Effect of oral creatine supplementation on skeletal muscle

phosphocreatine resynthesis
P. L. GREENHAFF, K. BODIN, K. SODERLUND, AND E. HULTMAN
Queens Medical Center, Department of Physiology and Pharmacology, University Medical School,
NG7 2UH Nottingham, United Kingdom; and Department of Clinical Chemistry,
Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden

Greenhaff, P. L., K. Bodin, K. Soderlund, and E. ported by the lower accumulation of plasma ammonia
Hultman. Effect of oral creatine supplementation on skeletal and hypoxanthine, which was observed during exercise
muscle phosphocreatine resynthesis. Am. J. Physiol. 266 after Cr ingestion.
(Endocrinol. Metab. 29): E725-E730, 1994.-Biopsy samples The aim of the present experiment was therefore to
were obtained from the vastus lateralis muscle of eight investigate the effect of oral Cr ingestion on muscle PCr
subjects after 0, 20, 60, and 120 s of recovery from intense

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electrically evoked isometric contraction. Later (10 days), the resynthesis after a contraction-induced depletion of
same procedures were performed using the other leg, but muscle PCr stores.
subjects ingested 20 g creatine (W/day for the preceding 5
METHODS
days. Muscle ATP, phosphocreatine (PCr), free Cr, and lactate
concentrations were measured, and total Cr was calculated as Eight male subjects volunteered to take part in the present
the sum of PCr and free Cr concentrations. In five of the eight experiment. All undertook some form of recreational exercise,
subjects, Cr ingestion substantially increased muscle total Cr but none was highly trained. Their physical characteristics
concentration (mean 29 t 3 mmol/kg dry matter, 25 t 3%; were as follows (mean * SE): age 29.1 t 1.6 yr; weight 80.0 t
range 19-35 mmol/kg dry matter, 15-32%) and PCr resynthe- 4.7 kg, and height 184 t 3 cm. Before the commencement of
sis during recovery (mean 19 t 4 mmol/kg dry matter, 35 t the study, all subjects gave voluntary consent to take part in
6%; range 11-28 mmol/kg dry matter, 23-53%). In the the experiment; all were informed of the experimental proce-
remaining three subjects, Cr ingestion had little effect on dures to be undertaken and were aware that they were free to
muscle total Cr concentration, producing increases of 8-9 withdraw from the study at any point. The study was approved
mmol/kg dry matter (5-7%), and did not increase PCr resyn- by the Ethics Committee of the Karolinska Institute, Hud-
thesis. The data suggest that a dietary-induced increase in dinge, Sweden.
muscle total Cr concentration can increase PCr resynthesis Each subject reported to the laboratory in a normal fed
during the 2nd min of recovery from intense contraction. state on the morning of the study, having abstained from
energy metabolism; recovery strenuous physical exercise the previous day, and had their
nude body weight recorded. The experiment began with each
subject lying in a semi-supine position on a bed with both legs
flexed over one end at an angle of 90. The leg to be investi-
gated on this visit was chosen at random and was attached via
CREATINE (Cr) is a naturally occurring compound found
an ankle strap to a strain gauge built into the frame of the bed.
principally in skeletal muscle, and in-its free and phos- The subject was then asked to perform three maximal volun-
phorylated forms it plays a pivotal role in the regulation tary contractions to determine the maximal voluntary isomet-
and homeostasis of skeletal muscle energy metabolism ric force of the knee extensors. The isometric force produced
(5, 6, 24, 31). It is now generally accepted that the was measured with a strain gauge (AB Bofors, Karlskoga,
maintenance of phosphocreatine (PCr) availability is Sweden) and, after amplification (direct current amp Medelec
important to the-continuation of muscle force produc- AD6; Medelac, Surrey, UK), was displayed on an oscilloscope
tion (19,23). Endogenous synthesis of Cr occurs in liver, and recorded on ultraviolet paper (Medelec). The leg was then
kidney, and pancreas. However, it has been known for prepared for electrical stimulation, as described previously
some time that oral ingestion of Cr in the form of meat (20). Briefly, the muscles of the anterolateral portion of the
thigh were stimulated to contract with square wave impulses
and fish or supplements will add to the whole body Cr
of 0.5 ms duration, at a frequency of 50 Hz, and at a voltage
pool (7,8,18). It has recently been shown that ingestion sufficient to elicit maximal contraction. Approximately 35% of
of 20-30 g Cr/day for several days can lead to a > 20% the musculature that extends the knee is activated in this way
increase in human skeletal muscle total Cr content, of (20). Stimulation was intermittent with 20 trains of 1.6 s
which N 20-30% is in the form of PCr (16). It also stimulation being separated by rest periods of 1.6 s. The total
appears that muscle Cr uptake is augmented if submaxi- contraction time was therefore 32 s. Before initiation of
mal exercise is performed during the period of supple- stimulation (30 s), a cuff surrounding the proximal portion of
mentation (16). Further recent evidence of our own (11, the thigh was inflated (250 mmHg) to occlude limb blood flow
17) and others (1) demonstrates that Cr ingestion can and remained inflated until a muscle biopsy had been obtained
significantly increase the amount of work that can be from the vastus lateralis immediately after the final contrac-
performed during repeated bouts of maximal exercise. It tion (4). This stimulation protocol was chosen because it has
been previously shown to result in almost total degradation of
was postulated in these studies that the ergogenic effect muscle PCr stores (12). The cuff was then deflated, and
of Cr ingestion may be attributable to an increased further muscle biopsy samples were obtained after 20,60, and
muscle Cr content, accelerating PCr resynthesis be- 120 s. During this time, subjects remained resting in a
tween exercise bouts. As a result, the required rate of semi-supine position on the bed.
ADP rephosphorylation would have been sustained Subjects reported back to the laboratory 10 days later for
longer during contraction. This suggestion was sup- the second part of the study. However, for the 5 days preceding

0193-1849/94 $3.00 Copyright o 1994 the American Physiological Society E725

E726 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION

their second visit, each ingested 4 x 5 g (total 20 g) creatine

60 A
monohydrate (Chemi, Linz, Austria). Subjects were instructed 1
to ingest a single 5-g dose in the early morning, at noon, in the
late afternoon, and in the evening. They were also instructed
1
MO-
to totally dissolve each 5-g dose in warm-hot tea or coffee
before ingestion. Upon reporting back to the laboratory, .
subjects underwent exactly the same experimental procedures
that they had performed 10 days previously, but on this E MO-
occasion their other leg was electrically stimulated and biop- W
.
sied. g
Upon removal from the muscle, all biopsy samples were 0 130-
immediately frozen by plunging the biopsy needle into liquid E
nitrogen. The time delay between the insertion of the biopsy 5
needle and freezing of the sample ranged from 3 to 5 s. All
p lzo-
samples were freeze dried and stored at -80C until analyzed
at a later date. .
After fat extraction with petroleum ether, a portion of each

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freeze-dried muscle sample was dissected free of all visible llO-
blood and connective tissue and was pulverized. The powdered
muscle was used for the spectrophotometric
ATP, PCr, free Cr, and lactate concentrations
determination
(15). Muscle
of
100
1
total Cr concentration was calculated by summing PCr and
Pre-Ingestion Post-Ingestion
free Cr concentrations.
Unless stated otherwise, values presented in the text,
Tables 1 and 2, and Figs. 1 and 2 refer to means t SE. B
Comparison of metabolite levels during recovery before and 30
after Cr ingestion was performed by using repeated-measures 03
two-way analysis of variance with respect to treatment (with/
G i l 7
without Cr) and time (O-120 s). When a significant F value
was achieved (P < 0.05), Fishers test for pairwise compari-
sons was used to locate the difference between paired means
across treatments. On all occasions, statistical difference was 02 01
declared at P < 0.05. 04

RESULTS

Mean body weight before Cr ingestion was 80.0 t 4.7

kg. After Cr ingestion, body weight had increased to
81.6 t 4.8 kg (P < 0.05). A body weight increase was
observed in seven of the eight subjects.
Table 1 shows mean muscle ATP, PCr, free Cr, total
Cr (sum of PCr + free Cr), and lactate concentrations
for all eight subjects during recovery from contraction,
-20 . .
d 10 2'0 - 3'0 4'0

Table 1. Muscle ATP, PCr, free Cr, total Cr, and lactate Increase in TCr after Cr ingestion (mmol/kg dm)
concentrations in muscle biopsy samples Fig. 1. A. individual values for muscle total creatine (TCr) concentra-
tion before (CI) and after (N) creatine (Cr) ingestion. Subjects have
Seconds been numbered 1-8, based on their initial muscle TCr content, which
was calculated to be equal to sum of PCr and free Cr. dm, dry matter.
0 20 60 120
B: individual increases in muscle TCr after Cr ingestion for same
Before Cr Ingestion subjects in A, plotted against change in PCr resynthesis during
recovery after Cr ingestion. Values on y-axis were calculated by
ATP 20.6 + 0.4 22.0 2 0.5 21.6 + 0.4 22.5 _+ 0.4 subtracting PCr resynthesis during 2 min of recovery before Cr
PCr 8.8 2 0.6 27.7 + 2.6 49.4 2 3.5 62.0 + 4.3 ingestion from corresponding values after Cr ingestion.
Free Cr 115.3 2 3.1 96.3 + 4.7 74.4 + 2.3 60.2 2 2.8
Total Cr 125.5 + 3.3 124.0 2 3.3 123.9 + 3.2 122.12 3.4
Lactate 96.3 2 4.3 82.0 2 7.7 70.12 5.7 49.7 f 5.6
before and after Cr ingestion. As expected, electrical
After Cr ingestion stimulation resulted in a decline in muscle ATP, almost
ATP 19.0 2 0.6 20.2 2 0.5 22.0 2 0.7 22.5 k 0.6 total degradation of muscle PCr, and marked increases
PCr 8.9 2 1.0 24.6 + 2.4 52.2 + 2.1 70.12 3.3 in muscle free Cr and lactate. All variables returned
Free Cr 134.12 3.0 118.6 +, 3.9 90.7 t 4.0 73.0 + 2.4 toward resting values during recovery. On average, Cr
Total Cr 142.9 t 2.3 143.2 + 2.4 142.8 + 2.4 143.0 + 2.2
Lactate 102.6 + 4.4 89.12 6.8 75.5 t 6.8 49.9 + 8.0 ingestion resulted in an - 15% increase in muscle total
Cr concentration. PCr resynthesis was similar during
Values are means * SE; n = 8 subjects. Muscle ATP, phosphocre- the 1st min of recovery when comparing subjects before
atine (PCr), free creatine (Cr), total Cr, and lactate concentrations
(mmol/kg dry matter) in muscle biopsy samples obtained at 0, 20, 60,
and after Cr ingestion. However, during the 2nd min,
and 120 s after intense electrically evoked isometric contraction, both the mean rate of PCr resynthesis was increased by
before and after oral Cr ingestion. -42% after Cr feeding. Despite this trend, the mean
 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION E727

Table 2. Muscle ATP, PCr, free Cr, total Cr, and lactate concentrations (mmollkg dry matter) in muscle biopsy
samples obtained from responders and nonresponders
Before Cr Supplementation, s After Cr Supplementation, s

0 20 60 120 0 20 60 120

Responders
ATP 20.12 0.2 22.2 2 0.7 20.9 t 0.4 22.2 + 0.5 18.4 iI 1.0 19.7 + 0.6 21.0 * 0.6 22.3 + 0.8
PCr 7.8 + 0.9 26.3 + 0.3 42.9 f 2.4 55.1 k 2.3 7.4 + 0.9 22.12 3.3 50.8 + 3.0 71.8 k 4.7*
Free Cr 111.6 + 4.1 93.0 * 5.4 76.4 t 4.3 62.3 + 3.8 138.7 + 2.7f 124.6 + 5.0-F 95.4 + 4.1* 74.12 4.4*
Total Cr 119.4 k 3.5 119.2 4 3.5 119.3 + 3.4 117.4 + 3.7 146.12 2.1$ 146.7 + 2.0$ 146.12 2.1$ 145.8 2 1.7$
Lactate 95.7 2 8.3 88.3 2 7.3 77.8 + 8.5 54.7 + 7.2 110.3 2 5.0* 101.8 2 5.8 85.4 AI 7.8 53.2 + 12.0
Nonresponders
ATP 21.4 + 0.7 21.6 +_ 0.8 22.5 f 0.4 23.1 f 0.5 19.8 + 0.2 20.9 t 1.0 23.2 +_ 1.2 22.9 +_ 1.0
PCr 10.2 ?.z0.1 29.7 f 5.4 58.12 4.4 73.5 + 7.6 10.6 IL 1.6 28.0 2 3.9 54.0 + 3.6 67.2 + 4.8
Free Cr 120.2 f 4.8 100.8 + 10.1 72.0 + 0.6 56.6 + 4.0 127.9 + 5.2 110.6 ~fr 3.6 84.4 f 7.8 71.1 t 0.5
Total Cr 130.4 * 4.7 130.4 + 4.9 130.0 f 5.0 130.1 t 4.9 138.6 f 4.2-f 138.6 f 4.4.f 138.4 f 4.4f 138.3 t 4.3t

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Lactate 97.12 3.5 79.7 t 13.6 59.9 + 4.2 41.3 2 5.2 92.2 * 3.2 72.12 6.8 62.5 + 9.4 44.5 + 9.3

PCr concentration was not significantly different at the muscle total Cr after oral Cr supplementation (respond-
end of recovery when comparing treatments. ers, n = 5) and those who showed only a small change
Figure lA shows the change in muscle total Cr (nonresponders, n = 3). For the sake of clarity, mean
concentration with Cr feeding for each subject. Subjects muscle PCr and free Cr concentrations during recovery
have been numbered one to eight, based on their initial in the group of responders have been plotted in Fig. 2.
muscle total Cr concentration. As can be seen, five Figure 2 shows data obtained before (open symbols) and
subjects experienced a marked 29 t 3 mmol/kg dry after (closed symbols) Cr feeding, and metabolite levels
matter (25 t 3%) increase in total Cr concentration are represented as millimoles per kilogram dry matter
(subjects l-4 and 7), which ranged from 19 to 35 and millimoles per liter intracellular water. Figure 2
mmol/kg dry matter or 15 to 32% of the initial total Cr illustrates that muscle free Cr concentration declined
content. In particular, the four subjects with the lowest during recovery on both occasions but that the free Cr
initial total Cr concentration (120 mmol/kg dry matter; concentration was higher throughout recovery after Cr
subjects l-4) experienced the most dramatic increase in ingestion. As expected, PCr resynthesis began soon after
total Cr (25-35 mmol/kg dry matter), which was equiva- exercise, and concentrations were almost identical dur-
lent to 20-32% of their initial total Cr concentration. ing the first 40 s of recovery when comparing treat-
The remaining three subjects (subjects 5, 6, and S), each ments. However, during the remainder of recovery, the
had an initial total Cr concentration of > 125 mmol/kg rate of PCr resynthesis was greater after Cr ingestion,
dry matter, and each experienced a relatively small resulting in the mean muscle concentration being 30%
increase in total Cr concentration with Cr ingestion higher at the end of recovery (P < 0.05). Table 2 also
(7-9 mmol/kg dry matter), which was equivalent to demonstrates that muscle lactate concentration was
- 5% of their initial total Cr concentration. significantly higher immediately after contraction in the
Figure 1B shows, for each subject, the increase in group of responders.
muscle total Cr after Cr ingestion plotted against the With the exception of the reported small increase in
change in PCr resynthesis during the 2 min of recovery muscle total Cr concentration, dietary Cr supplementa-
after Cr ingestion (the latter being PCr resynthesis tion had no influence on muscle metabolite concentra-
before Cr ingestion - PCr resynthesis after Cr inges- tions in the nonresponders.
tion). As can be seen, the same five subjects who
experienced a substantial increase in muscle total Cr DISCUSSION
concentration with Cr ingestion also showed an in-
creased rate of PCr resynthesis during recovery after Cr The major finding of the present experiment is that
ingestion (mean 19 t 4 mmol/kg dry matter, 35 t 6%; oral Cr ingestion markedly influences muscle Cr uptake
range 11-28 mmol/kg dry matter, 23-53%). Con- in those individuals who have a total muscle Cr concen-
versely, the three subjects who had a < 10 mmol/kg dry tration of close to or < 120 mmol/kg dry matter before
matter increase in muscle total Cr concentration with ingestion (Fig. lA), and these same individuals demon-
feeding showed very little change or even a lower rate of strate an accelerated rate of PCr resynthesis after 1 min
PCr resynthesis during recovery after Cr feeding. of recovery from intense muscular contraction (Fig. 1B).
As in Table 1, Table 2 shows mean muscle metabolite Cr ingestion has for some time been known to result
concentrations during recovery: but on this occasion in an increase in the body Cr pool in humans (7, 8, 18).
subjects have been categorized into the following two Only recently has it been shown that the ingestion of a
groups: those who demonstrated a marked increase in dose similar to that used in the present experiment can
E728 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION

strate that Cr loading is achievable in normal healthy

individuals. However, they also suggest that increases
may be most dramatic in vegetarians, who obviously will
have a low dietary Cr intake and have been shown to
have a reduced total body Cr pool (9), and in patients
with diseased or atrophied skeletal and heart muscle,
where low total Cr concentrations have been observed
(10, 22). We are unclear about the factors that will
dictate muscle Cr content under normal dietary condi-
tions. A comparison of the subjects who participated in
the present study showed no obvious differences in
lifestyle or eating habits.
As far as we are aware, the present experiment is the
first to show that an increase in muscle Cr concentra-
tion, resulting from dietary Cr supplementation, can
accelerate the rate of muscle PCr resynthesis during

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recovery from exercise. We have recently demonstrated
that a regimen of Cr ingestion similar to that used in the
present experiment can significantly increase perfor-
mance during repeated bouts of 400 and 1,000 m
running (17) and can reduce the decline in muscle
torque production seen during repeated bouts of maxi-
mal isokinetic contraction in humans (11). More re-
cently, these findings relating to exercise performance
have been confirmed by others (1). The authors showed
that Cr ingestion significantly improved work capacity
during the 4th-6th s of ten 6-s sprints when each sprint
was interspersed with 30 s of rest. The depletion of
muscle PCr stores is generally considered to be one of
the limitations to muscle force production during maxi-
mal exercise (19, 23). In the above study (ll), it was
suggested that the increase in work capacity observed
after Cr ingestion may have been attributable to the rate
of PCr resynthesis being accelerated during recovery
between exercise bouts, possibly as a result of Cr
availability displacing the equilibrium reaction cata-
Recovery (s) lyzed by creatine kinase (CK). Thus it was postulated
that, after Cr ingestion, subjects began each bout of
Fig. 2. Phosphocreatine (PCr; O,O) and free creatine (Cr; q I,~)
concentrations measured in muscle biopsy samples obtained after 0, exercise with PCr levels at a relatively higher level and
20,60, and 120 s of recovery from intense contraction before (0,~) and thereby may have delayed the depletion of muscle PCr
after (~,m) Cr ingestion. Values (means IT SE) are represented as stores during exercise. In general, PCr resynthesis
mmol/l intracellular water and mmol/kg dry matter (dm). Differences follows an exponential curve after intense muscle con-
between treatments are indicated as follows: **P < 0.01, *P < 0.05.
traction (14, 25, 26) and the half-time for resynthesis in
mixed-fibred human skeletal muscle is - 30-40 s (14). It
result in a substantial increase in muscle total Cr is generally accepted that the resynthesis of PCr during
concentration, of which -2O-30% was in the form of recovery is mediated by mitochondrial membrane-
PCr (16). In agreement with the present experiment, it bound CK, thus linking oxidative ATP production to
was found that the greatest increases were in those cytoplasmic PCr resynthesis (5, 24, 31). Factors that
individuals with the lowest prefeeding levels and that will undoubtedly influence the rate of resynthesis will
muscle total Cr concentration did not increase above include free ATP, ADP, H+, and Cr concentrations, due
- 155 mmol/kg dry matter with ingestion. Although not to their role in the CK equilibrium reaction. The in vitro
commented on at the time, the largest increases were Michaelis constant (K,) values of CK for ATP and ADP
also found in those individuals with a total muscle Cr are relatively low, being -0.6 and 1 mmol/l, respec-
concentration close to or < 120 mmol/kg dry matter. tively (3). Conversely, the K, of CK for Cr is compar-
The total Cr concentration of human skeletal muscle atively high, being close to 19 mmol/l. It is known that
has been shown to be 124.4 t 11.2 mmol/kg dry matter muscle free Cr can range in concentration from - 13
(mean -+ SD of 81 biopsy samples; see Ref. 15) and to mmol/l intracellular water at rest (16) to - 40 mmolll
follow a normal distribution ranging from - 90 to 170 intracellular water after maximal exercise (29). Thus,
mmol/kg dry matter (13). A muscle total Cr concentra- during the initial stages of recovery from maximal
tion of - 120 mmol/kg dry matter should therefore not exercise, when the rate of mitochondrial ADP rephos-
be viewed as appreciably low. The present results demon- phorylation to ATP will be at its highest, it is unlikely
 PHOSPHOCREATINE RESYNTHESIS AFTER CREATINE INGESTION E729

that the rate of PCr formation by mitochondrial CK will matter of PCr were still present in the muscle. Thus the
be dependent on the availability of free Cr, as its pattern of PCr resynthesis in our previous study (11)
concentration will be far in excess of the K, value. would have probably been more similar to that recorded
However, as PCr resynthesis continues and as the between 60 and 120 s in the present experiment.
muscle free Cr concentration declines toward 19 mmol/l, On the basis of the lactate data shown in Table 2,
it is suggested that free Cr concentration may then another explanation for the increase in exercise perfor-
begin to be a determinant of the rate of PCr resynthesis. mance could be that anaerobic glycolysis made a greater
The results of the present experiment support this contribution to ATP production during exercise after Cr
suggestion. Figure 2 demonstrates that, in those sub- ingestion ( - 10 mmol/kg dry matter). However, the
jects who responded to Cr feeding during the initial 20 s similarity in blood lactate concentrations in our previ-
of recovery, the rates of PCr resynthesis were almost ous study (11) and the lower lactate accumulation
identical when comparing values obtained before and observed after Cr ingestion by Balsom et al. (1) is not in
after Cr ingestion. This, based on the above explanation, agreement with this suggestion. In the present experi-
might be expected, as the free Cr concentration was at ment, muscle force production was not measured with
all times in excess of 27 mmol/l intracellular water. sufficient precision to enable it to be accurately related
However, as recovery proceeded beyond 60 s, it can be to lactate accumulation.

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seen that Cr feeding was associated with a higher rate of In conclusion, the results demonstrate that oral Cr
PCr resynthesis, producing a 30% higher PCr concentra- ingestion markedly increases the muscle total Cr concen-
tion at the end of recovery (P < 0.05). This, we suggest, tration of those individuals who have a concentration of
was a result of Cr ingestion maintaining the muscle free close to or < 120 mmol/kg dry matter before ingestion.
Cr concentration throughout the second half of recovery Furthermore, these same individuals show an acceler-
higher than the K, of CK for Cr (19 mmol/l) and ated rate of PCr resynthesis in the 2nd min of recovery
thereby sustaining a high flux rate through the CK from intense muscular contraction, which has depleted
reaction in favor of PCr resynthesis and ADP formation. muscle PCr stores.
The latter, in turn, will have provided mitochondria
Present addresses: K. Bodin, Dept. of Clinical Physiology, Karolin-
with substrate to maintain a high rate of ATP forma- ska Institute, Huddinge University Hospital, S-141 86 Huddinge,
tion. The role of Cr as an acceptor of mitochondrial ATP Sweden; K. Soderlund, Department of Physiology III, Karolinska
has been discussed in a series of previously published Institute, Box 5626,114 86 Stockholm, Sweden.
papers (2,5,6,24,31). Address for reprint requests: P. L. Greenhaff, Queens Medical
Center, Dept. of Physiology & Pharmacology, University Medical
The rate of muscle PCr resynthesis during recovery School, Nottingham NG7 2UH, UK.
from intense contraction has been suggested to be at
Received 22 October 1993; accepted in final form 22 December 1993.
least partly limited by intracellular pH (14, 27). How-
ever, it is unlikely that a difference in intracellular pH
between treatments could explain the faster PCr resyn- REFERENCES
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