J Appl Physiol 94: 1751–1756, 2003.
First published January 3, 2003; 10.1152/japplphysiol.01070.2002.
Phosphocreatine content of freeze-clamped muscle:
influence of creatine kinase inhibition
Jeffrey J. Brault, Kirk A. Abraham, and Ronald L. Terjung
Department of Physiology, College of Medicine, Department of Biomedical Sciences, College of Veterinary
Medicine, and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211
Submitted 21 November 2002; accepted in final form 23 December 2002
where ⌬G°⬘ is the standard free energy, R is the gas
constant, and T is absolute temperature. Intramuscular ATP is relatively abundant and considered readily
available (“free”) to react in solution. Therefore, concentrations that are determined by various methods
are similar and thought to be directly applicable to the
calculations. On the other hand, the majority of ADP is
bound, particularly to actin; therefore the total amount
measured in acid-extracted muscle does not represent
that which participates in the creatine (Cr) kinase (CK)
reaction. It has not been possible to measure the free
ADP (ADPf) concentration, because its concentration
within skeletal muscle is far below the sensitivity limits of measurements in vivo [nuclear magnetic resonance (NMR)]. Rather, ADPf concentration has been
calculated by using the CK reaction (PCr ⫹ ADPf ⫹
H⫹ 7 Cr ⫹ ATP), because the equilibrium constant is
known, the reaction is fast and near equilibrium under
most conditions, and the relative concentrations of Cr
and phosphocreatine (PCr) are high. In substituting
this estimate of ADPf into Eq. 1, it is apparent that
changes in ⌬GATP scale with changes in the Cr/PCr
ratio and Pi. Therefore, accurate determinations of
PCr, Cr, and Pi are vital for correct energetic measurements in skeletal muscle.
In contrast to measurements of ATP, striking differences are found in the measurements of skeletal muscle PCr and Pi depending on the method employed. For
example, chemically determined PCr and Pi content in
mammalian fast-twitch muscle from extracts of muscle
quick-frozen at liquid nitrogen temperature expressed
as the ratio of PCr/ATP (⬃2–3:1) and Pi/ATP (⬃0.8–
1.0:1) (24, 26, 30, 33) is far different than corresponding values determined by NMR in vivo (PCr/ATP ratio
of ⬃3–4:1; Pi/ATP ratio of ⬃0.2–0.3:1) (1, 22, 26, 33),
although this has not always been found (35). The
cause of the systematically higher PCr and lower Pi
values from NMR measurements in vivo have not been
identified experimentally. A large quantity of bound
intracellular Pi would make it “invisible” during NMR
analyses and reconcile the difference for Pi; however,
the same argument applied to PCr requires that the
smaller value in muscle extracts must be attributed to
a pool of PCr that is seen by NMR but is not extracted.
This seems unlikely. On the other hand, the differences
in Pi and PCr could be due to PCr hydrolysis during the
process of muscle isolation (30) and/or quick-freezing
(26). Calcium release during quick-freezing could activate actomyosin ATPase, hydrolyze ATP to ADP, and
prompt phosphotransfer between PCr and ADP via the
CK reaction. If this were the case, there would be little
Address for reprint requests and other correspondence: R. L.
Terjung, Biomedical Sciences, College of Veterinary Medicine, E102
Vet. Med. Bldg., Univ. of Missouri, Columbia, MO 65211 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
iodoacetamide; muscle fiber type; contractions
as contraction and calcium regulation is critically dependent on the free
energy (⌬G) available from ATP hydrolysis (ATP 3
ADP ⫹ Pi). The ⌬G is determined by the ratio of the
product and reactant concentrations of those metabolite pools readily available to react in solution. The free
energy of ATP hydrolysis (⌬GATP) is defined as
SKELETAL MUSCLE FUNCTION SUCH
⌬G ATP ⫽ ⌬G°⬘ ⫹ R ⫻ T ⫻ ln
http://www.jap.org
ADP ⫻ P i
ATP
(1)
8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society
1751
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Brault, Jeffrey J., Kirk A. Abraham, and Ronald L.
Terjung. Phosphocreatine content of freeze-clamped muscle:
influence of creatine kinase inhibition. J Appl Physiol 94:
1751–1756, 2003. First published January 3, 2003; 10.1152/
japplphysiol.01070.2002.—The study of cellular energetics is
critically dependent on accurate measurement of high-energy
phosphates. Muscle values of phosphocreatine (PCr) vary
greatly between in vivo measurements (i.e., by nuclear magnetic resonance) and chemical measurements determined
from muscles isolated and quick-frozen. The source of this
difference has not been experimentally identified. A likely
cause is activation of ATPases and phosphotransfer from PCr
to ADP. Therefore, rat hindlimb skeletal muscle was perfused either with or without 2 mM iodoacetamide, a creatine
kinase inhibitor, and muscle was freeze-clamped either at
rest or after contraction. Creatine kinase inhibition resulted
in ⬃6 ␮mol/g higher PCr and lower creatine in the freezeclamped soleus, red gastrocnemius, and white gastrocnemius. This PCr content difference was reduced when the
initial PCr content was decreased with prior contractions.
Therefore, the amount of PCr artifact appears to scale with
initial PCr content within a fiber-type section. This artifact
directly affects the measurement and, thus, the calculations
of muscle energetic parameters from studies using isolated
and frozen muscle.
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PHOSPHOCREATINE CONTENT AND MUSCLE FREEZING
difference expected in ATP concentration, whereas
there would be a decline in PCr and a stoichiometric
increase in Cr and Pi.
The purpose of this study was to evaluate whether
chemical analysis of muscle PCr is confounded by the
isolation and quick-freezing process due to ⬃Pi transfer through the CK reaction. Employing an isolated
perfused rat hindlimb preparation, we inhibited CK
with iodoacetamide (12) in muscle with a high PCr
(resting) content and a relatively low PCr content produced by contractions. We hypothesized that muscle
collected after iodoacetamide treatment would have a
higher PCr and lower Cr content and that this effect
would be lessened when PCr content was decreased.
METHODS
J Appl Physiol • VOL
RESULTS
Preliminary experiments established that perfusion
of the hindlimb for 10 min with medium containing 2
mM iodoacetamide was effective at inhibiting CK activity. As illustrated in Fig. 1, the typical ⬎80% reduction in PCr that occurs with intense contraction conditions (5 Hz twitch for 30 s; Ref. 9) did not occur with
prior exposure to iodoacetamide. Muscle PCr content
remained high (⬃30 ␮mol/g) even though the muscle
force profile was reasonably similar to stimulated muscle in the absence of iodoacetamide. Therefore, we had
a useful perfusion condition that essentially eliminated
CK activity.
As shown in Table 1, resting muscle PCr concentrations of 16–21 ␮mol/g, depending on the muscle fiber
section, were higher by ⬃6 ␮mol/g when freezeclamped with iodoacetamide. Cr concentrations in
these same muscle sections were stoichiometrically
lower. Thus total Cr (PCr ⫹ Cr) content of the muscles
was not affected by iodoacetamide. Interestingly, these
same iodoacetamide-treated muscles showed evidence
of ATP degradation with lower ATP and higher IMP,
AMP, and ADP concentrations. There were no changes
in inosine, hypoxanthine, or adenine contents in the
muscles (data not shown). The difference in total phosphate equivalents among the phosphate pools was determined for the mixed-fiber plantaris (Fig. 2). After
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Animal care. Male Sprague-Dawley rats (Taconic, Germantown, NY), weighing 325–375 g, were housed two per
cage in a temperature- (20–22°C) and 12:12-h light-dark
cycle-controlled environment. All animals were provided unrestricted food and water. This study was approved by the
University of Missouri-Columbia Animal Care and Use Committee.
Hindquarter perfusion. The standard perfusion medium
consisted of 5% bovine serum albumin in Krebs-Henseleit
buffer, 5 mM glucose, 100 ␮U/ml bovine insulin, and typical
plasma concentrations of amino acids (3). Immediately before
use, the perfusate was filtered (0.45 ␮m), warmed to 37°C,
and adjusted to a pH of 7.40. A portion was used to prime the
perfusion apparatus, which included, in series, a peristaltic
pump, a filter, a heating and oxygenating chamber supplied
with 95% O2-5% CO2, and a bubble trap. The entire apparatus was located inside a Plexiglas cabinet maintained at
37°C. Perfusion pressure and temperature were monitored
continuously throughout the experiment.
Rats were anesthetized with pentobarbital sodium (60
mg/kg ip) and administered 100% oxygen during surgical
preparation as described previously (14). The hind feet and
tail were tied with umbilical tape to limit blood flow to the
hindlimb tissues. After catheters were secured in the descending aorta and inferior vena cava and flow was begun,
the rats were humanely killed with an overdose of pentobarbital into the carotid artery.
The flow rate was increased gradually over ⬃20 min until
aortic perfusion pressure (⬃45 mmHg) was stable at a flow of
50 ml/min. The initial ⬃150 ml of effluent was discarded to
clear the system of essentially all red cells, after which the
perfusate was recirculated. For studies of resting muscle,
samples of the right hindlimb were taken first (see below).
Then, the perfusate was immediately switched to an identical medium containing 2 mM iodoacetamide, and muscles of
the left hindlimb were collected 8.5 min later. For studies of
contracting muscle, we employed an intense isometric tetanic
contraction sequence, known to greatly reduce muscle PCr
concentration (27). The sciatic nerve was exposed, cut, and
electrically stimulated with supramaximal square-wave electrical pulses (6–8 V, 0.1 ms, 100 Hz) eliciting 60 tetani/min,
each 100 ms in duration. Control muscles were stimulated for
a total of 9 min. The contralateral leg was stimulated for 1.5
min to permit muscle PCr to decrease, and then the perfusate
was switched to an identical medium containing 2 mM iodoacetamide. Contractions and iodoacetamide treatment
continued for an additional 7.5 min. Thus inhibition of CK
was achieved by perfusion of the muscle with 2 mM iodo-
acetamide for either 8.5 min (resting muscle) or 7.5 min
(contracting muscle).
Muscle sections were rapidly resected and quick-frozen
with compression between aluminum tongs cooled in liquid
nitrogen. From the time of resection and/or loss of blood flow
to the time of freezing, we estimate that it took 4–5 s for the
soleus (predominantly slow-twitch red fibers), 8–10 s for the
plantaris (mixed fibers), 1–2 s for the superficial medial
white gastrocnemius (predominantly fast-twitch white fibers), 12–14 s for the deep lateral red gastrocnemius (predominantly fast-twitch red fibers), and ⬃16 s for the remainder of the gastrocnemius (mixed fibers) (2). Each section
weighed ⬃125–300 mg, except for the remainder of the
gastrocnemius, which weighed ⬃2 g. Frozen tissue samples
were stored at ⫺80°C until analyzed.
Metabolite analyses. Metabolites from muscle sections
were extracted in cold ethanolic (20% vol/vol) perchloric acid
(3.5% wt/vol) and neutralized with tri-n-octylamine and
1,1,2-trichlorotrifluoroethane (7). Extracts were stored at
⫺80°C until analyzed.
Cr and PCr concentrations were determined by use of
ion-exchange HPLC as described by Wiseman et al. (36).
Adenine nucleotides and bases were determined by reversephase HPLC (32). Inorganic phosphate was determined by
cation-exchange HPLC (23).
The glycolytic intermediate fructose 1,6 bisphosphate, the
primary glycolytic Pi sink (9), was measured by standard
enzymatic technique (28).
To determine the muscle water content, an 150- to 250-mg
portion of each gastrocnemius mixed fiber section was dried
at 60°C to a stable weight. Metabolite concentrations were
calculated to a common water content of 76%, typical for
rested rat skeletal muscle (20).
Statistics. Analysis of variance was used to identify main
treatment effects, with significance accepted at P ⬍ 0.05.
Values are given as means ⫾ SE.
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PHOSPHOCREATINE CONTENT AND MUSCLE FREEZING
found only in the soleus as a result of iodoacetamide
with prior contractions.
DISCUSSION
iodoacetamide treatment, phosphate is lower in the
total nucleotide pool ([⌬ATP] ⫻ 3 ⫺ [⌬ADP] ⫻ 2 ⫺
[⌬AMP] ⫺ [⌬IMP]; 4.45 ⫾ 1.51 ␮mol/g) and Pi pool
(2.73 ⫾ 0.33 ␮mol/g) for a total decrement of 7.18 ⫾
1.38 ␮mol/g. This is in excellent stoichiometry with
increased phosphate in the PCr pool (6.50 ⫾ 1.32
␮mol/g) and fructose 1,6 bisphosphate pool (0.58 ⫾ 0.26
␮mol/g) for a total increase of 7.08 ⫾ 1.26 ␮mol/g.
After depressing PCr content to 4–6 ␮mol/g, by prior
muscle contractions, iodoacetamide reduced the magnitude of the higher PCr and lower Cr to 1–5 ␮mol/g
whereas total Cr remained essentially unchanged (Table 1). A significant difference in ATP content was
Table 1. Phosphocreatine, creatine, ATP, and IMP content of freeze-clamped muscle perfused
with or without iodoacetamide
Resting
No iodoacetamide
Phosphocreatine
Soleus
Red gastrocnemius
White gastrocnemius
Creatine
Soleus
Red gastrocnemius
White gastrocnemius
ATP
Soleus
Red gastrocnemius
White gastrocnemius
IMP
Soleus
Red gastrocnemius
White gastrocnemius
2 mM Iodoacetamide
Contracted
Difference
No iodoacetamide
2 mM Iodoacetamide
Difference
15.70 ⫾ 0.68
21.34 ⫾ 1.24
18.15 ⫾ 1.49
22.18 ⫾ 1.09
27.18 ⫾ 1.24
24.22 ⫾ 1.42
6.48 ⫾ 1.25*
5.84 ⫾ 1.24*
6.07 ⫾ 0.95*
3.77 ⫾ 0.45
6.48 ⫾ 0.79
6.08 ⫾ 0.82
5.53 ⫾ 0.76
11.25 ⫾ 0.95
8.81 ⫾ 0.90
14.91 ⫾ 0.83
18.69 ⫾ 0.81
26.08 ⫾ 1.02
8.17 ⫾ 0.81
11.38 ⫾ 0.72
22.46 ⫾ 0.86
⫺6.74 ⫾ 0.61*
⫺7.31 ⫾ 0.58*
⫺3.62 ⫾ 0.85*
23.11 ⫾ 1.24
29.49 ⫾ 1.20
36.93 ⫾ 2.38
20.23 ⫾ 1.42
27.44 ⫾ 1.44
36.30 ⫾ 1.81
⫺2.88 ⫾ 1.95
⫺2.05 ⫾ 1.86
⫺0.62 ⫾ 1.19
4.54 ⫾ 0.09
6.88 ⫾ 0.32
7.15 ⫾ 0.24
3.30 ⫾ 0.22
4.55 ⫾ 0.30
6.36 ⫾ 0.33
⫺1.24 ⫾ 0.23*
⫺2.33 ⫾ 0.57*
⫺0.79 ⫾ 0.52
4.57 ⫾ 0.25
5.09 ⫾ 0.47
4.36 ⫾ 0.72
3.29 ⫾ 0.27
4.73 ⫾ 0.16
4.05 ⫾ 0.40
⫺1.28 ⫾ 0.35*
⫺0.36 ⫾ 0.49
⫺0.31 ⫾ 0.75
0.025 ⫾ 0.005
0.030 ⫾ 0.006
0.024 ⫾ 0.006
0.490 ⫾ 0.143
0.872 ⫾ 0.177
0.301 ⫾ 0.095
0.083 ⫾ 0.022
1.627 ⫾ 0.360
2.852 ⫾ 0.510
0.306 ⫾ 0.098
1.823 ⫾ 0.223
2.794 ⫾ 0.519
0.223 ⫾ 0.100
0.196 ⫾ 0.339
⫺0.058 ⫾ 0.522
0.465 ⫾ 0.145*
0.842 ⫾ 0.178*
0.277 ⫾ 0.096*
Values are means ⫾ SE expressed as ␮mol/g; n ⫽ 8 for resting condition; n ⫽ 8 for contracted. * P ⬍ 0.05.
J Appl Physiol • VOL
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1.76 ⫾ 0.97
4.77 ⫾ 0.85*
2.73 ⫾ 1.49
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Fig. 1. Phosphocreatine (PCr) content of fast-twitch white muscle
fiber sections stimulated (5 Hz for 30 s) in the presence or absence of
2 mM iodoacetamide (n ⫽ 4).
To our knowledge, this is the first study to experimentally establish that PCr degradation via CK occurs
with tissue sampling, even with great care to rapidly
resect and quick-freeze the muscle. When CK was
inhibited by iodoacetamide in resting muscle, PCr concentration was ⬃6 ␮mol/g greater and Cr was ⬃6
␮mol/g lower than when CK was not inhibited. Because
total Cr remains constant, this lower PCr content indicates that an energy-utilizing process was activated,
a suggestion that has been made by Meyer et al. (26)
and Soderlund and Hultman (30) and is consistent
with the data of Curtin and Woledge (8). Therefore, an
artifact exists in the isolation and freezing process that
consumes high-energy phosphates and substantially
effects chemical measurement of PCr and Cr. Interestingly, the observation of a greater PCr content (⬃5.5
␮mol/g) in the muscle of CK-deficient compared with
wild-type mice (33) could characterize the same process
as demonstrated here.
It is curious that the higher PCr amount in the
presence of iodoacetamide was similar among the different skeletal muscle fiber sections. This implies that
the total Pi exchange in the absence of iodoacetamide
was similar for each of the muscles. The precipitating
event for this hydrolysis is likely Ca2⫹ release from the
sarcoplasmic reticulum as a result of tissue damage
(30), rapid cooling (21), or freezing (13). This in turn
would activate ATPases and hydrolyze ATP to ADP
(26, 30). Given the high capacity of CK activity and the
high equilibrium constant, there is expected to be a
rapid decrease in PCr content with little to no change
in ATP content (6). If the ATP hydrolysis rate was the
limiting process, then the amount of the PCr artifact
might be expected to vary with fiber type (26), because
significant differences in the rates of sarcoplasmic re-
1754
PHOSPHOCREATINE CONTENT AND MUSCLE FREEZING
Fig. 2. Difference in phosphate equivalents between
muscle treated with iodoacetamide and muscle in its
absence (plantaris; n ⫽ 8). F1,6BP, fructose 1,6
bisphosphate. See text for calculation of the composite
change in phosphate.
J Appl Physiol • VOL
gastrocnemius suggests that this PCr value may be
aberrant. Nonetheless, we interpret our findings to
support the expectation that the magnitude of the PCr
artifact is directly proportional to the PCr pool within
the muscle.
If our findings have general application, then the
chemically determined PCr and Cr measurements, typically presented in many animal or human studies, do
not represent the true in vivo values. This can have
several implications. First, it is apparent that any
calculation of ADPf would be incorrect because of the
large error in the PCr/Cr ratio used. Recalculated ADPf
values, corrected for the PCr artifact described herein,
would be considerably lower for resting muscle but
increase over a wider-fold range as PCr content declines experimentally within the muscle. Thus any
reliance on the uncorrected value(s) of ADPf would be
misleading. Fortunately, typical interpretations of results in those studies are based on the general pattern
or direction of response, not on absolute value(s) of
ADPf. Thus even though errors are found in published
work, the interpretation of results in the affected studies remain generally unchanged (e.g., Refs. 10, 24).
Second, the observed phenomenon reported here may
help clarify previous results implicating changes in
and/or the distribution of PCr and Cr pools within
muscle. For example, some studies involving Cr depletion in rats (25, 31) have reported a surprisingly low
PCr/Cr ratio in the quick-frozen muscle. As predicted
by Meyer et al. (25), these aberrant values can be
reconciled if a freezing artifact is considered. Recalculations of PCr and Cr contents to account for the
expected PCr hydrolysis return the PCr/Cr ratios to
values more consistent with the expected energy state
of the rested muscle (25). Similarly, the general inconsistency of low PCr/Cr ratios often observed with oral
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ticulum Ca2⫹ ATPase (16) and myosin ATPase exist
among the different fiber types (4, 5). Obviously, this
was not the case. Two alternatives may be pertinent.
First, there may not be a sufficient difference in
ATPase rates, relative to the variation in artifact (e.g.,
due to differences in collection time and/or freezing
rate), to manifest a difference in PCr artifact among
the fiber types of this study. Second, the extent of
activation (e.g., the amount of calcium released) may
be such that a fixed amount of ATP hydrolysis occurs in
spite of different rates. During contractions when the
PCr is low, the freeze artifact is expected to be smaller,
possibly related to a reduced sensitivity of the involved
ATPase(s) in the extreme cellular environment. Furthermore, in the presence of the high demand for ATP
resynthesis, the CK rate should be influenced by the
PCr concentration available to hydrolyze. In other
words, the lower the initial PCr content (or PCr/Cr
ratio), the smaller should be the change in PCr content
that occurs during the quick-freezing process. Indeed,
there can be no difference in PCr in muscles frozen
with and without CK activity when the initial PCr
content is zero. We confirmed this expectation, but not
with absolute precision in all fiber sections. The size of
the PCr artifact remained 20–30% of the existing PCr
pool within the muscle, at the time of the isolation and
freezing, regardless of whether the muscle was resting
or during intense contractions, in two of the muscle
sections (soleus and white gastrocnemius), but not in
the third (red gastrocnemius). We cannot account for
the larger (42%) PCr artifact in the red gastrocnemius
muscle when PCr was initially lower, other than related to the complexity of the experiment requiring
iodoacetamide inhibition of CK during contractions
over the 8.5 min of perfusion. Furthermore, the relatively small change in Cr in relation to PCr in the red
PHOSPHOCREATINE CONTENT AND MUSCLE FREEZING
J Appl Physiol • VOL
We gratefully acknowledge the excellent technical assistance of
Hong Song and Jackie Love.
This study was supported by the National Institute of Arthritis
and Musculoskeletal and Skin Diseases Grant AR-21617.
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Cr supplementation (15, 17) could be reconciled, if the
variability in PCr hydrolysis that occurs during quickfreezing were similar in magnitude to the induced
change in muscle PCr brought about by Cr supplementation. Thus true PCr changes would be lost in the
measurement variability. It is unlikely, however, that
the observed phenomenon reported here can clarify
previous results that implicate the existence of different pools of PCr and/or Cr within muscle. For example,
evidence for a large discrete pool of Cr, suggested by
differential radiolabeling of Cr and PCr in fish muscle
(19), is further strengthened if their results for rested
muscle are recalculated taking into account the ⬃6
␮mol/g PCr isolation/freezing artifact. Similarly, there
is little quantitative relationship between the PCr that
was degraded during freezing and the putative PCr
pool contained within the mitochondria (29, 34). The
⬃6 ␮mol/g PCr measured here for the white gastrocnemius is far greater (more than sixfold) than a liberal
estimate of the mitochondrial PCr pool, on the basis of
reasonable estimates of mitochondrial volume (11) and
a maximal mitochondrial Cr concentration of 20 mM
(34). Finally, it is important to emphasize that the
artifact characterized in this study pertains to data
obtained by using chemical determinations of PCr and
Cr. In this regard, studies employing 31P-NMR spectroscopy for high-energy phosphates are not confounded; in fact, this in vivo method identified the inconsistencies in PCr measurement that our study helps
reconcile experimentally.
Similar to the findings demonstrated in isolated perfused hearts (12, 18), we confirmed that CK was inhibited by iodoacetamide in skeletal muscle in our case by
eliminating PCr degradation during contractions (cf.
Fig. 1). In contrast, iodoacetamide does not effectively
inhibit mechanical function at moderate work intensity or ATPases in vitro in the heart (12, 18). ATP
hydrolysis also proceeds in skeletal muscle of this
study, because there was a net loss of ATP and an
increase in ADP, AMP, and IMP contents in iodoacetamide-treated muscles, compared with muscles in the
absence of iodoacetamide (Fig. 2). This loss of highenergy bonds from ATP, presumably caused by the lack
of contribution from PCr, represents a net ATPase
activity of ⬃2.5 ␮mol/g, an amount seemingly less than
the ⬃6 ␮mol/g represented in the PCr that was retained. Whether this represents a reduction in ATPase
rate with iodoacetamide is unclear. Fortunately, this
does not impact on the calculation of the isolation/
freeze artifact, because evidence of the artifact is solely
due to differences in PCr content.
In summary, PCr content of inactive muscle was
appreciably higher when isolated and frozen in the
presence of iodoacetamide, a CK inhibitor. This difference was lessened as PCr was reduced by prior muscle
contractions. Our results provide quantitative evidence for an isolation/freeze artifact that occurs in the
chemical measurement of PCr, Cr, and Pi in all fiber
types. This artifact directly affects the measurement
and, thus, the calculations of muscle energetic parameters from studies using isolated and frozen muscle.
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