2015912 9402 Increases+in+human+skeletal+muscle+Na%2b K%2b ATPase

Prévia do material em texto

Increases in human skeletal muscle Na+-K+-ATPase 
concentration with short-term training 
H. J. GREEN, E. R. CHIN, M. BALL-BURNETT, AND D. RANNEY 
Department of Kinesiology, University of Waterloo, Waterloo, Ontario NZL 3Gl, Canada 
Green, H. J., E. R. Chin, M. Ball-Burnett, and D. 
Ranney. Increases in human skeletal muscle Na+-K+-ATPase 
concentration with short-term training. Am. J. Physiol. 264 
(Cell Physiol. 33): C1538-C1541, 1993.-To investigate the ef- 
fect of short-term training on Na+-K+-adenosine triphos- 
phatase (ATPase) concentration in skeletal muscle and on 
plasma K+ homeostasis during exercise, 9 subjects performed 
cycle exercise for 2 h per. day for 6 consecutive days at 65% of 
maximal aerobic power (VO, max). Na+-K+-ATPase concentra- 
tion determined from biopsies obtained from the vastus lateralis 
muscle using the [ 3H] ouabain-binding technique increased 
13.6% (P < 0.05) as a result of the training (339 t 16 vs. 385 k 
19 pmol/g wet wt, means t SE). Increases in Na+-K+-ATPase 
concentration were accompanied by a small but significant in- 
crease in VO, max (3.36 & 0.16 vs. 3.58 t 0.13 l/min). The 
increase in arterialized plasma K+ concentration and plasma 
K+ content determined during continuous exercise at three dif- 
ferent intensities (60, 79, and 94% i702 max) was depressed (P < 
0.05) following training. These results indicate that not only is 
training capable of inducing an upregulation in sarcolemmal 
Na+-K+-ATPase concentration in humans, but provided that 
the exercise is of sufficient intensity and duration, the upregu- 
lation can occur within the first week of training. Moreover, our 
findings are consistent with the notion that the increase in 
Na+-K+-ATPase pump concentration attenuates the loss of K+ 
from the working muscle. 
training; skeletal muscle; potassium homeostasis 
SARCOLEMMAL Na+-K+-ATPase pump concentration in 
skeletal muscle appears to be regulated by a number of 
factors including thyroid status, caloric intake, and K+ 
deficiency (2, 3). However, the effect of physical activ- 
ity, particularly in humans, remains uncertain. The 
most compelling evidence for an effect of regular activity 
on Na+-K+ pump regulation has been obtained on adult 
rats subjected to a 6-wk program of prolonged swim 
training for up to 6 h per day, five times per week (12). 
Using vanadate-facilitated binding of [ 3H] ouabain to 
estimate the concentration of Na+-K+-ATPase (12), an 
increase of between 22 and 46% was found in selected 
locomotor muscles. With detraining, the concentration 
of Na+-K+-ATPase decreased. Training has also been 
postulated to increase Na+-K+-ATPase pump concen- 
tration in human skeletal muscle, based on cross- 
sectional differences observed between differentially 
trained older subjects and age-matched controls (13). 
However, such an effect has not been demonstrated 
longitudinally, at least in response to a moderate phys- 
ical conditioning program of lo-wk duration (10). 
Among the hypotheses that might explain these con- 
tradictory results in humans, one is most inviting, 
namely that physical activity can induce an upregula- 
tion in Na+-K+-ATPase pump concentration, provided 
that the sampled muscle groups are appropriately re- 
cruited during the training activity and that the inten- 
sity of the activity is sufficient to challenge Na+-K+ 
sarcolemmal exchange and Na+-K+ pump capacity. 
Cl538 0363-6143/93 $2.00 Copyright 0 1993 
To examine this question, we have used a training 
model consisting of 2 h of daily cycling at 60-70% of 
maximal aerobic power (VO 2 ,,,). At this intensity, elec- 
tromyographic recordings indicate extensive activation 
of the quadriceps, including the vastus lateralis (7) and, 
based on glycogen depletion patterns of fiber-specific 
types and subtypes, recruitment of 43590% of the fi- 
ber pool (8). Our results indicate that this program of 
physical activity not only increases Na+-K+-ATPase 
concentration in humans but does so during the first 
week of training. 
METHODS 
Subjects. The subjects who volunteered for this study were 
healthy male university students between 19 and 20 years of age 
[mean 19.7 t 0.2 (SE) yr]. Although all subjects were periodi- 
cally active, none were engaging in exercise on a regular basis. 
Before acceptance into the study, the subjects were fully 
informed of the experimental procedures and the associated 
risks. Official approval of the Ethics Committee, University of 
Waterloo, and written consent from each subject were obtained 
as required. 
Experimental design. To investigate the effects of training on 
VO 2 max and the changes in fluid and electrolyte balance, both a 
progressive and a prolonged cycle exercise challenge were uti- 
lized. The progressive exercise test was conducted at least 1 wk 
before the start of training and within 3 days of the last training 
session. Progressive exercise was performed to fatigue on a 
Quinton cycle ergometer beginning with 4 min of loadless ped- 
aling followed by ramp increases in power output of 15 W/min. 
VO 2 max was taken as the peak value obtained averaged over at 
least a 15-s time period. Techniques and procedures are as pre- 
viously outlined (6). The prolonged exercise test was performed 
l-2 days before training and -48 h after the last training ses- 
sion. This period of time was designed to ensure full recovery 
from the last training session. Before training, the prolonged 
exercise test was performed until fatigue or for a maximum of 60 
min at three different exercise intensities corresponding to 60, 
79, and 94% of 7j0, max, respectively. Each intensity was per- 
formed for a maximum of 20 min. With this protocol, all sub- 
jects were able to complete 20 min of exercise at each of 60 and 
79% of Vo2 max. However, no subject was able to complete 20 
min at 94% i702. The average time to fatigue was 50.6 & 1.6 
(SE) min. After the training, the subjects performed a protocol 
as nearly as identical as possible as the pretraining test with 
regard to both absolute power output and exercise time. 
For the determination of sarcolemmal Na+-K+-ATPase 
pump concentration, a muscle sample was harvested from the 
vastus lateralis muscle using the needle biopsy technique (1). 
Small pieces of tissue were extracted from each of the left and 
right legs before and after training. Care was taken to ensure 
that both the location and depth of the sampling sites were 
comparable before and after the training. Tissues were imme- 
diately frozen in liquid nitrogen and stored at -80°C until 
analysis. 
Training consisted of 6 consecutive days of cycling at -60% 
VO 2 max for 2 h/day. Where the exercise could not be performed 
continuously due to fatigue, brief rest periods were provided. 
the American Physiological Society 
TRAINING AND MUSCLE NA+-K+ ATPASE CONCENTRATION Cl539 
Normally, rest periods were only needed during the first 2-3 
days of training. Training and testing were performed at tem- 
peratures between 22 and 24°C with relative humidity between 
40 and 50%. Water only was provided during the training and 
on an ad libitum basis. 
Analytic procedures. For estimation of the changes in plasma 
volume and plasma K+, arterialized venous blood samples were 
obtained before the exercise, after the subject had been posi- 
tioned on the cycle for at least 15 min, and at 20,40, and 60 min 
of exercise. Blood was obtained without stasis from a catheter 
(Angiocath 21) inserted into a hand vein ~60 min before the 
beginning of exercise. During the sampling, the arm was ex- 
tended and positioned at a level slightly below the heart. Trip- 
licate determinations of microhematocrit were made on each 
blood sample, and the hematocrit (Hct) values were used to 
estimate the changes in plasma volume (PV) according to the 
procedures of van Beaumont (17, 18).Before calculating the 
percentage change in PV, all Hct were multiplied by a combined 
correction factor of 0.874 to adjust for trapped plasma (0.96) 
and for conversion of venous Hct to whole body Hct (0.91). 
Plasma K+ concentration was measured in duplicate with a 
flame photometer. Changes in plasma K+ content were deter- 
mined using the plasma K+ concentration and the change in PV 
according to van Beaumont et al. (18). 
Quantification of skeletal muscle Na+-K+-dependent 
ATPase concentration was made using the procedure of Nplr- 
gaard et al. (16) and Kjeldsen (9), which has been adapted to 
small tissue samples. With this procedure, two tissue samples 
from each biopsy weighing between 2 and 8 mg are prewashed 
twice for 10 min periods in a tris(hydroxymethyl)ami- 
nomethane (Tris) -sucrose buffer containing sodium metavana- 
date (NaV03) at 0°C. The buffer (pH 7.3) contained 10 mM 
Tris HCl, 3 mM MgS04, 1 mM Tris-vanadate and 250 mM 
sucrose. With the same buffer but with the addition of [3H]- 
ouabain (1.8 &i/ml) and unlabeled ouabain (1 FM final con- 
centration), the samples were then incubated two times for 60 
min at 37°C. This concentration of ouabain has previously been 
shown to produce saturable binding with similar size samples 
(16). After the unbound ouabain was removed by washing four 
times for 30 min in ice-cold buffer (see above), the samples were 
blotted, weighed, placed in 1.5-ml Eppendorf tubes, soaked in 1 
ml 5% trichloroacetic acid for 16 h at room temperature, and 
then 0.5 ml of sample was counted for 3H radioactivity in a 
scintillation mixture. All determinations of [3H]ouabain bind- 
ing capacity were multiplied by a factor of 1.05 to correct for the 
loss of specifically bound [3H]ouabain during the washout and 
the unspecific uptake and retention of [3H]ouabain (16). The 
isotopic purity of the [3H]ouabain was 99% as determined by 
the supplier (New England Nuclear-Du Pont Canada) using 
chromatographic techniques. The procedures are as published 
by Ntirgaard et al. (16). 
To determine the effect of training on Na+-K+-ATPase 
pump concentration and i’o, m8X, a Studentized t test for cor- 
related samples was used. Where serial measurements were per- 
formed as during the prolonged exercise tests, a two-way anal- 
ysis of variance for repeated measures was used. Where 
significance was found (P < 0.05), post hoc tests were appro- 
priately performed using Newman-Keuls procedures. 
RESULTS 
The short-term training program resulted in 6.5% in- 
crease (P < 0.05) in \io, m8X when expressed in liters per 
minute (Table 1). Since body weight was not altered by 
the training, a comparable and significant increase in . 
vo 2 max also occurred when expressed in milliliters per 
kilogram per minute. The increase in Vo2 max was not 
accompanied by increases in either maximal heart rate 
(HRmax) or in maximal minute ventilation (VEmaxBTPS; 
Table 1). 
Concentrations for Na+-K+-ATPase from vastus later- 
alis muscle before and after training were 339 t 16 and 
385 t 19 pmol/g wet wt, respectively. This represented an 
increase of 13.6% (P < 0.05). 
Since it has been suggested that increases in Na+-K+- 
ATPase concen tration may be important in 
the loss of K+ from the working muscles 
quently in a ttenuating the increase in plasma 
tration and content, we have also examined 
minimizing 
and conse- 
K+ concen- 
changes in 
K+ in arterialized plasma samples during a given absolute 
amount of exercise before and after training. With the 
exercise challenge used in this study, which involved 
three step increases in exercise intensity, progressive in- 
creases (P < 0.05) in K+ concentration occurred regard- 
less of the training state (Table 2). After training, plasma 
K+ concentration was persistently lower (P < 0.05). 
Since exercise and training are known to alter plasma 
volume, the change in plasma K+ concentration could 
conceivably be due to alterations in vascular fluid con- 
tent. Under the conditions of this study, changes in Hct 
reflect changes in plasma volume. As expected, exercise 
resulted in an increase (P < 0.05) in Hct both before and 
after training. Following training, Hct was depressed (P 
< 0.05) (Table 2). 
Percent changes in PV and in plasma K+ content from 
rest to exercise before and after training are provided in 
Figs. 1 and 2, respectively. For comparison, all changes 
are calculated relative to the rest values prior to training. 
On the basis of Hct changes, exercise before training 
resulted in progressive PV losses (P < 0.05) for the first 
two step increases in power output (Fig. 1). Thereafter, 
no further losses of PV were observed. At fatigue, the PV 
loss amounted to 7.3%. Training induced a 10.7% in- 
crease in resting PV. Training did not alter the percent 
PV loss during exercise. 
higher when averaged o 
untrained state. 
. 
Wl th training, however, PV was 
ver rest and exercise than in the 
in 
Large increases in K+ content occurred during exercise 
the u ntrained state (Fig. 2). Increases in K+ content 
occurred during the first two exercise stages (P < 0.05), 
ultimately plateauing at a level 23.5 % above rest. Al- 
though resting K+ concentration was not affected by 
training, K+ content was. K+ content was 10.7% higher 
at rest after training. Training blunted the increase in K+ 
content with exercise particularly during the first 20 min. 
After training, differences in K+ content were found at 20 
min of exercise (P < 0.05) but not during the final two 
steps. 
Table 1. Maximal aerobic power and 
changes before and after training 
related 
VO 2 max 
H&ax, VEmax BTPS, 
l/min ml-kg-l-min-1 beats/min l/min 
Pre 3.36t0.16 47.5t2.0 198t0.2 156t0.8 
Post 3.58t0.13* 50.2k1.2’ 189t0.2 155kO.4 
Values are means t SE; n = 9. \io, max, maximal O2 uptake; Hkax, 
maximal heart rate; ~~~~~ BTPS, maximal minute Ventilation. Pre, pre- 
training; Post, posttraining. * Significantly different from pretraining 
(P < 0.05). 
Cl540 TRAINING AND MUSCLE NA+-K+ ATPASE CONCENTRATION 
Table 2. Changes in hematocrit and plasma K+ 
concentration with exercise and training 
Time, min 
Fatigue 
0 20 40 
HGorr, % 
Pre 40.4t0.89 40.9tl.l 41.6kO.88 42.2t0.91 
\:;:*b , *ab 
post 38.0t0.84 38.9t0.95 39.8H.O 40.Otl.O 
K+, meq/l 
Pre 4.45kO.12 4.91-+0.14* 5.50+0.08*t 5.89aO.l8*t$ 
post 4.47t0.14 4.78t0.11* 5.25+0.08*t 5.55+0.12*?$ 
y-; _r:. 
Values are means t SE; n = 9-10. Hct,,,,, corrected hematocrit. For II 
HGorr and K+, main effects (P < 0.05) for both time and training were 
-10 - I I I II f 
found. For K+, an interaction effect (P c 0.05) was also found. For 
0 20 40 Fatigue 
HcLrr, pre > post; 0 min < 20 min < 40 min and fatigue. For K+, pre 
Time (mid 
> post (P < 0.05). * Significantly different from 0 min (P < 0.05). Fig. 1. Percent changes in plasma volume with exercise and training. 
t Significantly different from 20 min (P c 0.05). $ Significantly differ- Percent changes are expressed relative to the rest value prior to training. 
ent from 40 min (P < 0.05). Pre, pretraining; Post, posttraining. * Significantly different from Pre 
(PC 0.05);" significantly different from 0 min (P < 0.05); b significant- 
ly different from 20 min (P < 0.05). 
DISCUSSION 
As hypothesized, the training program utilized in this 
study, which involved 2 h of prolonged cycle exercise at 
-65% %702 max daily for 6 days, increased sarcolemmal 
Na+-K+-ATPase concentration in the vastus lateralis 
muscle by 13.6%. Although training-induced increases in 
Na+-K+-ATPase activity (14) and in Na+-K+-ATPase 
concentration (12) have previously been demonstrated in 
dogs and rats, respectively, to the authors’ knowledge this 
is the first study to demonstrate a similar effect of short- 
term training in humans. An upregulation in Na+-K+- 
ATPaseas a result of regular exercise has previously been 
suggested, at least in older trained humans, based on 
cross-sectional comparisons (13). However, in the only 
published longitudinal training study, no effect was found 
(10) 
The discrepancy between the results of this study and 
previous work that failed to demonstrate a change in 
Na+-K+-ATPase (10) probably relates to differences in 
the exercise challenge and the strain imposed on Na+-K+ 
exchange in the muscle fibers of the vastus lateralis, the 
muscle sampled for measurement of Na+-K+ pump con- 
centration. In contrast to previous work (10) that em- 
ployed a diversity of moderately intense physical activi- 
ties in male army recruits undergoing military training, 
we have used a highly specific cycle protocol known to 
substantially challenge the quadriceps muscle, including 
the biceps femoris, the rectus femoris, and the vastus 
lateralis (7). On the basis of glycogen depletion patterns, 
the prolonged cycle exercise used for training appears to 
recruit virtually all of the type I and type IIA fiber pool 
and a part of the type IIB fiber pool (8). Typically, type I 
and type IIA fibers represent 40435% of the total fiber 
pool (8). 
The measurement of Na+-K+-ATPase concentration is 
based on the vanadate-facilitated binding of [3H]ouabain 
in muscle tissue samples. Previous work-has been able to 
validate the procedure as a measurement of Na+-K+- 
ATPase concentration (9) and to demonstrate that the 
procedure can be applied to small amounts of tissue har- 
vested from human muscle (16). Our results fall within 
the range of what has been published previously using 
healthy subjects of similar age (10, 16). Increases in the 
30 - 
25 - 
-5 * 
Post 
PM 
0 20 40 
Time (min) 
Fatigue 
Fig. 2. Percent changes in K+ content with exercise and training. Per- 
cent changes are expressed relative to the rest value prior to training. 
* Significantly different from Pre (P c 0.05); a significantly different 
from 0 min (P < 0.05); b significantly different from 20 min (P < 0.05). 
Table 3. Percent change in plasma volume and K+ 
content from rest to exercise before and after training 
0 
Time, min 
20 40 
Fatigue 
PV, % 
P= 0 -1.89t1.5 -4.99t1.2 -7.30t0.99 
post 
K+ content, % 
Pre 
post 
0 -3.52k1.2 -7.16t1.4 -7.95k1.3 
0 8.38t2.8 17.2t2.7 23.5t4.4 
0 4.14t2.3 11.2t4.2 13.3t4.2 
Values are means t SE; n = 9-10; values represent changes from 0. 
A main effect (P < 0.05) for training was found for plasma K+ content 
only. Main effects for time (P < 0.05) were found for both plasma 
volume (PV; %) and K+ content (%). No interaction effects were found. 
For both PV (%) and K+ content (%) 0 min < 20 < 40 and fatigue. 
actual concentration of Na+-K+-ATPase with training is 
strongly suggested, since it has been previously demon- 
strated that the increase in Na+-K+-ATPase observed in 
swim-trained rats was not due to an increase in the af- 
finity of the receptors binding ouabain or in changes in 
unspecific uptake and retention of [3H]ouabain binding 
during incubation (12). 
TRAINING AND MUSCLE NA+-K+ ATPASE CONCENTRATION Cl541 
At present it is not clear whether muscle activity di- 
rectly induces an intracellular signal leading to the en- 
hanced expression of Na+-K+-ATPase, or whether some 
other signal that changes as a byproduct of exercise is 
involved. At present, several hormones (notably thyrox- 
ine) are known to induce an upregulation in Na+-K+- 
ATPase. The secretion of many candidate hormones 
including thyroxine are elevated with training in hu- 
mans (5) and may be implicated in the training-induced 
adaptations. 
Consistent with many previous studies [for review, see 
Lindinger and Sjprgaard (15)], we have been able to dem- 
onstrate that exercise results in a pronounced increase in 
both plasma K+ concentration and plasma K+ content. It 
is generally believed that most K+ originates from the 
working muscle as a consequence of an inadequate sar- 
colemmal Na+-K+-ATPase activity and a failure to re- 
store Na+ and K+ gradients across the sarcolemma during 
excitation (15). Increases in Na+-K+-ATPase pump con- 
centration have been viewed as a beneficial adaptation, 
necessary for reducing exercise-induced hyperkalemia 
(3). However, it has been reported that training-induced 
reductions in exercise plasma K+ concentration can occur 
in the absence of increases in Na+-K+-ATPase concen- 
tration (10). Measurements of changes in plasma K+ con- 
centration, however, without correction for possible 
changes in PV (6) do not necessarily indicate a reduction 
in K+ loss from the working muscle (4, 6). Both exercise 
and training are known to alter PV (4, 6). Similar to 
Kjeldsen et al. (11) we have also found a reduction in 
plasma K+ concentration following training. However, as 
in previous work employing this type of training model, 
we have found that PV was expanded during rest and 
exercise, a finding that could by itself lower plasma K+ 
concentration. When changes in K+ content were calcu- 
lated, it was found that plasma K+ accumulation during 
exercise was significantly blunted following training. Al- 
though many factors are undoubtedly involved in regu- 
lating plasma K+ homeostasis following training, these 
results do suggest that the loss of K+ from the working 
muscle is attenuated with training and that increases in 
Na+-K+-ATPase activity may be implicated. The fact 
that significant increases were found in Na+-I(+-ATPase 
pump concentration following training may be important 
in mediating changes in Na+-K+-ATPase activity. 
We gratefully acknowledge the technical expertise of Guy Milton. 
This study is supported by the Natural Science and Engineering 
Research Council of Canada. 
Address renrint reauests to H. J. Green. 
Received 8 July 1992; accepted in final form 3 February 1993. 
REFERENCES 
1. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
9. 
10. 
11. 
12. 
13. 
14. 
15. 
16. 
17. 
18. 
Bergstrom, J., G. Guarnieri, and E. Hultman. Carbohydrate 
metabolism and electrolyte changes in human muscle tissue during 
heavy work. J. Appl. Physiol. 30: 122-125, 1971. 
Clausen, T. Regulation of active Na+-K+ transport in skeletal 
muscle. Physiol. Reu. 66: 542-580, 1986. 
Clausen, T. Significance of Na+-K+ pump regulation in skeletal 
muscle. News Physiol. Sci. 5: 148-151, 1990. 
Convertino, V. A. Blood volume: its adaptation to endurance 
training. Med. Sci. Sports Exercise 23: 1338-1348, 1991. 
Galbo, H. Hormonal and Metabolic Adaptation to Exercise. New 
York: Thieme-Stratton, 1983, p. 5-56. 
Green, H. J., L. L. Jones, R. L. Hughson, D. C. Painter, 
and B. W. Farrance. Training-induced hypervolemia: lack of an 
effect on oxygen utilization during exercise. Med. Sci. Sports Ex- 
ercise 19: 202-206, 1987. 
Green, H. J., and A. E. Patla. Maximal aerobic power: neuro- 
muscular and metabolic considerations. Med. Sci. Sports Exercise 
24: 38-46, 1992. 
Green, H. J., D. Smith, P. Murphy, and I. Fraser. Training- 
induced alterations in muscle glycogen utilization in fibre-specific 
types during prolonged exercise. Can. J. Physiol. Pharmacol. 68: 
1372-1376, 1990. 
Kjeldsen, K. Complete quantification of the total concentration 
of rat skeletal muscle Na+-K+ dependent ATPase by measure- 
ments of [3H]ouabain binding. Biochem. J. 240: 725-730, 1986. 
Kjeldsen, K., A. Ngrgaard, and C. Hau. Human skeletal mus- 
cle Na, K-ATPase concentration quantified by 3H-ouabain bind- 
ing to intact biopsies before and after moderate physical condi- 
tioning. Int. J. Sports Med. 11: 304-307, 1990. 
Kjeldsen, K., A. Ngrgaard, and C. Hau. Exercise induced 
hyperkalemia can be reduced in human subjects by moderate 
training without change in skeletal muscle Na, K-ATPase con- 
centration. Eur. J. Clin.Invest. 20: 642-647, 1990. 
Kjeldsen, K., E. A. Richter, H. Galbo, G. Lortie, and T. 
Clausen. Training increases the concentration of [3H]ouabain 
binding sites in rat skeletal muscle. Biochim. Biophys. Acta 860: 
708-712, 1986. 
Klitgaard, H., and T. Clausen. Increased total concentration 
of Na-K pumps in vastus lateralis muscle of old trained human 
subjects. J. Appl. Physiol. 67: 2491-2494, 1989. 
Knochel, J. P., J. D. Blachley, J. H. Johnson, and N. W. 
Carter. Muscle cell electrical hyperpolarization and reduced hy- 
perkalemia in physically conditioned dogs. J. Clin. Invest. 75: 
740-745, 1985. 
Lindinger, M. I., and G. Sjggaard. Potassium regulation dur- 
ing exercise and recovery. Sports Med. 11: 382-401, 1991. 
Ngrgaard, A., K. Kjeldsen, and T. Clausen. A method for the 
determination of the total number of 3H-ouabain binding sites in 
biopsies of human skeletal muscle. Stand. J. Clin. Lab. Invest. 44: 
509-518, 1984. 
Van Beaumont, W. Evaluation of hemoconcentration from he- 
matocrit measurements. J. Appl. Physiol. 31: 712-713, 1972. 
Van Beaumont, W., J. C. Strand, J. S. Petrofsy, S. G. 
Hipskind, and J. E. Greenleaf. Changes in total plasma con- 
tent of electrolytes and proteins with maximal exercise. J. Appl. 
Physiol. 34: 102-106, 1973.