Download Tricarboxylic Acid Cycle Intermediates during

Clinical Science (1995) 88, 687-693 (Printed in Great Britain)
687
Tricarboxylic acid cycle intermediates during incremental
exercise in healthy subjects and in patients with
McArdle’s disease
K. SAHLIN, L. ]ORFELDT*, K.-G. HENRIKSSONP, S. F. LEWIS$ and R. G . HALLERS
Department of Physiology and Pharmacology, Karolinska Institute and Department of Sport and
Health Sciences, University College of Physical Education and Sports, Stockholm, Sweden,
*Department of Thoracic Physiology, Karolinska Hospital, Stockholm, Sweden, tNeuromuscular
Unit, Linkwing University Hospital, Linkoping, Sweden, $Department of Health Sciences, Boston
University, Boston, Massachusetts, U.S.A., and §Department of Neurology, University of Texas
Southwestern Medical Center and the Institute for Exercise and Environmental Medicine,
Presbyterian Hospital, Dallas, Texas, U.S.A.
1. The importance of the level of tricarboxylic acid
cycle intermediates (malate, citrate and fumarate) for
energy transduction during exercise has been investigated in six healthy subjects and in two patients with
muscle phosphorylase deficiency (McArdle’s disease).
2. Healthy subjects cycled for 10min at low (50W),
moderate [130+6W (mean+SEM)] and high
(226f12W) work rates, corresponding to 26, 50 and
80% of their maximal O2 uptake, respectively.
Patients with McArdle’s disease cycled for 11-13 min
at submaximal (40 W) rates, and to fatigue at maximal work rates of 60-90 W.
3. In healthy subjects, phosphocreatine was unchanged
during low work rates, but decreased to 79 and 32%
of the initial level during moderate and high work
rates. In patients with McArdle’s disease, phosphocreatine decreased to 82 and 34% of the initial level
during submaximal and peak exercise. Muscle lactate
increased in healthy subjects during exercise at
moderate and high work rates, but remained low in
patients with McArdle’s disease.
4. In healthy subjects, tricarboxylic acid cycle intermediates were similar at rest and at low work rates
(0.48 f 0.04 mmol/kg dry weight), but increased to
1.6 f 0.2 mmol/kg dry weight and 4.0 f 0.3 mmol/kg
dry weight at moderate and high work rates. The
tricarboxylic acid cycle intermediate level in patients
with McArdle’s disease was similar to that in healthy
subjects at rest, but was markedly reduced during
exercise when compared at the same relative intensity. The peak level of tricarboxylic acid cycle intermediates in patients with McArdle’s disease was 22%
of that in healthy subjects. However, when compared
at the same absolute workload, tricarboxylic acid
cycle intermediates were similar in patients with
McArdle’s disease and in healthy subjects.
5. The decrease in glutamate and increase in alanine
suggest that the alanine aminotransaminase reaction
was the major anaplerotic process in healthy subjects.
However, in patients with McArdle’s disease (n= l),
muscle alanine remained unchanged and the purine
nucleotide cycle may instead be the route of a limited
anaplerosis during maximal exercise. The muscle
content of glutamate and glutamine (n=1) was markedly reduced in patients with McArdle’s disease.
6. It is concluded that the tricarboxylic acid cycle
intermediate level is related to the work rate in
healthy subjects, and that the attenuated peak level in
patients with McArdle’s disease may be a limitation
for aerobic energy transduction.
INTRODUCTION
In skeletal muscle, the rate of aerobic energy
production can increase more than 80-fold during
exercise. An essential part of cellular aerobic energetics is the transfer of reducing equivalents from fuel
to oxygen. The tricarboxylic acid (TCA) cycle has a
crucial role in this process, producing the cofactor
NADH which is a substrate of the respiratory chain.
The pathway substrate of the TCA cycle is acetylCoA, which, after fusion with oxaloacetate, is oxidized to C 0 2 in the TCA cycle. The inherent
enzyme activities, the concentration of TCA cycle
intermediates (TCAIs) and the availability of substrate (acetyl-CoA) and cofactors, determine the
maximal activity of the TCA cycle. Increases in
CaZ+, ADP and NAD+/NADH are important for
allosteric activation of different enzymes in the TCA
cycle [l]. Increases in Ca2+ and ADP are intimately related to exercise intensity and can thus
serve to adjust the TCA cycle activity to the
metabolic demands. In addition to allosteric regulation of enzyme activities, the actual flux rate will be
Key words: amino acids, anaplerosir, citrate, fumarate, glycogen storage disease, glycogenolyris, malate, metabolic control, muscle contraction, oxidative metabolism.
Abbreviations: HWR. high work rate; LWR, low work rate; MWR, moderate work rate; TCA, tricarboxylic acid; TCAlr, TCA cycle intermediates.
Correspondence: Dr K. Sahlin, Department of Physiology and Pharmacology, Division of Physiology 111, Karolinska Institute, Box 5626, S-114 86 Stockholm, Sweden.
688
K. Sahlin
et
al.
Linkoping University Hospital and Karolinska
Hospital.
dependent on the level of TCAIs and acetyl-CoA.
Increases in the muscle levels of TCAIs [2], and in
the availability of acetyl units [3-51, have been
observed after strenuous exercise. These metabolic
changes could be prerequisites for attaining high
rates of oxidative energy transduction. Whether the
increase in TCAIs is related to VO, and thus also
occurs at low intensity exercise is not known.
Replenishment of TCAIs (anaplerosis) is dependent on the reactions catalysed by alanine aminotransferase, pyruvate carboxylase, malic enzyme and
phosphoenolpyruvate carboxykinase, but cannot
occur through pyruvate dehydrogenase. The directions and/or the rates of these reactions are dependent on pyruvate concentration, and therefore are in
part related to the rate of glycogenolysis. If an
increase in pyruvate is essential for anaplerosis, one
would not expect an increase in TCAIs at low
exercise intensities, since muscle pyruvate and lactate remain low [4-61. Alternatively, anaplerosis can
occur through catabolism of certain amino acids
(glutamate, isoleucine, valine and aspartate).
Patients with glycogen phosphorylase deficiency
(McArdle’s disease) exhibit a marked exercise intolerance, which, during dynamic exercise, is considered mainly to be caused by impaired oxidative
metabolism due to substrate deficiency [7]. Patients
with McArdle’s disease are unable to utilize muscle
glycogen, and, since availability of carbohydrate is
essential for attaining increases in pyruvate, it is
possible that these patients also have low levels of
TCAIs during exercise. To our knowledge, no
reports of the TCAI levels in the muscle of patients
with McArdle’s disease are available.
The purpose of the present study was (i) to
determine TCAIs in muscle during incremental exercise in healthy subjects and in patients with
McArdle’s disease, and (ii) to elucidate potential
mechanisms of anaplerosis.
Subjects and patients were instructed to abstain
from exercise (other than walking short distances)
for 2days before the experiment. A short catheter
was inserted in an antecubital vein and incisions
were made over the lateral aspect of the quadriceps
femoris muscle of both thighs before exercise.
Healthy subjects participated in a pretest (at least
3 days before the experiment), during which peak
pulmonary Vo, was determined by graded cycling
on an electrically braked ergometer (Siemens-Elema,
60rpm) to fatigue. The peak VO, was considered to
be equal to Vo,max. During the experiment, healthy
subjects exercised at 50 W and at work rates calculated to correspond to 50 and 80% of Vo,max. Each
work rate was sustained for lOmin, with a short
break (approximately 30 s) in between, during which
a muscle biopsy was taken. Muscle biopsies and
blood samples were obtained at rest and after each
work rate. The patients were familiarized with the
procedures on a separate day before the experiment.
In the morning, approximately 2 h after a light
breakfast, the patients exercised in the sitting
position on an ergometer. Patient no. 1 cycled for
13rnin at 40 W, rested for 20 rnin and continued the
exercise at 60 W until fatigue (1 1 min). Patient no. 2
cycled at 20, 40 and 60 W for 11 min at each work
rate and thereafter at 70 ( 1 min), 80 (1 min) and 90
(1min)W until fatigue. Except for a short break
(approximately 1 min) after exercise at 40 W, during
which a muscle biopsy was taken (see below),
exercise in patient no. 2 was not interrupted by rest.
Muscle biopsies were obtained at rest, after exercise
at 40W and at fatigue. VO,, heart rate and blood
lactate were measured during exercise.
MATERIAL A N D METHODS
Analytical methods
Subjects and patients
Muscle samples were quick-frozen (within 10 s
after the needle was inserted into the muscle) in
liquid nitrogen and stored at -80°C until analysis.
Biopsies were freeze-dried, dissected free of solid
non-muscle constituents (connective tissue and
blood), powdered, extracted with perchloric acid
(0.5 mol/l) and neutralized with KHCOJ (2.2 mol/l).
The extract was analysed for phosphocreatine,
creatine, lactate, malate, citrate, fumarate, alanine,
glutamate and glutamine by NAD( P)H-coupled
enzymic reactions [8, 91 adapted for fluorimetry.
ATP, ADP, AMP and I M P were analysed by
HPLC [lo]. Muscle metabolites, except for lactate
(due to its extracellular presence), were adjusted to
the peak total creatine (phosphocreatine plus creatine) content in muscle for each subject, to correct
for variability in solid non-muscle constituents
between biopsies. Total creatine in healthy
subjects was 114.5k 2.9, 114.6 k2.6, 11 1.7 f4.7 and
Six healthy male subjects, whose mean age
(range), height, weight and Vo,max. were, respectively, 26 (23-31)yeaq 182 (168-192)cm, 76 (64.587) kg and 3.87 (3.17-4.44) l/min, participated in the
study. Two patients with myophosphorylase deficiency were studied: patient no. 1 (male, age
42 years, weight 72 kg, height 169 cm), and patient
no. 2 (female, age 34years, weight 63.4kg, height
162cm). The diagnosis was based on the following
criteria: absence of histochemical staining for glycogen phosphorylase in skeletal muscle biopsy sample,
absence of increased lactate release during ischaemic
exercise performance, and exercise-induced stiffness
and pain. The patients and the subjects were
informed of the possible risks involved before giving
their voluntary consent. The experimental protocol
was approved by the Ethics Committee of
Experimental procedures
Tricarboxylic acid cycle and McArdle’s disease
689
Table I. Cardiorerpiratory and blood lactate data
Work rate
0
Duration
(min)
Heart rate
beatslmin
Healthy subjects
At rest
Exercise (LWR)
Exercise (MWR)
Exercise (HWR)
Patient no. I
At rest
Exercise
Patient no. 2
At rest
Exercise
0
50
130f6
226k 12
0
40
60
0
20
40
60
7&90
-
60+4
10
10
10
92f4
126+4
173f4
yo*
(l/min)
Blood lactate
(mmol/l)
1.0+0.1
0.7kO.1
0.7 +O.l
2.0 0.I
3.I f0.2
4.6 -I- 0.5
% of maximum
31 +4
48f I
66f2
91 f2
*
1.0+0.1
-
-
-
-
0.8
13
136
I70
0.94
I.20
-
II
74:
93*
-
86
141
I57
43
71
74
0.23
0.63
0.84
I80
200
90
1.1
loo
-
-
II
II
II
3
I
0.4
*Calculated from ageestimated maximal heart rate.
106.3 f.3.9 mmol/kg dry weight at rest, low (LWR),
moderate (MWR) and high (HWR) work rates,
respectively.
Lactate was analysed in neutralized perchloric
acid extracts of blood by an enzymic method applying fluorimetry (see above). VO, was determined in
patients with McArdle’s disease with the Douglas
bag procedure, and in healthy subjects with the
argon dilution technique, in both cases applying MS
for gas analysis.
Data on adenine nucleotides and IMP from one
of the patients (patient no. 1) have been reported in
a separate study [l 11.
Statistical methods
For statistical evaluation in healthy subjects, a
one-way analysis of variance with repeated measures
was employed. When the analysis of variance
resulted in a significant F value (P<0.05), the
difference was located with the Newman-Keul test.
Values are reported as meansfSEM, unless indicated otherwise. Due to the rarity of McArdle’s
disease, only two patients were able to be studied,
which precludes analysis of statistical significance. A
parameter with a normal distribution has 96% of
the values within meank2SD. When a value from
patients with McArdle’s disease was outside this
range in healthy subjects, we have considered it to
be different.
RESULTS
The heart rate in healthy subjects after exercise at
50W, 50% (MWR) and 80% (HWR) of Vo2 max.
was 92 & 4, 126k4 and 173& 4 beats/min, respectively (Table l), which corresponds to 48, 66 and
91% of maximal heart rate measured during the
pretests. The heart rate in the patients with McArdle’s
disease averaged 147 (136, 157)beats/min at 40 W
and 185 (170, 200)beats/min at fatigue. The heart
rate in these patients during exercise at 40 W corresponded to 79% of their peak heart rate. The high
stress on the circulatory system, despite the relatively low absolute work rate, demonstrates a hyperkinetic response and is a well-known feature of
McArdle’s disease [7].
Lactate and high-energy phosphates (Figs. I and 2)
Muscle lactate was unchanged at LWR but
increased in all healthy subjects during MWR, and
even more during HWR (50+7mmol/kg dry
weight). Blood lactate was low at LWR and MWR
but above the lactate threshold (4mmol/l) in four of
the six subjects at HWR. Patients with McArdle’s
disease showed no increase in muscle lactate, which
is in accordance with their inability to utilize muscle
glycogen.
Total creatine at rest was similar in patients with
McArdle’s disease (1 16 and 127mmol/kg dry
weight) and healthy subjects (1 14.5 f2.9 mmol/kg
dry weight). There was no significant change in
phosphocreatine in healthy subjects after exercise at
50 W (compared with at rest), but a decrease to 79
and 32% of the initial level was observed during
MWR and HWR, respectively. Phosphocreatine in
patients with McArdle’s disease was similar to that
of healthy subjects at rest, but, in contrast to
healthy subjects, phosphocreatine decreased after
exercise already at 40 W. After exercise to fatigue,
phosphocreatine in patients with McArdle’s disease
was similar to that in healthy subjects during HWR.
ATP showed no significant change in healthy
subjects during exercise, although a tendency
towards lower values was observed at 80% of
V0,max. ATP was lower in patients with McArdle’s
disease than in healthy subjects at rest, and both
K. Sahlin et al.
690
5T
Y
.-
4-
-c
M
5
e
-0
3-
M
1
,
-5.”
3
2-
I -
0-
I
I
I
I
I
I
0
50
loo
60
200
250
Work rate (w)
Fig. 3. Muscle contents of TCAls at rest and after exercise. Symbols
are the same as in the legend t o Fig. I.
I
I
I
I
I
i
0
50
100
I50
200
250
Fig. I. Muscle content of lactate and phosphocreatine at rest and
after exercise. Values in healthy subjects (0)are means+SEM (n=6).
Filled symbols denote individual values in two patients with McArdle’s
patient no. 2. Statistical significance:
disease: 0 , patient no. I,
*P<O.O5 compared with values at rest.
v,
-.-
patients showed a decrease in ATP after exercise to
iatigue. I M P was close to or below the detection
level (0.01 mmol/kg dry weight) in healthy subjects
at rest and after exercise at the two lower work
rates (LWR and MWR), but increased at HWR
(0.9 f0.23mmol/kg dry weight). The increase in
I M P was related to muscle lactate (r=0.96) and
inversely related to phosphocreatine (I = -0.83),
which is in accordance with previous findings [6].
In patients with McArdle’s disease, I M P increased
already at 40 W, and the muscle content (0.13 mmol/
kg dry weight; average of both patients with McArdle’s disease) was higher in both patients with
McArdle’s disease ( > mean 2SD) than in healthy
subjects at LWR. I M P increased markedly at fatigue (2.6mmol/kg dry weight; average of both
patients with McArdle’s disease), and was higher in
one patient (>mean + 2SD) than in healthy subjects
at HWR.
+
4-
,T
,
c
M
5 3-
e
g
- 2TI
,,
B
El-
Amino acids and TCAls
z
030
1
0’
I
I
I
0
50
100
I
150
I
t
200
250
Work rate (w)
Fig. 2. Muscle contents of IMP and ATP at rest and after exercise.
Symbols are the same as in the legend to Fig. I.
In healthy subjects, TCAIs (malate, citrate and
fumarate) were unchanged during LWR but
increased 3-fold and 8-fold during MWR and HWR,
respectively (Fig. 3). The increase in TCAIs was
mainly due to an increase in malate, which corresponded to 25, 30, 61 and 77% of the measured
TCAIs at rest, LWR, MWR and HWR, respectively
(Fig. 4). The malate/citrate ratio increased from 0.5
at rest to 2.2 and 6.1 at MWR and HWR. The
increase in TCAIs in healthy subjects was related to,
but not proportional to, the increase in muscle
lactate. In patients with McArdle’s disease, levels of
TCAIs at rest were similar to those in healthy
subjects, and no increase occurred during exercise at
40 W, the average value being about half of that in
healthy subjects at 50 W. The peak value of TCAIs
reached in patients with McArdle’s disease at fatigue
was about 2-fold higher than that at rest, but
markedly lower than that in healthy subjects during
Tricarboxylic acid cycle and McArdle’s disease
69 I
*
1
T
4
3
2
pl
.-M
E
t-
-v
M O
-r
2
-zPE
Rest
sow
130W
226W
LE
ME
HE
ReSt
6OW
4OW
*
fatigue
Rest
9ow
4OW
fatigue
Fig. 4. TCAls in healthy subjects (means+SEM, n=6) and in two
patients with McArdle’s disease.
Malate +citrate fumarate: m,
w,
+
citrate; 0. fumarate: @, malate. Statistical significance: *P<O.OS compared with values at rest .
MWR and HWR. However, in relation to the
absolute workload, the increase in TCAIs was similar in patients with McArdle’s disease and healthy
subjects (Fig. 3). At fatigue, malate corresponded to
39% of TCAIs in patients with McArdle’s disease,
and the malate/citrate ratio was 0.9.
The muscle content of alanine was unchanged in
healthy subjects during LWR, but increased during
MWR and HWR (Fig. 5). At the end of exercise
(HWR), the alanine content was about 6mmol/kg
dry weight higher than at rest. Muscle glutamate
decreased progressively when the exercise intensity
increased, and was about 9 mmol/kg dry weight
lower at the end of exercise (HWR) than at rest.
Muscle glutamine remained unchanged during exerand
cise in
healthy
subjects (49.1 f3.6
49.5f 1.2mmol/kg dry weight at rest and during
HWR, respectively).
Due to a shortage of muscle extract, alanine,
glutamate and glutamine could not be measured in
all samples from patients with McArdle’s disease. At
rest, alanine in patients with McArdle’s disease was
similar to that in healthy subjects, but no increase
was observed during exercise. Glutamate at rest
(6.4mmol/kg dry weight, n = 1) was less than 50% of
that in healthy subjects (<mean-2SD), and a
further decrease was observed during exercise. In
I
I
I
I
I
0
50
100
150
200
I
150
Work rate (w)
Fig. 5. Muscle content of glutarnine, glutamate and alanine at rest
and after exercise. Symbols are the same as in the legend t o Fig. I. Note
that the ordinate for glutamine is cut and the scale is different from that of
glutamate and alanine.
one of the patients with McArdle’s disease, glutamine was 38mmol/kg dry weight at fatigue, which is
lower (<mean-2SD) than that in healthy subjects.
DISCUSSION
TCAls
All of the TCAIs were not measured, but since
previous studies have shown that the concentration
of the remaining TCAIs (succinate, 2-oxoglutarate,
isocitrate and oxaloacetate) is less’ than 30% of the
total pool size [12], the bulk of TCAIs have been
accounted for in the present study. The present
study demonstrates that TCAIs remain unchanged
at LWR but increase progressively at MWR and
HWR. The magnitude of the increase in TCAIs after
HWR was similar to that obtained previously
during prolonged exercise at 75% Vo,max. [2]. The
increases in malate at MWR and HWR are consistent with previous results, where increases in malate
were observed both in type I and in type I1 fibres at
exercise intensities below and above the lactate
threshold [131.
692
K. Sahlin et al.
Previous studies in healthy subjects have shown
that the initial increase in TCAIs is partially
reversed during prolonged exercise when low muscle
glycogen levels are reached [2]. It was suggested
that this attenuation of TCAIs could result in TCA
cycle dysfunction, with energetic failure and fatigue
as consequences [2]. This hypothesis was supported
by the finding that oral supplementation with carbohydrate during exercise resulted in higher levels
of TCAIs, an attenuated increase in IMP and
delayed fatigue compared with control conditions
[14). When comparing patients with McArdle’s
disease with healthy subjects at similar relative
exercise intensities, it is evident that the increase in
TCAIs is attenuated in patients with McArdle’s
disease. By analogy, this could be explained by the
absence of glycogenolysis, resulting in low levels of
pyruvate and impaired anaplerosis. However, comparison at the same absolute workload, demonstrates a similar TCAI response in patients with
McArdle’s disease (at fatigue) and healthy subjects.
Anaplerotic reactions and amino acid metabolism
Studies with isotopes [lS] have shown that the
level of TCAIs is the net effect of a continuous
influx (anaplerotic reactions) and efflux (cataplerotic
reactions) of carbon substances to the TCA cycle.
Several of these reactions (catalysed by pyruvate
carboxylase, alanine aminotransferase, phosphoenolpyruvate carboxykinase and malic enzyme) are directly or indirectly dependent on the level of pyruvate, where a high level will promote anaplerosis.
The parallel between increases in muscle lactate and
TCAIs in healthy subjects, and the attenuated
increase in TCAIs in patients with McArdle’s disease at fatigue, are presumably reflections of the
dependency on pyruvate in anaplerosis.
Pyruvate and glutamate are converted to alanine
and 2-oxoglutarate (one of the TCAIs) in the aminotransferase reaction catalysed by alanine aminotransferase. In healthy subjects, muscle alanine
increased in parallel with the increase in TCAIs, and
since the total increase in alanine and the decrease
in glutamate during exercise were larger than the
corresponding increase in TCAIs, it seems likely
that the alanine aminotransferase reaction is the
most important anaplerotic reaction under the
present conditions. Similar conclusions were
obtained in a previous study [2], where changes in
glutamate and alanine were temporally and quantitatively related to the increase in TCAIs.
In patients with McArdle’s disease, there was a
small increase in TCAIs at fatigue. Since muscle
alanine remained unchanged during exercise (Fig. 5 ) ,
and since previous studies [16] have shown an
uptake of alanine, anaplerosis is unlikely to occur
through the alanine aminotransferase reaction in
patients with McArdle’s disease. Patients with
McArdle’s disease have an augmented release of
NH, [ l l , 17, 181. The increase in muscle IMP
demonstrates that part of the NH, formation in
patients with McArdle’s disease originates from
catabolism of adenine nucleotides. In addition, NH,
may also be derived from catabolism of amino acids
via the purine nucleotide cycle, or through the
glutamate dehydrogenase reaction. Formation of
NH, in patients with McArdle’s disease seems to
originate primarily from the AMP deaminase reaction, since a patient with McArdle’s disease with
additional deficiency in AMP deaminase showed no
increase in NH, during exercise [19].
Several lines of evidence demonstrate that the
metabolism of branched-chain amino acids (leucine,
isoleucine and valine) is augmented during exercise
in patients with McArdle’s disease [16, 181. It is at
present unclear whether this will increase anaplerosis or, as argued by Wagenmakers et al. [18],
provide a drain on TCAIs.
Control of the TCA cycle activity: implications for the
oxidative defect in patients with McArdle’s disease
Maximal rates of energy transduction by the TCA
cycle require the availability of a sufficient supply of
substrate (acetyl-CoA), allosteric activation and
increases in the substrates of the non-equilibrium
reactions (i.e. oxaloacetate, isocitrate and 2oxoglutarate). Increases in total TCAIs provide
information on the capacity to increase substrate
concentration, and thus the flux rate, in these nonequilibrium reactions. We have postulated that the
block in glycogen breakdown in patients with
McArdle’s disease limits oxidative phosphorylation
by limiting TCA flux and the production of reducing equivalents [7, 111. Impaired TCA cycle function may relate both to limited substrate and low
levels of TCAIs. Comparison of TCAIs in patients
with McArdle’s disease and healthy subjects at the
same relative exercise intensity, supports the
hypothesis that the level of TCAIs limits aerobic
energy transduction in patients with McArdle’s disease, whereas comparison at the same absolute
workload speaks against this hypothesis.
Alternatively, the impairment of aerobic exercise
capacity in patients with McArdle’s disease could be
the consequence of a limited rate of acetyl-CoA
formation. This hypothesis is supported by the
finding that an increased substrate availability,
through either infusion of carbohydrate [20-22] or
elevation of circulating free fatty acid levels [21-231,
results in enhanced exercise capacity in patients
with McArdle’s disease, and by the discovery of low
levels of TCA cycle substrate (acetylcarnitine) in
patients with McArdle’s disease at rest and during
exercise [2].
ADP is an important allosteric regulator of
several enzymes in the TCA cycle [l]. Total muscle
ADP increased during exercise by about 20% in
both healthy subjects and patients with McArdle’s
disease. Most of the cellular ADP, however, is
Tricarboxylic acid cycle and McArdle’s disease
bound to proteins and metabolically inactive [24].
The concentration of free ADP, which is the metabolically active form, is conventionally calculated
[24] from the mass action ratio of the creatine
kinase reaction (phosphocreatine + ADP + H + t t
creatine + ATP). Phosphocreatine decreased during
exercise, and reached similar values at fatigue in
patients with McArdle’s disease to those after HWR
in healthy subjects. The decrease in phosphocreatine
corresponds to increases in free ADP and/or H’. In
contrast to healthy subjects, where H + increases
during exercise due to lactic acid accumulation,
there is a decrease in H + in patients with McArdle’s
disease during exercise [25]. Hence, for a given
decrease in phosphocreatine, the increase in free
ADP will be larger in patients with McArdle’s
disease than in healthy subjects [25]. The augmented increase in free ADP in patients with
McArdle’s disease at low absolute work rates could
be a compensatory mechanism to increase the
activity of the TCA cycle at low levels of TCAIs and
acetyl units. In healthy subjects, there was no
change in TCAIs or in ADP (as judged by the
constancy of phosphocreatine) during LWR. The
increased oxidative metabolism at LWR must therefore be a consequence of some other control signal,
such as increases in Ca2+ [l] or increased availability of oxygen.
In summary, the present study demonstrates that
there is a progressive increase in TCAIs in healthy
subjects during exercise at 50 and 80% of Vo,max.,
which may be essential for attaining high rates of
aerobic ATP production. When compared at the
same relative intensity, a markedly reduced TCAI
level was observed in patients with McArdle’s disease, and the peak level was only 22% of that in
healthy subjects. However, when compared at the
same absolute workload, TCAIs were similar in
patients with McArdle’s disease and healthy subjects. The alanine aminotransferase reaction appears
to be one of the major anaplerotic reactions in
healthy subjects, but not in patients with McArdle’s
disease. The frequently quoted expression ‘fat burns
in the glow of carbohydrate’ [26] may be related to
the requirement of carbohydrate for expansion of
TCAIs.
ACKNOWLEDGMENTS
This work was supported by grants from The
Swedish Sport Research Council, Swedish Medical
Research Council (project no. 8671 and 4139),
National Heart, Lung and Blood Institute (NHLBI
grant HL-06296) and The Muscular Dystrophy
Association.
693
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