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Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy.

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Investigation of Human Mitochondrial
Myopahes by Phosphorus Magnetic
Resonance Spectroscopy
D. L. Arnold, MD," D. J. Taylor, DPhil, and G. K. Radda, DPhil, FRS
Abnormal mitochondria are an increasingly recognized cause of neuromuscular disease. We have used phosphorus
magnetic resonance spectroscopy to monitor noninvasively the metabolism of high-energy phosphates and the intracellular p H of human skeletal muscle in vivo in 12 patients with mitochondrial myopathy. At rest, an abnormality could
be demonstrated in 11 of 12 patients. Ten patients had evidence of a reduced muscle energy state with at least one of
the following abnormalities: low phosphorylation potential, low phosphocreatine concentration, high adenosine
diphosphate concentration, or high inorganic phosphate concentration. Two patients had abnormal resting muscle
intracellular pH. In some patients phosphocreatine concentration decreased to low values during exercise despite
limited work output. This was not accompanied by particularly severe intracellular acidosis. Evidence of impaired
rephosphorylation of adenosine diphosphate to adenosine triphosphate during recovery from exercise was found in
approximately half the patients. Phosphorus magnetic resonance spectroscopy is useful in the noninvasive diagnosis of
mitochondrial myopathies and in defining the pathophysiological basis of these disorders.
Arnold DL, Taylor DJ, Radda GK Investigation of human mitochondrial rnyopathies by phosphorus
magnetic resonance spectroscopy. Ann Neurol 18:189-196, 1985
Mitochondrial abnormalities are an uncommon but increasingly recognized cause of disease (for reviews see
14, 151). Skeletal muscle, which may increase its energy turnover more than 100-fold during exercise, is
the most common clinically affected tissue. However,
the central nervous system and other highly oxidative
organs such as the heart and retina may also be affected. Manifestations of the muscle disease are extremely varied. There may be no symptoms or signs,
the clinical abnormality may be restricted to the extraocular muscles (chronic progressive external ophthalmoplegia), or there may be a severe proximal myopathy with fatigability and exercise intolerance.
Occasionally encephalopathy, cardiomyopathy, retinopathy, or lactic acidosis may be the presenting problem. Diagnosis is usually based on muscle biopsy demonstrating the presence of ragged red fibers, which are
now known to have this appearance as the result of the
subsarcolemmal accumulation of abnormal mitochondria.
The biochemical lesion in patients with mitochondrial myopathy may be located at one of many different steps in the oxidation of substrates. Abnormalities
have been reported involving the cytochromes, the
iron-sulfur centers, the adenine nucleotide trans-
locator, the adenosine triphosphate (ATP) synthetase
itself, and various components of the carnitine transport system 1151. The nature of the link between these
lesions and the clinical manifestations of disease remains to be defined. Because mitochondria are the
major source of energy for muscle cells, impaired energy metabolism is a logical candidate for being the
final common pathway leading to cell injury. Because
of the need for rapidly frozen biopsy specimens, little
information is available on high-energy phosphate metabolism in vivo in these patients. Most studies relate
to blood lactate and pyruvate as indirect indices of
abnormal oxidative metabolism. In some cases,
mitochondria have been isolated and the site of the
metabolic block identified. We have previously reported alterations in high-energy phosphate metabolism in 3 patients with mitochondrial myopathy 19,
181. These patients are included in this review (Patients 2, 6, and 7). 31P magnetic resonance spectroscopy (NMRd findings have been reported recently in
another patidnt {5).
Most of the energy (i.e., ATP) required by muscle
cells at rest is supplied by mitochondrial oxidative
phosphorylation. During exercise, there is an increased
demand for ATP that is largely met by an increase in
From the Department of Biochemistry, University of Oxford, and
Clinical Magnetic Resonance Laboratory, Radcliffe Infirmary, Oxford, United Kingdom.
"Current address: Montreal Neurological Institute, 380 1 University
Street, Montreal, Quebec, Canada H3A 2B4.
Address reprint requests to Dr Arnold, Montreal Neurological Institute, 3801 University St, Montreal, Quebec, Canada H3A 2B4.
Received Oct 4, 1984, and in revised form Dec 19. Accepted for
publication Dec 27, 1984.
189
the rate of mitochondrial oxidative metabolism. This is
augmented by phosphoryl transfer to adenosine
diphosphate (ADP) from phosphocreatine (PCr) in the
reaction catalyzed by creatine kinase (PCr
ADP +
H+
ATP
creatine) and by an acceleration of
anaerobic glycolysis. 31PNMR, which can be used for
continuous noninvasive monitoring of ATP, inorganic
phosphate (Pi), PCr, and intracellular p H (pH,) in vivo
[b, 71, provides a unique opportunity to study the metabolic consequences of human mitochondrial disorders.
In this report we review our experience using 31P
NMR to examine muscle energy metabolism in 12
patients with biopsy-proved non-carnitine-related mitochondrial myopathy. Many of these patients have
undergone extensive biochemical investigation arid defects have been localized to specific components of the
electron transport chain.
+
+
Methods
Magnetic Resonance Spectroscopy
The methods used in this laboratory for studying the metabolism of human forearm muscle by 31P NMR have been
described [21]. Spectra were obtained using a Fourier transform spectrometer (TMR-32; Oxford Research Systems) interfaced with a 1.89-tesla, 20-cm bore superconducting magnet operating at resonant frequencies of 32.5 iind 80.285
MHz for 31Pand 'H, respectively. Subjects lay on a bed with
one arm abducted 90 degrees and inserted into the bore of
the magnet such that a 2.5-cm diameter surface coil was
positioned over the flexor digitorum superficialis muscle.
Radiofrequency pulses were applied at 2-second intervals
and accumulations were time-averaged over 32, 64, or 512
seconds, depending on the desired time resolution. The
pulse width was chosen to maximize efficiency of data accumulation and partially saturated the spin systems observed
[b]. Therefore, the observed signal intensities were corrected for differential saturation 121). The arms of Patient 12
were too short to permit study as described. Her results
were obtained from study of the gastrocnemius muscle in a
60-cm bore magnet. A brief report 011the method used to
study this patient has been given [lo]. These studies were
approved by the local ethics committee and informed consent was obtained in all cases.
biopsy and estimated the [ATP] to be 5.78 p,moYgm wet
weight. This is in reasonable agreement with the assumption
for [ATP) above. It has been shown in animal studies that all
of the ATP measured in conventional biochemical analyses
of muscle extracts is observed in high-resolution 31P NMR
spectra 13, 13). We have assumed that the same is true in
human muscle 121) and have calculated concentrations of
PCr ([PCr)) and Pi ([P,)) from the signal intensity of these
compounds relative to ATP. Intracellular concentrations in
millimoles per liter of intracellular water (mM) were estimated assuming the compounds were uniformly distributed
throughout the 670 cm3 of intracellular water per kilogram
wet weight of muscle [20). These concentrations were then
used with the measured pH, to calculate the cytosolic free
ADP concentration ([ADP')), assuming equilibrium of the
creatine kinase reaction. The equilibrium constant, I(eq =
[CATP)[creatine)/[ZADP)[H )[PCr], where 2 refers to
the sums of all free and metal-complexed species, was taken
as 1.66 x lo9, which is valid at p H 7, 38"C, ionic strength,
0.25, and 1 mM free Mg2+ [22). It was also assumed that the
total amount of creatine and PCr together remained constant
at 28.5 mmol per kilogram wet weight of muscle 112). The
enzymatically measured concentration of total creatine in the
quadriceps biopsy of the one patient in whom this was determined (Patient 12) was 29.9 mmol per kilogram wet weight,
again in close agreement with the assumed value. For simphcity of presentation here, XATP and ZADP are referred to
simply as ATP and ADP, respectively.
+
Exercise Protocol
In all control subjects and in most patients, exercise was
performed by squeezing the rubber bulb of a sphygmomanometer 22 times per minute. The maximum pressure
that could be generated on emptying the bulb was preset at
100 mm H g for the first 5 minutes of exercise and then
increased to 300 mm Hg for an additional 2.5 minutes. Patients who could not achieve these pressures did the best
they could. Patients 6 and 7 were the most severely affected
and could not squeeze the bulb at all, but could only carry
out unopposed finger flexion. Patient 12, whose gastrocnemius muscle was studied, exercised by plantar flexion 30
times per minute against a load that was set equal to 20% of
her lean body mass for 7 minutes and then increased to 32%
of her lean body mass for a further 3 minutes.
Control Data
Determination ofpHi
pHi was calculated from the chemical shift of PI 114) according to the equation, p H = 6.75 -t log (a - 3.27)/(5.69 u),where u represents the chemical shift of Pi with respect
to PCr in parts per million 181.
Control subjects were healthy male and female volunteers
aged 20 to 80 years who were on ordinary mixed diets. N o
athletes were included. Results are presented as the mean -+
SD. Statistical significance was evaluated using Student's t
test.
Calcidztion of Intracellukzr Metabolite Concentrations
Results
Clinical details and results of laboratory investigations
of the 12 patients can be found in Table 1. All patients
had evidence of myopathy, but manifestations ranged
from mild external ophthalmoplegia without symptoms or signs of limb weakness to severe generalized
weakness and exercise intolerance.
For purposes of description, it is convenient to di-
Relative concentrations of intracellular PCr, PI, and ATP
were determined from their signal intensities corrected for
differential saturation as noted. To our knowledge, ATP
concentration ((ATP)) in human mitochondrial myopathies
has not been described in the literature. Therefore, in 11 of
12 patients we assumed a normal value of 5.5 mmol ATP per
kilogram wet weight 112). In our most recent patient (Patient
12) we measured total adenine nucleotides in the muscle
190 Annals of Neurology Vol 18 No 2 August 1985
Table 1. Clinical and Laboratory Findings in 12 Patients with Mitochondria1 Myopathy
Patient
No.
Age, Sex
Clinical Features
1
2
23, F
42, F
3
17, M
4
5
41, M
46, F
6
28, F
7
26, F
8
38, M
9
19, M
10
11
25, M
70, M
12
14, F
Mild proximal weakness
Mild proximal weakness,
deafness, epilepsy, neuropathy, intellectual impairment
Moderate proximal weakness, ptosis
Moderate proximal weakness
Moderate proximal weakness, ophthalmoplegia
Severe proximal weakness,
exercise intolerance
Severe proximal weakness,
exercise intolerance
Chronic progressive external
ophthalmoplegia
Mild proximal weakness, exercise intolerance, renal
calculi, anemia
Paroxysmal myoglobinuria
Chronic progressive external
ophthalmoplegia
Moderate proximal weakness, exercise intolerance,
encephalopathy, short stature, sensorineural hearing
loss
Biochemical Lesion
Resting
Lactate
Concentration
(mM)a
20-30
10
NADH-CoQ reductase
1.3
...
...
Presentb
Probable CoQ
3.0
32
10
Probable CoQ
Probable CoQ
0.5
1.5
70
NADH-CoQ reductase
2.0
70
NADH-CoQ reductasec
0.75
Ragged Red
Fibers
(% of Total)
...
5-10
Present‘
...
6.8
5-10
...
...
...
...
20
2.5
“Normal resting blood [lactate] < 1 m.
‘Percentage not specified.
‘Biochemical lesion assumed to be the same as in her sister, Patient 6.
NADH = reduced form of nicotinamide-adenine dinucleotide; CoQ = coenzyme Q.
vide the NMR findings into rest, exercise, and recovery periods.
Rest
Table 2 provides a summary of values at rest for parameters measured and derived by NMR spectroscopy
in patients and controls. Resting {PCr) was more than
2 SD below the control mean (i.e., less than 34.3 mM)
in 8 of 12 patients. In 2 of these patients the decrease
in [PCr} was accompanied by an elevation in [Pi}. Although resting blood lactate levels were elevated in 6
of 8 patients for whom this information was available,
no correlation was found between blood lactate and
pHi. One patient with a high resting lactate level (Patient 5) had a high pHi at 7.12, whereas the remaining
5 patients had pHi values within the normal range. The
lactate concentration was not known in Patient 2, who
had a low p H (6.90). Figure 1 shows a 31P NMR spectrum from resting forearm muscle of a normal subject and
from Patient 3, illustrating a low [PCr] and a high [Pi}.
From the measured ratios of ATP, PCr, and Pi, and
the pHi, we have calculated the cytosolic free {ADP),
as described in the Methods section, and the phosphorylation potential, defined as CATPl/{ADP}[Pi). In
keeping with impaired synthesis of ATP, free [ADP)
at rest was greater than 2 SD above the normal mean
(6 -+ 3 ) in 8 patients. The mean for all patients was 24
2 17 pM, significantly higher than in normal subjects
( p < 0.05).
We were able to examine Patient 3 on six occasions
over 2 2 months and Patient 9 on four occasions over 5
months. The NMR findings were consistent over this
period. The reproducibility of these measurements
lends further credence to the results of the group as a
whole even though most patients were examined only
once.
Metabolic control of oxidative phosphorylation has
been variously attributed to Pi or to ADP. Oxygen
consumption in Patients 3 and 9 was increased. Patient
3 had an [ADP) four times normal and a [Pi] almost
Arnold et al: 31P NMR of Mitochondrial Myopathy
191
Table 2. High-Energy Phosphates and Intracelldar pH at Rest in 18 Normal
Control Subjects and 12 Patients with Mitochondrial Myopathy
Phosphorylation
Potentid -(M X lo6)
'
Subject
PCr (mM)
Pi (mM)
PHi
Normals
(n = 18)
Patients
1
2
38.3
4.1 t: 0.7
7.03 t: 0.03
6 5 3
5.7
4.2
7.04
6.90
7.03 t: 0.03
7.00
7.12
6.97
7.04
7.02
7.04 -1- 0.02
7.02
7.06
7.02
29
3
17.5
25 t: 7
6
31
41
38
13
12 f 4
21
10
60
22.0 f 7.6
1.8
21.3
20.4
20.8
8.1
5.1 t: 1.7
7.2
5.4
23.3
-1-
2.0
28.5
39.7
29.3 ? 2.0
37.9
28.9
22.4
25.0
34.1
35.5 f 3.9
32.4
36.2
22.1
3"
4
5
6
7
8
9b
10
11
12
7.3
1.5
-fr
2.4
5.6
4.1
4.5
5.1
3.7
0.8
k
4.6
4.5
5.0
ADP (PM)
2.8
-1-
1.3
1.4
"Mean of 6 examinations.
bMean of 4 examinations
PCr
=
phosphocreatine; P, = inorganic phosphate; pH,
intracellular pH; ADP = adenosine &phosphate.
=
Kr
Cr
ij\
Y o r
s
o
,
-5
-10
-
-n
A
A
*
5
0
1
I
-5
-10
I
-15
ppm
B
Fig 1. j l P magnetic resonance spectruni of resting forearm muscle
in (A)a control subject and ( B ) Patient 3. Relative to adenosine
triphosphate (ATP), the concentration of phosphocreatine (PCr)
is decreased and inorganicphosphate (PJ is increased in this patient's muscle. Spectra are time averageJ of 128 transients (A)
and 256 transients (B) using a 2-secoti~dpulse repetition rate.
The Y axis represents signal intensity and the X axis the chemical shift from the PCr resonance in parts per million (ppm). (01,
p, and y refw to the three phosphates qf ATP.)
two times normal. His oxygen consumption was two
times normal. Patient 9 had a normal [Pi] but his mean
[ADPI was two times higher than, and significantly
different from, the normal mean ( p < 0.01). His oxygen consumption was 1.5 times normal. Results on
these patients are consistent with control of respiration
192 Annals of Neurology
Vol 18 NO 2
August 1985
in vivo by [ADP) rather than [Pi}. A similar conclusion
was drawn after an NMR study of phosphofructokinase-deficient muscle in which {Pi}was constant after
exercise even though the oxidative phosphorylation
rate was changing [ 2 ) .
Of all measured or calculated variables the most sensitive index of impaired mitochondrial oxidative metabolism was the phosphorylation potential. An index
of the amount of energy available within the cell, the
phosphorylation potential was lower in 10 of 12 patients than the lowest value from the 18 normal subjects (Table 2). The distribution of values for phosphorylation potential is skewed toward higher values,
making it inappropriate to describe in terms of a mean
and standard deviation. The main reason for the skew
is the presence in the denominator of this ratio of a
calculated [ADP] that becomes very small when the
[PCr) approaches the total creatine concentration,
which is usually assumed. Therefore, for statistical
evaluation, we used (phosphorylation potential) Values are shown in Table 2. Again 10 of 12 patients
were 2 SD or greater from the mean.
'.
Exercise
Measured and derived biochemical values at the end of
exercise in patients and controls are summarized in
Table 3. Despite the fact that patients often could not
achieve the usual pressures on squeezing the bulb or
could not keep exercising for the entire duration of the
exercise protocol, [PCr) was lower in the patient group
( p < 0.05) and [ADP] tended to be higher ( p < 0.10).
Table 3. Phosphorus Metabolites and IntracellukwpH at the End of Exercise
in 17 Control Subjects and 12 Patients with Mitochondrial Myopathy
Subjects
PCr (mM)
Normals
(n = 17)
Patients
(n = 12)
17.1
+ 6.6
10.6
+ 4.0
pi (mM)
PHi
ADP
+ 5.1
23.5 + 6.5
6.70
+ 0.24
6.72 + 0.21
+ 28
100 + 59
25.8
PCr = phosphocreatine; Pi = inorganic phosphate; pHi
=
47
Phosphorylation
Potentid-’ (M)
154
-t
102
229
-t
182
intracellular pH; ADP = adenosine diphosphate
Recovery
Resynthesis of PCr after exercise relies exclusively on
oxidative metabolism [11, 2 1). PCr “recovery” was
characterized by the time taken to replenish half the
difference in the [PCr) between the end of exercise
and rest. This was equal to 52 ? 16 seconds (n = 17)
in normal muscle. Of the 10 patients in whom we were
able to obtain adequate PCr recovery data, 3 showed
times for half recovery more than 2 SD above the
normal mean. Two patients (Nos. 6 and 7) have been
described previously in the literature [l6, 187; these
sisters have severe mitochondrial dysfunction and
times for half recovery of greater than 200 seconds.
The third patient (No. 9 ) showed slow recovery each
of the four times he was investigated (178 -+ 33 seconds (Fig 2).
We have previously shown that the rate of PCr resynthesis after exercise is in part dependent on pH,
during the recovery period, and we have suggested the
calculated cytosolic free [ADP) as an index of impaired mitochondrial oxidative metabolism [l). All of
the patients who had slow PCr recovery also had calculated free [ADP) that remained above resting values
for an abnormally long time after exercise. The results
pHi was similar in the two groups. Given the lactic
acidemia so often seen in patients with mitochondrial
myopathies, one would conclude that limitation of
aerobic ATP synthesis results in activation of anaerobic glycolysis and should be accompanied by an
abnormally rapid decrease in muscle pH; during
exercise. This was not seen, presumably because of a
compensatory increase in acid extrusion from the cell.
Experiments using the exercise protocol described
have demonstrated a consistent relationship between
[PCr) and pHi in normal subjects [21). This normal
relationship between [PCr) and pHi was maintained in
all these patients.
Fig 2. Resynthesis of phosphocreatine (PCr) afer exercise in Patient 9 (0) and normal subjects (@) showing the slow recovery to
resting values in the patient. Each point on the normal curve
represents the mean SD of a single examination of 13 normal
subjects. Each point on the patient’s curve represents the mean &
SD offour examinations of the patient. The decrease in PCr
concentration during exercise was taken as 100%. i n control
subjects PCr concentration was reduced during exercise to 44 -t
17% of the resting value and in the patient it was reduced to
38 -+ 2%.
*
120.
z
PCr
80
6
Recovery
11
O‘
i
i
i
i
i
min. of recovery
i
i
s
i
io
Arnold et al: “P NMR of Mitochondrial Myopathy
193
3:
25
ADP
difference
from rest
(uM)
c
I
-c
1
2
3
5
6 0
1
min. o f recovery
4
3
4
5
6
7
B
A
from normal subjects and Patient 9 are shown in Figure 3A. In addition, 2 patients (Nos. 8 and 10) whose
PCr recovery times were normal (30 and 66 seconds,
respectively) and another (Patient 1) whose results
were inconsistent (141 and 72 seconds for two investigations) took at least twice as long as normal subjects
to return to their resting calculated cytoplasmic free
{ADP) (Fig 3B). Thus, of the patients for whom data
were adequate to determine ADP recovery, 6 had abnormal times.
pH, during the entire recovery period could be assessed in 5 patients and appeared to return to resting
values faster than in normal subjects. All of these patients had high resting blood lactate levels with either
normal or elevated pH, at rest. Thus, they appear to
have adapted to increased lactic acid production by
increasing rates of acid extrusion. The mean pH, recovery time for the four examinations in Patient 9 is
shown in Figure 4. The rate of recovery is more rapid
than that of the normal controls. It is also more rapid
than the rates of pH, recovery illustrated in [ l ) ,which
start from end exercise pH, values both above and
below that of this patient.
Discussion
The pathophysiological basis of human mitochondrial
myopathy is poorly understood. High-energy phosphates have a central role in the bioenergetics of skeletal muscle. Before the development of large-bore magnets and 31P NMR in vivo, almost no information was
available regarding the concentrations of these compounds and the metabolic response to exercise in patients with mitochondrial myopathy.
In this report we document the 31PNMR findings in
a diverse group of patients with mitochondrial disorders. We have found that, at rest, the {PCr] is often
low with respect to (ATP] and the calculated free
194 Annals of Neurology Vol 18 No 2
2
August 1985
Fig 3. Return to preexercisefree adenosine diphosphate concentration (ADP) in: (A) 1 1 normal subjects (* = phosphocreatine
half recovery time, 52 +- 16 seconds) and Patient 4, (o),who
had sloui phosphocreatine recovery (half recovery time, 1 78 2 33
seconds, mean of four examinations IT SD), and (B) Patients 8
(X) and 10 (A),who had normal phosphocreatine half recovery
tames (30 and 66 seconds, respectively),and Patient 1 (0,
a),
whose phosphocreatine half recovery time was abnormal in the
first examination (141 seconds) and normal in the second examination (72 seconds). Error bars are I SD.
*
I
6 01
0
1
2
3
L
5
6
7
8
I
9
10
Min of recovery
Fig 4. pH, recovery after exercise in: (0) Patient 9 (mean of four
examinations ? SO) and (*) 6 normalsubjects (mean ? SD).
The mean phosphocreatine concentration at the end of exercise
was 10.1 ? 4.2 mM in the controls and 13.9 ? 1.5 mM in the
patient.
{ADP) is often high. Occasionally, {Pi) is increased or
pHi is abnormal. In contrast to observations in control
subjects, the [PCr) may decrease to low values during
exercise without an accompanying severe intracellular
acidosis. This means that the free [ADP) is high. After
exercise, evidence of a slowed rate of rephosphorylation of ADP to ATP is seen in approximately half the
patients and the rate of pH, recovery may be accelerated.
The primary role of mitochondria is to provide energy to the cell in the form of ATP. The ratio {ATP)/
[ADP)[P,) is defined as the phosphorylation potential.
The higher this is, the more free energy is available
from hydrolysis of ATP in the cell. The resting phosphorylation potential appears to be an early casualty in
mitochondrial disorders, since it was abnormal in 10 of
12 patients (every patient but 1 in whom an abnormality was demonstrated). This is a surprising finding
when one considers that in some patients only a small
percentage of fibers demonstrated a morphological abnormality on biopsy.
During exercise the increased demand on mitochondrial oxidative phosphorylation might be expected to
elicit abnormalities not evident at rest in patients with
mitochondrial myopathies. However, changes in phosphorus-containing metabolites and pH, in response to
exercise show large variation among normal subjects.
The differences arise because many factors such as glycolytic flux, oxidative ATP synthesis, blood flow, and
muscle fiber types all contribute to the final response.
As a result of this, we have had difficulty defining
diagnostic criteria for this disorder during exercise.
The most severely affected patients demonstrated features of the metabolic response to exercise that we
believe may be important in the group as a whole.
These features are exemplified by Patients 6 and 7.
These two sisters were too weak to squeeze the rubber
bulb normally used for exercise. Instead they simply
flexed and extended their fingers 22 times per minute
for 5 minutes. This minimal work resulted in severe
reduction of [PCr) but only a modest decrease in pH,.
We can calculate from the creatine kinase equilibrium
that the cytosolic free [ADP) was high, presumably as
a result of the block in oxidative phosphorylation. In
spite of this, the [ATP} was unchanged. This must
mean that anaerobic glycogenolysis supplied the necessary additional energy for ATP synthesis. The exceptionally high blood lactate level is consistent with increased glycolytic flux. The fact that the decrease in
pH, during exercise was relatively small suggests an
adaptation of lactic acid extrusion mechanisms. The
increased rate of pH, recovery after exercise is compatible with this interpretation.
After exercise, PCr is resynthesized by phosphoryl
transfer from ATP generated by mitochondrial oxidative phosphorylation. The oxidative dependence of
this reaction has been demonstrated in human subjects
performing ischemic exercise. No resynthesis of PCr
takes place under these conditions until circulation is
restored Ell, 21). Recovery of PCr can therefore be
used as an index of mitochondrial oxidative metabolism 118). We have previously shown that the rate of
PCr resynthesis depends on pH, recovery in addition
to oxidative metabolism and have suggested the rate of
free ADP recovery as a more specific index of mitochondrial oxidative function [1). Free ADP recovery
after exercise appears to be more sensitive and specific
than PCr recovery as an index of impaired oxidative
metabolism, as seen in Patients 8 and 10. However,
the rates of PCr and ADP recovery are not as sensitive
an index of abnormal mitochondrial function as the
resting phosphorylation potential. This may be because
ATP resynthesis throughout most of the recovery
phase does not require maximally stimulated mitochondrial response. Phosphorylation potential, being
an equilibrium property, appears to be sensitive even
at very low rates of oxidative metabolism, i.e., at rest.
The biochemical data presented show that the weakness of patients with mitochondrial myopathy does not
result from depletion of ATP stores or from intracellular acidosis. The pathophysiological basis of the weakness in this disorder requires further investigation.
In summary, 11 of 12 patients with mitochondrial
myopathy have been found to have abnormalities in
phosphate metabolism as studied by 31P NMR. These
abnormalities are compatible with a reduced energy
state of the cell, impaired rephosphorylation of ADP,
and adaptation of acid extrusion mechanisms. The only
other muscle disease in which we have consistently
found a reduced energy state is Duchenne muscular
dystrophy El?], which is also associated with a mitochondrial abnormality C19). This disorder is easily distinguished from mitochondrial myopathy. 31PNMR in
vivo thus appears to provide reasonable sensitivity and
specificity in the diagnosis of human mitochondrial
myopathies. This technique has a role in defining the
pathophysiological features of these conditions and
may prove useful in evaluating therapeutic intervention.
~~
~
Supported by grants from the British Heart Foundation and the
Medical Research Council. D. L. A. is grateful to the Medical Research Council of Canada for personal support.
The authors are grateful to their colleagues in the Clinical Magnetic
Resonance Laboratory, especially Drs P. J. Bore and P. Styles,
whose help with these studies has been invaluable. We wish to thank
Drs J. Morgan-Hughes, B. Ross, J. Hockaday, N. Hyman, and C.
Davis for allowing us to investigate their patients and for making
available results of clinical investigations. Drs B. D. Ross and D.
Hilton-Jones gave additional clinical assistance. Technical help was
provided by Miss J. Harley and Mrs Y.Green. We wish to thank Dr
D. Hayes, who carried out most of the isolated mitochondrial studies, for helpful discussions and for measuring adenine nucleotides in
Patient 12.Total creatine concentration in this patient was measured
by Dr E. A. Shoubridge.
References
1. Arnold DL, Matthews PM, Radda GK Metabolic recovery after
exercise and the assessment of mitochondrial function in human
skeletal muscle in vivo by means of 31PNMR. Magn Reson Med
1:307-315, 1984
Arnold et al: 31PNMR of Mitochondria1 Myopathy
195
2. Chance B, Eleff S, Bank W, et al: 3'P NMR studies of control of
mitochondrial function in phosphofructokinase-deficient human
skeletal muscle. Proc NatI Acad Sci USA 79:7714-7718, 1982
3. Dawson MJ, Gadian DG, Wilkie DR: Contraction and recovery
of living muscles studied by 31P nuclear magnetic resonance. J
Physiol (Lond) 267:703-735, 1977
4. DiMauro S: Metabolic myopathies. In Vinken PJ, Bruyn GW,
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