle damage, we have studied the release of glutathione
by, and the glutathione content of, isolated rat muscles
subjected to various forms of experimental damage. In
addition, to clarify the relevance of this work to human
patients we have analysed the glutathione content of
muscle biopsy specimens from patients with Duchenne
muscular dystrophy. Since one important consequence of
a reduction in the muscle content of reduced glutathione
could be the oxidation of protein-thiol groups, we have
also measured these in the samples from patients with
Duchenne muscular dystrophy and from control subjects.
METHODS
Experimental damage to normal rat muscles
Female Wistar rats (100-200 g), fed on a standard
laboratory diet, were killed by cervical dislocation and
soleus muscles were carefully and rapidly removed. The
muscles were mounted on special holders and incubated
in 4 ml of an oxygenated, bicarbonate-buffered
mammalian Ringer solution at 37°C as previously
described [19]. After 30 min of incubation, the medium
was removed and replaced by fresh Ringer solution.
Damage to the muscle was then induced by either incorporation of various agents into the medium for 30 min or
30 min of repetitive electrically stimulated tetanii (for
0.5 s every 2 s at 100 Hz and 30 V ) under nitrogen. After
a further 30 min in the oxygenated medium, it was again
renewed and this was repeated every 30 min until the end
of the experiment. All media were analysed immediately
for creatine kinase activity (as a measure of the extent of
the damage to the muscle) and for glutathione content. In
all cases one of the pair of muscles from each animal
provided the control tissue. At the end of the experiment
muscles were rapidly frozen in liquid nitrogen and stored
at - 70°C before analysis of the glutathione content.
Human biopsy samples from control subjects and patients
with Duchenne muscular dystrophy
Samples were obtained from 11 patients with
Duchenne muscular dystrophy (aged 4-1 1 years) and six
control non-dystrophic subjects (aged 7-35 years), who
were biopsied for investigational purposes and were
subsequently found to have no biochemical evidence of
muscle damage or degeneration on histological and histochemical analysis. All patients suffering from Duchenne
muscular dystrophy showed no staining for dystrophin on
immunocytochemical staining of muscle biopsies and had
grossly elevated plasma creatine kinase activities (in the
range 1000-20 000 unitsll), while control subjects all
showed normal dystrophin staining and had normal
plasma activities of creatine kinase ( < 100 units/l).
Biopsies were obtained from the biceps muscle using the
‘open’-biopsy technique under local anaesthesia. A small
piece of muscle (approximately 30 mg) was rapidly frozen
in liquid nitrogen and stored at - 70°C for approximately
1 week, until analysed for glutathione and protein-thiol
contents. Preliminary studies indicated that total glutathione, oxidized glutathione and protein-thiol contents
were unchanged after storage at - 70°C for 2-3 weeks.
The local ethical committee gave their approval for this
study.
Biochemical analyses
The total glutathione content of all muscle samples and
incubation media were analysed by using the glutathione
reductase recycling method, as described by Anderson
[ 201. Analyses of the oxidized glutathione content were
also undertaken after derivitization using 2-vinylpyridine
[20]. However, analyses of the oxidized glutathione
content of the incubation media were found to be unreliable owing to oxidation of reduced glutathione in the
incubation media during the 30 min incubation periods;
therefore only the total glutathione contents of incubation
media are presented. Muscle protein-thiol groups were
analysed by titration of the sulphosalicylic acid precipitate
from the glutathione assay with 5,5-dithiobis-2-nitrobenzoic acid essentially as described by Di Monte et al.
(211. Creatine kinase activities of incubation fluids were
analysed as previously described (191.
All reagents used were of Analar grade or of the
highest grade commercially available. The calcium
ionophore, A23 187, was obtained from Sigma Chemical
Co. (Poole, Dorset, U.K.).
Statistical analysis
The statistical significance of results was assessed by
using Student’s t-test, a P value of less than 0.05 being
considered significant.
RESULTS
Experimental damage to normal rat muscle
The creatine kinase activity and total glutathione
content of the incubation media surrounding the control
muscles, the muscles treated with 2,4-dinitrophenol
(DNP, 1 mmol/l) for 30 rnin and the muscles subjected to
repetitive tetanic stimuli for 30 min are shown in Fig. 1.
The release of both creatine kinase and glutathione was
initially low and the release from control untreated
muscles remained low throughout the experiment, but
DNP treatment and stimulation induced substantial efflux
of both creatine kinase and glutathione after the treatment
period, accounting for the loss of approximately 70% of
the muscle glutathione in both cases. The pattern of efflux
of the two substances induced by the two damaging protocols differed in that a greater appearance of creatine
kinase activity occurred after DNP treatment than after
Glutathione and muscle damage
561
DNP or
(a)
DNP or
stimulation
/
b* l**
L
I
150
180
30
60
,
90
120
0
.
23
Y
0
30
0
0
It
DNP or
60
90
120
150
180
Time of incubation (min)
Fig. 2. Total glutathione content of rat soleus muscles
incubated in vitro. Muscles were either untreated (m),
treated with 1 mmol/l DNP (A ) or subjected to electrically stimulated excessive contractile activity ( 0 ) during
the period shown. Values are means, with bars indicating
SEM.
Statistical significance: *P<0.01, t P < 0.001
compared with untreated muscles at the same time point.
~
30
60
90
120
150
180
Time of incubation (min)
Fig. 1. Efflux of creatine b a s e (CK) ( a ) and total
glutathione ( b ) from isolated rat soleus muscles. Muscles
were either untreated (m), treated with 1 mmol/l DNP ( A)
or subjected to electrically stimulated excessive contractile activity ).( during the period shown. Values are
means, with bars indicating SEM. Statistical sigmficance:
*P<0.05, **P<0.01, t P < 0.001 compared with
untreated muscles at the same time point.
400
200
&+P
30
60
90
120
150
180
150
180
DNP
stimulation, whereas the reverse was the case for glutathione efflux.
Analysis of the total glutathione content of the muscles
(Fig. 2) revealed a minor decrease in the control muscles
during the course of the experiment and a much more
substantial decrease in both the stimulated and DNPtreated tissues. Immediately at the end of DNP treatment
the total glutathione content was not significantly different
from the untreated controls (Fig. 2). The proportion of
the glutathione in the oxidized form was unaffected by the
various manipulations, remaining at 2-5% throughout.
The glutathione lost from the muscles could be entirely
accounted for by that detected in the incubation fluids
(Fig. 1).
Effect of manipulation of external and intracellular
calcium on muscle glutathione content
The calcium content of the external medium has previously been shown to have a marked influence on the
damage to and release of cytosolic enzymes from skeletal
muscles subjected to certain forms of experimental stress
[ 5 ] . The effect of treatment of the muscles with DNP
O' 30
60
90
120
Time of incubation (min)
Fig. 3. Effect of DNP (200 pmol/l) on the efflux of
creatine kinase (CK)( a ) and glutathione ( b )from isolated
rat soleus muscles in the presence ( 0 )or absence (0)
of
extracellular calcium. Values are means, with bars
indicating SEM. Statistical significance: *P<0.05,
**P<0.01, tP< 0.001 compared with muscles in calciumcontaining media at the same time point.
(200 pmol/l) in media with no added external calcium is
shown in Fig. 3. This media (nominally calcium-free) produced a dramatic reduction in the creatine kinase efflux
from DNP-treated muscles, the reduction being sigmfi-
562
M. J. Jackson et al.
cant at all times from 30 min after the DNP treatment, but
had a relatively minor effect on the glutathione efflux, the
reduction in the release of glutathione being significant at
only 30 min after DNP treatment with muscles from both
calcium-containing and calcium-free media releasing
identical amounts of glutathione at the end of the incubation. The effect of this manipulation of the extracellular
calcium content of the muscle glutathione content is
shown in Table 1 and demonstrates that the omission of
external calcium had no protective effect on the reduction
in muscle glutathione content.
The effect of an elevation of intracellular calcium
content by treatment of muscles with the calcium ionophore, A23187 is shown in Fig. 4.This induced a substantial efflux of creatine kinase from the muscles, but the
release of glutathione was not significantly elevated compared with the control muscles, although the mean value
was always higher than that of the controls. The muscle
glutathione content was also not significantly influenced
by the calcium ionophore treatment when compared with
the control muscles (Table 1).
Human muscle analyses
The total glutathione content of the biopsy specimens
from 11 patients with Duchenne muscular dystrophy are
shown in Table 2, together with the protein-thiol content.
Glutathione is expressed both as per g of muscle protein
and p e r g of creatine in order to standardize for the
amount of non-muscle material present in the analysed
samples. Both ways of expressing the results show a
significant increase in the glutathione content of the
dystrophic muscle. Oxidized glutathione represented
2-5% of the total glutathione content in the muscles from
both control subjects and patients with Duchenne
muscular dystrophy. The protein-thiol content was
identical in the patient and the control groups.
DISCUSSION
Little work has been undertaken on the glutathione status
of skeletal muscle, although the glutathione content of
cardiac muscle has been extensively studied as an index of
Table 1. Total glutathione content of rat soleus muscle
Results are presented as means k SEM ( n= 6). Statistical
significance: *P< 0.002, **P< 0.01 compared with
muscles which were incubated for 3 h but not treated.
Values for muscles treated in the presence or absence of
external calcium were not significantly different from each
other.
Total glutathione content
(pmollg wet wt.)
Fresh unincubated muscle
Incubated, untreated muscle
DNP-treated ( + Ca’+) muscle
DNP-treated ( - CaZ+ ) muscle
A23187-treated muscle
1.79 f0.18
1.37+0.14
0.79 If.0.02*
0.83 f0.06**
1.10 f0.10
.- 4001
-E zoo
O ’ 30
60
90
150
180
O ’ 30
60
90
120
160
Time of incubation (min)
180
120
Fig. 4. Efflux of creatine kinase (CK) ( a ) and total
glutathione ( b )from untreated ( 0 ) and A23187-treated
( w ) isolated rat soleus muscles. Values are means, with
bars indicating SEM. Statistical significance: * P < 0.05,
**P< 0.01, t P < 0.001 compared with untreated muscles
at the same time point.
Table 2. Total glutathione and protein-thiol contents of
biopsy samples from patients with Duchenne muscular
dystrophy and control subjects
Results are presented as means k SEM. Abbreviation:
DMD, Duchenne muscular dystrophy. Statistical significance: * P < 0.001 compared with control subjects.
Control subjects
(n=6)
Patients with DMD
( n = 11)
Glutathione content
pmol/g of protein
pmol/g of creatine
1 1.2 f0.8
523 k 54
20.3 f4.0*
1206 k 21 3*
Protein-thiol content
pmol/g of protein
118.2+8.1
103.3f 10.8
oxidative stress to the heart [22-241. This work has shown
that, during ischaemia, there is an oxidation of intracellular glutathione and after reperfusion of ischaemic
cardiac tissue there is a dramatic release of both oxidized
and reduced glutathione [22]. These authors have
suggested that the extent of the oxidation of glutathione
during ischaemia is crucial in determining the vulnerability of the heart to damage due to oxygen radicals
during the reperfusion process.
Glutathione and muscle damage
It has been suggested that in skeletal muscle substantial
oxidation and release of glutathione may occur during
severe exercise [25], reflecting an increased flux of
oxidizing free-radicals in this situation. Oxidative soleus
muscles were used for the present work because preliminary results indicated that the total glutathione content of
rat soleus muscles (2.19 k 0.2pmol/g wet wt; mean+ SEM,
n = 6 ) was much higher than that of fast, glycolytic
extensor digitorum longus muscles (0.84 k 0.16 pmol/g
wet wt.) or gastrocnemius muscles of mixed-fibre-type
composition (0.97 k 0.06 pmol/g wet wt.).
The results presented demonstrate that certain forms
of damage to skeletal muscle can induce a substantial
efflux of glutathione from the muscle cells. The extent of
this release is not directly related to that of an indicator of
damage to the muscle, release of creatine kinase (Fig. l),
although it occurs with a similar time course and does not
appear to precede the cytosolic enzyme efflux. Cytosolic
enzyme efflux in this experimental system is dependent
upon the presence of external calcium (Fig. 3), although
the addition of calcium chelators (e.g. EGTA) to the
incubation media causes damage to the muscle (results
not presented in detail).However, removal of the external
calcium had little effect on the release of glutathione by
DNP-treated muscles (Fig. 3 ) or on the intracellular
muscle glutathione content (Table 1).In a further attempt
to study the calcium dependency of glutathione release
from skeletal muscle we have treated soleus muscles with
the calcium ionophore, A23187, in order to raise intracellular calcium levels. This compound precipitated a
substantial efflux of creatine kinase (Fig. 4) from the
muscles, but had only a minor effect on glutathione
release (Fig. 4) or the total glutathione content of the
muscle (Table 1).On balance, it therefore appears that the
cytosolic enzyme creatine kinase and glutathione are
released from damaged isolated skeletal muscles by
different mechanisms, that leading to creatine kinase
efflux involving a step dependent on the presence of
external calcium which is not involved in glutathione
release. In addition, neither the time courses of release
(Fig. 1) nor the muscle glutathione content during the
course of the experiment (Fig. 2) support a primary role
for glutathione release or depletion in the processes
leading to creatine kinase release.
DNP is a known inhibitor of normal mitochondria1
function and we have previously shown that treatment of
isolated soleus muscles with this compound leads to a
total loss of cellular ATP and phosphocreatine within 6
min [26]. Likewise, measurements of the force produced
by muscles subjected to the stimulation protocol used
here suggests that these muscles suffer (at least) a transient
failure of energy supply [19].It is therefore possible that a
failure of energy supply causes the release of glutathione
from the cell in both of these situations, although there is a
time-lag between the failure of energy supply and the loss
of glutathione from the muscles. Unfortunately, we were
unable to determine the proportion of the glutathione
released from the muscle which was present in the
oxidized form. so we are unable to further consider the
possibility that the efflux resulted from active transport of
563
oxidized glutathione [17]. However, since this would
require consumption of scarce energy supplies in energydepleted cells, it seems an unlikely mechanism.
Previous studies have suggested that although cytosolic
enzyme efflux from DNP-treated or electricallystimulated
muscle occurs via a mechanism that is dependent on the
presence of external calcium, this may not be true of the
ultrastructural damage which occurs due to these stresses
[27]; it is therefore possible that the loss of cellular
glutathione may be important in the initiation of these
processes.
Analysis of the glutathione and protein-thiol contents
of muscle biopsy samples from patients with Duchenne
muscular dystrophy have revealed an increase in the
glutathione content when expressed on a protein or
creatine basis and have demonstrated no s i m c a n t difference in the protein-thiol content compared with muscle
from control subjects. This lack of evidence of substantial
oxidation of muscle thiol groups essentially confirms the
finding of other workers, who have examined animal
models of muscular dystrophy [28], and is unsurprising in
the light of the above results concerning the mechanisms
of glutathione release from damaged skeletal muscle.
Muscle from patients with Duchenne muscular dystrophy
does not seem to be characterized by any sigruilcant
depletion of energy supply [29], but current theories suggest that the lack of dystrophin, the protein thought to be
the defective gene product in this disorder [30], may
induce cellular damage by a failure of calcium homoeostasis [12,30,31].
We are unable to offer an explanation for the increase
in the total glutathione content of dystrophic muscle. One
possibility is that there is an increased release of arachidonic acid metabolites, such as prostaglandins, from the
muscle of patients with Duchenne muscular dystrophy
[32, 331, which may lead to an increased oxidative stress
on the muscle. However, glutathione peroxidase activities
are essentially normal in muscle from patients with this
disorder [34], which argues against an increased glutathione requirement as a substate for this pathway.
ACKNOWLEDGMENTS
We are grateful for financial support from the Muscular
Dystrophy Group of Great Britain and Northern Ireland,
the Muscular Dystrophy Association of America and the
NATO Scientific Programme. We thank Mr J. Planar,
Washington University School of Medicine, and Mrs S.
Page, Mrs N. Lowe, Miss A. McArdle and Miss A. Swift,
University of Liverpool, for expert technical assistance.
REFERENCES
1. Wrogemann, K. & Pena, SJ.G. Mitochondrial overload: a
general mechanism for cell necrosis in muscle diseases.
Lancet 1976; ii, 672-4.
2. Duncan, CJ. Role of intracellular calcium in promoting
muscle damage: a strategy for controlling the dystrophic
condition.Experientia 1978; 34,1531-5.
3. Soybell, D., Morgan, J. & Cohen, L. Calcium augmentation
of
enzvme leakage
from mouse skeletal muscle and its
.~
u
~~
564
M. J. Jackson et al.
possible site of action. Res. Commun. Chem. Pathol.
Pharmacol. 1978; 20,317-29.
4. Anand, R. & Emergy, A.E.H. Verapamil and calciumstimulated enzyme efflux from skeletal muscle. Clin. Chem.
1982; 28,1482-84.
5. Jones, D.A., Jackson, M.J., McPhail, G. & Edwards, R.H.T.
Experimental muscle damage: the importance of external
calcium. Clin. Sci. 1984; 66, 317-22.
6. Dillard, C.J., Litov, R.E., Savin, W.M., Dunelin, E.E. &
Tappel, A.L. Effects of exercise, vitamin E and ozone on
pulmonary function and lipid peroxidation. J. Appl. Physiol.
1978; 45,927-32.
7. Brady, P.S., Brady, L.J. & Lillrey, D.E. Selenium, vitamin E
and the response to swimming stress in the rat. J. Nutr.
1979; 109,1103-09.
8. Davies, K.J.A., Quintanilha, A.T., Brooks, G.E. & Packer, L.
Free radicals and tissue damage produced by exercise.
Biochem. Riophys. Res. Commun. 1982; 107, 1198-205.
9. Jackson, M.J., Edwards, R.H.T. & Symons, M.C.R. Electron
spin resonance studies of intact mammalian skeletal muscle.
Biochim. Biophys. Acta 1985; 847, 185-90.
10. Bodenstein, J.B. & Engel, A.G. Intracellular calcium
accumulation in Duchenne dystrophy and other
myopathies: a study of 567,000 muscle fibres in 114 biopsies. Neurology 1978; 28,439-46.
11. Maunder-Sewry, C.A., Gorodetsky, R., Yarom, R. &
Dubowitz, V. Elemental analyses of skeletal muscle in
Duchenne muscular dystrophy using X-ray fluorescence
spectrometry. Muscle Nerve 1980; 3,502-8.
12. Turner, P.A., Westwood, T., Regen, M. & Steinhardt, R.A.
Increased protein degradation results from elevated free
calcium levels found in muscle from mdx mice. Nature
(London) 1988; 335,735-8.
13. Kar, N.C. & Pearson, C.M. Catalase, superoxide dismutase,
glutathione reductase and thiobarbituric acid-reactive
products in normal and dystrophic muscle. Clin. Chim. Acta
1979; 94,277-80.
14. Jackson, M.J., Jones, D.A. & Edwards, R.H.T. Techniques
for studying free ra.dical damage in muscular dystrophy.
Med. Biol. 1984; 62, 135-8.
15. Murphy, M.E. & Kehrer, J.P. Free radicals: a potential
pathogenic mechanism in inherited muscular dystrophy.
Life Sci. 1986; 39,227 1-8.
16. Meister, A. & Anderson, M.E. Glutathione. Annu. Rev.
Biochem. 1983; 52,7 1 1-60.
17. Chance, B., Sies, H. & Boveris, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 1979; 59, 527-605.
18. Gohil, K., Viguie, C.A., Stanley, W.C., Packer, L. & Brooks,
G.A. Blood glutathione oxidation during human exercise. J.
Appl. Physiol. 1988; 64, 1 15- 19.
19. Jones, D.A., Jackson, M.J. & Edwards, R.H.T. The release
of intracellular enzymes from an isolated mammalian
skeletal muscle preparation. Clin. Sci. 1983; 65, 193-201.
20. Anderson, M. Determination of glutathione and glutathione
disulphide. Methods Enzymol. 1985; 113,548-55.
21. Di Monte, D., Rose, D., Bellomo, G., Eklow, L. & Orrenius,
S. Alterations in intracellular thiol homeostasis during the
metabolism of menadione by isolated rat hepatocytes. Arch.
Biochem. Biophys. 1984; 235,334-42.
22. Ferrari, R., Ceconi, C., Curello, S. et al. Oxygen-mediated
myocardial damage during ischaemia and reperfusion: role
of the cellular defences against oxygen toxicity. J. Mol. Cell.
Cardiol. 1985; 17,937-45.
23. Ferrari, R., Ceconi, C., Curello, S. et al. Intracellular effects
of myocardial ischaemia and reperfusion: role of calcium
and oxygen. Eur. Heart J. 1986; 7 (Suppl. A), 3-12.
24. Curello, S., Ceconi, C., Cargnoni, A., Connacchian, A.,
Ferrari, R. & Albertini, A. Improved procedure for
determining glutathione in plasma as an index of myocardial
oxidative stress. Clin. Chem. 1987; 33,1448-9.
25. Packer, L. Oxygen radicals and antioxidants in endurance
exercise. In: Benzi, G., Packer, L. & Siliprandi, N.. eds.
Biochemical aspects of physical exercise. Amsterdam:
Elsevier, 1986: 73-92.
26. West-Jordan, J.A., Martin, P.A., Abraham, R.J., Edwards,
R.H.T. & Jackson, M.J. Energy dependence of cytosolic
enzyme efflux from rat skeletal muscle. Clin. Chim. Acta
1990; 189,163-72.
27. Duncan, C.J. & Jackson, M.J. Different mechanisms
mediate structural changes and intracellular enzyme efflux
following damage to skeletal muscle. J . Cell Sci. 1987; 87,
183-8.
28. Shatz, L. & Hollinshead, M.B. Developmental aspects of
glutathione levels in dystrophic mice. Proc. SOC.Exp. Biol.
Med. 1967; 126,624-7.
29. Griffiths, R.D., Cady, E.B., Edwards, R.H.T. & Wilkie, D.R.
Muscle energy metabolism in Duchenne dystrophy studied
by "P NMR: controlled trials show no effect on allopurinol
or ribose. Muscle and Nerve 1988; 8,760-7.
30. Hoffmann, E.P., Brown, R.H., Jr. & Kunkel, L.M.
Dystrophin. The protein product of the Duchenne muscular
dystrophy locus. Cell 1987; 51,919-28.
3 1 . Franco, A. & Lansman, J.B. Calcium entry through stretchinactivated ion channels in mdx myotubes. Nature (London)
1990; 334,670-3.
32. Tagesson, C. & Henriksson, K.G. Elevated phospholipase
Az in Duchenne muscle. Muscle Nerve 1984; 7,260- 1.
33. Jackson, M.J., Brooke, M.H., Kaiser, K. & Edwards, R.H.T.
Creatine kinase and prostaglandin E, release from isolated
Duchenne muscle. Neurology 1991; 41, 101-4.
34. Jackson, M.J., Coakley, J., Stokes, M., Edwards, R.H.T. &
Oster, 0. Selenium metabolism and supplementation in
patients with muscular dystrophy. Neurology 1989; 39,
655-9.