Summary. The glycogen depletion pattern in human muscle fibers was followed throughout the course of prolonged exercise at a work load requiring 67% of the.
Pflfigers Arch. 344, 1--12 (1973) 9 by Springer-Verlag 1973
Glycogen Depletion Patterns in Human Skeletal Muscle Fibers during Prolonged Work P. D. Gollnick, R. B. A r m s t r o n g * , C. W . S a u b e r t IV, W. L. Sembrowich, R. E. Shepherd, a n d B. Saltin** Department of Physical Education for Men, Washington State University Pullman, Washington, U.S.A. Received April 11, 1973
Summary. The glycogen depletion pattern in human muscle fibers was followed throughout the course of prolonged exercise at a work load requiring 67% of the subjects' maximal aerobic power. Biopsy samples were taken from the vastus lateralis muscle at rest and after 20, 60, 120, and 180 (or when unable to continue at the prescribed load) rain of exercise. Muscle fibers were identified as fast twitch (FT) or slow twitch (ST) on the basis of myofibrillar ATPase activity. The glycogen content of muscle samples was determined biochcmically. At the end of the exercise total muscle glycogen content was very low. Glycogen was also estimated in the fibers with the PAS stain. ST fibers were the first to become depleted of their glycogen but as the exercise progressed, the FT fibers were also depleted. These data may suggest a preferential utilization of ST fibers during prolonged, intense exercise, with a secondary recruitment of FT oeeuring as the ST fibers became depleted of their glycogen stores. Key words: Slow Muscle Fibers -- Fast Muscle Fibers -- Plasma F F A -- Blood Lactate -- Metabolism during Exercise -- Fiber Recruitment -- Fiber Types. Muscle g l y c o g e n declines p r o g r e s s i v e l y d u r i n g bicycle exercise r e q u i r i n g 60--700/0 of m a x i m a l o x y g e n u p t a k e u n t i l it is c o m p l e t e l y d e p l e t e d a t e x h a u s t i o n [5,14]. F r o m d a t a r e l a t i n g t h e force e x e r t e d on t h e bicycle p e d a l s to t h e r e l a t i v e m e t a b o l i c r a t e [15], it w o u l d a p p e a r t h a t o n l y 1 0 - - 2 0 ~ of m a x i m a l v o l u n t a r y contractile s t r e n g t h is r e q u i r e d to s u s t a i n such exercise. This implies t h a t this w o r k can be a c c o m p l i s h e d b y t h e u t i l i z a t i o n of o n l y a small p e r c e n t a g e of t h e m o t o r units in t h e w o r k i n g muscles. H o w e v e r , t h e c o m p l e t e d e p l e t i o n of muscle glycogen t h a t occurs dm'ing p r o l o n g e d e x h a u s t i v e w o r k suggests t h a t all muscle fibers are used a t some t i m e d u r i n g t h e exercise. T h e r e l a t i v e glycogen c o n t e n t of muscle fibers can be e s t i m a t e d h i s t o c h e m i c a l l y w i t h t h e periodic acid Schiff's (PAS) reaction. This m e t h o d has been used as a * A National Science Foundation predoctoral fellow. ** Present address: Department of Physiology, Gynmastik- och idrottshSgskolan, Stockholm, Sweden. I
Pflfigers Arch., Vol. 344
2
P.D. Gollnick et al.
m e t h o d for identifying previously active muscle fibers [19]. Stein a n d P a d y k u l a [26] also suggested t h a t it would be worthwhile to follow glycogen depletion i n a biochemical a n d histochemical m a n n e r to determine whether a preferential depletion occurs i n the fibers of skeletal muscle during contraction. Using these techniques a differential glycogen depletion has been observed i n h u m a n skeletal muscle after 30 m i n of bicycle work t h a t required 74~ of the s u b j e c t ' s m a x i m a l oxygen uptake [13]. I n the p r e s e n t s t u d y a n a t t e m p t has been made to use the PAS stain to follow the glycogen depletion p a t t e r n i n h u m a n muscle fibers from rest to e x h a u s t i o n d u r i n g m o d e r a t e l y intense bicycle exercise.
Materi~Is and Methods Ten men between the ages of 24 and 4~0 years were studied. Six subjects had completed a training program on the bicycle ergometer whereas 4 had little experience with bicycle exercise. The subjects reported to the laboratory in the morning or early afternoon after a light (uncontrolled) meal and rested in the supine position for at least 30 min. During this period heart rate was taken and oxygen uptake (17o2) determined from expired air collected in a 6001 wet spirometer. A sample was taken from the vastus lateralis muscle with the needle biopsy technique [4]. A catheter was inserted into the anticubital vein and a blood sample taken. The body weight of the subjects was recorded before and after the exercise. Each subject then exercised on a bicycle ergometer with 60 rpm at a work load that required approximately 650/0 of his maximal aerobic power (bicycle test). The objective was for each subject to work at this load for 3 h or until unable to continue. The subjects were encouraged to drink water throughout the work period. With this procedure an average weight loss of only 1 kg occurred during the work. After 20, 60, and 120 min the work was interrupted (< 15 sec) and a biopsy sample taken from the vastus lateralis muscle. Expired air was collected and heart rate and blood samples were taken in the 2-rain interval prior to the muscle biopsy. Five subjects, all of whom had trained on the bicycle ergometer, completed the 3 h of exercise. The remaining subjects exercised from 100 to 120 rain. Sampling procedures as described above were performed at the termination of the exercise. The concentrations of 02 and COswere determined in expired air with a Beckman E2 oxygen analyzer with the method described by Coreoran and Brengelmann [7]. The validity of this procedure was verified each day by analysis of the expired air with the micro Scholander technique. In all eases there was no difference between the results obtained by the two methods. Heart rate was determined by palpation of the carotid artery. Glucose (Glueostar, Worthington Biochemical Corp., Freehold, N. J.) determination were made on whole blood. An 0.05 ml sample of blood was pipetted into 4.0 ml of ice-cold 0.6 N HCI04 immediately after the sample was drawn. This sample was centrifuged and a perchlorate-free extract prepared by the addition of 0.6 ml of ice-cold 1 3s KaPO ~ to 3.0 ml of the supernatant fluid. The tubes were placed in an ice bath for at least 10 rain and the KCIO4 removed by eentrifugation. A 1.0 ml aliquot of the supernatant fluid was analyzed for lactate with the StrSm [27] modification of the Barker and Summerson technique. The deproteinization of the blood with HCIO4 and the preparation of a perchlorate-free extract of blood produced lower blanks against water (mean 0.08 O.D., range 0.065 to 0.090) than normally obtained with the use of triehloroacetie acid.
Human Mnscle Fiber Glycogen Depletion during Work The remaining whole blood was centrifuged and plasma free fatty acids (FFA) determined with the method of Dole and Meinertz ~8] using Nile Blue A as the titration indicator. The muscle samples were immediately examined under a dissecting microscope to determine fiber orientation, mounted on specimen holders in OCT embedding medium (Ames Tissue-Tek), and frozen in isopentane cooled in liquid nitrogen. Serial sections 10 ix thick were cut in a cryostat at --20~ and mounted on cover glasses for histochemical analysis. Myofibrillar adenosine triphosphatase (ATPase) and reduced diphosphopyridine nucleotide-diaphorase (DPNH-diaphorase) activities were estimated as described by Padykula and Herman [22] and Novikoff and co-workers [20], respectively. The distribution of glycogen in a serial section 16 ix thick was estimated with the PAS reaction r23]. The tissue remaining after histochemical sections were cut was removed from the embedding medium and analyzed for total glycogen [18]. Less than 2 rain elapsed between removal of the sample from the muscle and freezing it. This short delay should not have influenced the results of this study since Hultman [16] has shown that no appreciable decline in glycogen content occurred in muscle samples that had been left at room temperature for up to 10 min. Muscle fibers were assigned to two major types on the basis of myofibrillar ATPase activity. Although fibers with low and high myofibrillar ATPase activities have been called type I and type II fibers [9], respectively, we have used the terminology of slow twitch (ST) and fast twitch (FT) fibers [12,13]. This is based on the finding that animal muscles with low and high myofibrillar ATPase activities, as determined with assays similar to that of this study, have slow and fast eontractiIe properties, respectively [3]. Furthermore, human seleus muscle has slow contractile characteristics [6] and it is composed predominately of fibers with low myofibrillar ATPase activity (unpublished data). The stability of contractile properties [10] and of myosin ATPase activity of skeletal muscle following training [1,10,12] also makes the use of myofibrillar ATPase activity an objective method for classifing fibers. In contrast, the oxidative capacity and histoehemical staining intensity for it are readily modified by training [10,12]. The relative distribution of glycogen in the muscle fibers was estimated from the intensity of the PAS stain as evaluated under the light microscope. Fibers were classified as PAS dark, moderate, light, or negative as previously described [13]. This is similar to the system used by t(ugelberg and Edstr6m [19], who also demonstrated that histochemically observed changes in glycogen content or phosphorylase activity are sufficiently distinct and constant to serve as markers of previous activity of muscle fibers. The muscle samples were evaluated in a random manner. Evaluation of samples by different people has produced similar results thereby demonstrating that it is an objective method. Statistical significance was determined using the t-test for paired samples.
Results Mean ~o~ a n d h e a r t rate a t rest were 0.33 1 a n d 64 b x rain -1, respectively. After 20 rain of exercise these values were 2.75 (range 1.63 to 3.64) 1 a n d 159 b • m i n -1 (P < 0.01), respectively. The Po2 a t this p o i n t represented 64~ (range 58--720/0 ) of the subjects' l?o~ m a x (Fig. 1). Thereafter a significant (P < 0.01) rise in l?o~ occurred to the t e r m i n a t i o n of work. This was also true for h e a r t rate. F o r the entire e x p e r i m e n t the L*
4
P, D. Gollnick et al, HEART RATE blmIn 200 180 160 140 i20 I00 80
~
% . MAX
v~
-V
60
0
20
120
60 TIME
IN
180
MINUTES
Fig. 1. I t e a r t rate and R-value a t rest a n d during exercise and percent of 17o2 m a x during exercise. Values are means • 1 SD, The final values are from only 5 of the 10 subjects MUSCLE GLYCOGEN 200
- L
-~ 160
9 TRAINED .
U.TRA,NE
=~ lao
r o
so
r E E
40
0
I 0
I 20
I 60 TIME
I 120 IN
I 180
MINUTES
Fig. 2. Glycogen depletion in the biopsy samples from ~he vastus lateralis muscle during t h e course of the exercise
Human Muscle Fiber Glycogen Depletion during Work 17o2 during the exercise represented 67~ (range 57--770/0) of the I?os max of the subjects. The average respiratory exchange ratio (R) at rest was 0.82. This rose to 0.94 (range 0.85 to 0.97) (P < 0.01) after 20 min of exercise. The 1% values progressively declined throughout the remainder of the work to 0.89 after 2 h of exercise (P < 0.01) and to a final mean of 0.85 for the five subjects who completed 180 rain of exercise (Fig. 1). The initial glycogen concentration in the muscle of the untrained and trained groups was 96 and 182 mmoles of glucose units • kg -1 wet weight, respectively (Fig. 2). Glycogen values declined in a parallel manner for the two groups to final concentrations of 11 and 34 mmoles of glucose units • kg -1, respectively, for the untrained and trained groups at the termination of exercise. As shown in Fig. 2, the glycogen remaining in the muscles of the trained subjects (I05 mmoles of glucose units • kg -1) after 1 h of exercise exceeded the pre-exereise level for the untrained subjects. The inability of any of the untrained subjects to work beyond 2 h was probably related to their lower initial muscle glycogen content [5]. The pattern of glycogen depletion in the ST and FT fibers of the vastus lateralis muscle at the different sampling times during the exercise is summarized in Fig. 3. These data were divided into untrained and trained groups since marked differences existed in initial muscle glycogen content of the two groups. At rest all of the fibers in the muscle samples from the trained group stained dark for glycogen by the PAS reaction. In contrast, only 93 ~ of the F T fibers and 86 ~ of the ST fibers of the untrained subjects were rated as dark, with 7o/0 of the FT and 12~ of the ST fibers rated moderate, and 20/0 of the ST fibers light. The large difference in initial glycogen content of the two groups was evident from PAS staining but it could not be evaluated quantitatively by this method. After 20 rain of exercise a significant change in PAS staining occurred in the muscle samples from the untrained subjects. The percent of darkstained ST fibers declined to 1 ~ with 64~ moderate, 32 ~ light, and 3 ~ being negative. For the F T fibers these values were 30, 69, and 1 ~ for the dark-, moderate-, and light-stained fibers, respectively. No F T fibers were PAS negative after 20 min of exercise. After 60 rain of work, 980/0 of the ST fibers of the untrained subjects were PAS negative and the remaining 20/0 were light. In contrast, 260/0 of the F T fibers were rated as dark, 11 ~ moderate, 40/0 light, and 59 ~ negative. At exhaustion (2 h) the pattern for glycogen depletion in ST fibers of the untrained subjects was essentially unchanged whereas a further depletion in glycogen had occurred in the F T fibers. For these latter fibers 86 ~ were PAS negative at exhaustion but there were still some fibers that were rated as dark (20/0) or moderate (70/0).
P. I). Gollnick et al. 0
20
40
60
I00
80
I
WORK TIME-
MLN
0
iiii
83-FT 69-ST! 269 - FT 154-ST
~ii
~
iiiiiii i ~ ;.
i
iili~i]I --~_ I 1
,,,1111,111:11--4
212 - FT ~ "159 - ST ~
[
I TRAINED
20
60
120
(6)
477-FT 536-ST 238-FT [50-ST
20
251 - FT 139 - S T
60
!NiiiiiNIiiIiiii@ii}H%!Niiiiliiiiii~
'373 - FT 207 - S T I ~ l ~ - .
~
277 - F T 255 -ST
.....,,,~r
- :~=
......~ -
,
0
I 1
=~@--~--'Y~
I
20
i
I
-~
I
40
,
[ I
60
,
I
80
,
120
180
I
I00
t~ig. 3. The peroentage of I~'Tand ST fibers ro~ted as ~PASdark l , moderate IN, light [], or negative [] for glycogen at rest and throughout the exercise. The total number of fibers is given in the left column. The values for the trained subjects after 180 min of work are from 5 subjects
There was no change in the PAS staining of either fiber type in the samples t a k e n Mter 20 rain from the trained subjects. After 60 rain of exercise 4 ~ of the ST fibers were PAS negative, 30 ~ light, 52 ~ moderate, and 14~ dark. For the F T fibers, 85.50/0 were r a t e d dark, 10~ moderate, 4~ light, and 0.50/0 negative. I n the samples t a k e n after 2 h of work 45 ~ of the ST fibers were PAS negative, 44 ~ light, 9 ~ moderate, and 20/0 dark. I n contrast, 63 ~ of the F T fibers were still dark after 2 h of exercise, 280/0 were moderate, 60/0 light, and 30/0 were PAS negative. Following 3 h of exercise 96.50/0 of the ST fibers were PAS negative and only 3.5o/0 light. This p a t t e r n was similar to t h a t of the untrained group at exhaustion. For the F T fibers, however, only 480/0 were PAS negative and there were still a significant n u m b e r of fibers stained dark (23 ~ ) and m o d e r a t e (16~
. . . .
'\
Fig.4. Photomicrographs (• 130) of the vast,us lateralis muscle from one subject showing the glycogen concentration in the ST and F T fibers at rest (A) and after 60 (B), 120 (C), and 180 (D) mh~ of exercise. The number 1 series of micrographs are for myofibrillar ATPa.se activity showing the dark-(FT) and light-(ST) stained fbers. The number 2 series of mierographs contains the PAS stain for glycogen. The fibers were rated as dark, moderate, light, or negative (see text). A summary of this ra~ing is contained in Fig. 3
P. D. Gollniek et al. PLASMA FFA #Eq/ml 1,6 1.4. I.E 1.0 0.8 0.6 0.4. 0.2 BLOOD LACTATE mM
r 2
BLOOD GLUCOSE mM 6 5 4
0
I ~-0
I 60 TIME
I L2O IN
l IBO
MINUTES
Fig. 5. Plasma FFA, blood lactate, and blood glucose at rest and during the course of the exercise. Points represent means • 1 SD. The last points are from 5 of the 10 subjects
Fig. 4 is a set of micrographs showing the glycogen depletion p a t t e r n in the ST a n d F T fibers from rest to 180 min of exercise in one trained subieet. The lactate concentration in venous blood at rest averaged 2.2 mM (Fig.5). This rose to 4.5 mM (P < 0.01) after 20 rain of exercise and thereafter declined slightly to a final value of 3.8 mM. The m e a n blood glucose and plasma F F A concentrations at rest were 4.7 mM and 0.6 ~ E q • m1-1, respectively. After the first 20 min of exercise blood glucose declined to 3.3 mM (P < 0.01) whereas plasma F F A was essentially unchanged. There were no significant changes in blood glucose from
Human Muscle Fiber Glycogen Depletion during Work the 20th min to the termination of the work. However, plasma FFA rose steadily to a final concentration of 1.48 ~Eq • m1-1 (P < 0.01). There were no differences in the response of the trained and untrained subjects regarding changes in blood lactate and glucose or plasma FFA during exercise. Discussion
The constant decline in muscle glycogen during exercise and similar rates of glycogen utilization by trained and untrained subjects working at the same relative metabolic rate as observed in this study have been demonstrated previously [5,14]. However, it should be noted that although both groups worked at the same relative work load the trained subjects worked at higher absolute loads. The new finding of this study was the differential glycogen loss in the muscle fibers throughout the exercise. This was characterized by an initial glycogen depletion in some ST fibers followed by additional ST fibers, and finally a sequential glycogen loss from FT fibers. One interpretation of these data is that those fibers losing PAS staining first were recruited initially and as they became depleted of glycogen their metabolic rate, and therefore ability to develop tension, was impaired to a point where additional motor units had to be activated to sustain the work. This interpretation would be consistent with the concept that the muscular force developed during bicycle exercise at the metabolic rate used in this study requires only a small percentage of the motor units in the muscles involved. From this standpoint expression of work loads in terms of percent of ~o2 max may be misleading since it relates to the capacity of the oxygen transport system rather than to the ability of the skeletal muscles to develop tension. One requisite for the above interpretation is that glycogen resynthesis during exercise did not mask the glycogen depletion pattern. This assumption seems reasonable since maximal rates of glycogen synthesis are only about 1 ~ of glycogenolysis [16] and a reciprocal relationship exists between activation and inhibition of the enzymes glycogen phosphorylase and synthesis [25]. A second basis for the above interpretation is that when all muscle fibers are activated, as during electricM stimulation, those fibers with the lowest oxidative capacity become PAS negative first [10,19]. From histochemieal data the ST fibers of human skeletal muscle appear to have 2 to 3 times the oxidative capacity of the F T fibers. Baldwin et al. [2] have reported a 5-fold difference in the oxidative capacity of red and white rat skeletal muscle. The greater number of capillaries [24] and thus blood flow to oxidative fibers would favor the uptake and oxidation of blood glucose and FFA by these fibers and accentuate this difference. This would have a glycogen sparing effect in
10
P.D. Gollnick st al.
those fibers which were the first to lose glycogen. The greater consumption of A T P per unit of contractile force by fast contracting muscles [11] also points to a more economical use of the body's resources by use of ST muscle fibers. Another interpretation of the glycogen depletion pattern observed in this study is t h a t all fibers were active and the PAS staining pattern was caused by a major difference in glycogen content of the two fiber types. This would be consistent with reports t h a t ST (type I) fibers of human skeletal muscle have less glycogen than F T (type II) fibers [9]. Although we [12] have also seen differences in the PAS staining intensity of ST and F T fibers when the glycogen content of h u m a n skeletal muscle was relatively low, there were no major or consistent differences in the PAS staining of the fibers from either the trained or untrained subjects. The overall staining intensity of the muscles from the trained group was higher and reflected the elevated muscle glycogen content of this group. Differences in the glycogen content and depletion rate of the two major fiber types could have existed since the PAS staining technique is only semiquantitative. However, it is unlikely t h a t a large enough difference in the glycogen content existed between the two fiber types to support the differences in glycogen consumption if both had worked at identical rates at the same time. The fact t h a t there were some PAS dark fibers in some subjects after 3 h of work suggests t h a t these fibers had not been active. These were always F T fibers. One explanation t h a t can accomodate the activation of both fiber types throughout the exercise would be an asynchronous activation of motor units with a very intermittent use of those containing FT fibers. Thus, the FT fibers might be activated for only short intervals at the point(s) of peak tension. The high values for glycogen content of the skeletal muscle of the trained subjects made it impossible to detect any change in PAS staining of this group after the first 20 min of exercise. The fact that a considerable decline in glycogen m a y be necessary before any difference in PAS staining becomes apparent is a major limitation of this method for detecting previously activity in muscle fibers. I t is important to note, however, t h a t after 1 h of work the average glycogen content in the muscle of the trained subjects was 105 mmoles of glucose units • kg -1 wet weight. This is above the limit where the PAS technique can detect differences between the glycogen content of h u m a n muscle fibers when the glycogen is uniformly distributed among the fibers [13]. However, at this point there were major changes in the PAS staining intensity of the ST fibers. From this point to the point of exhaustion changes in the PAS staining intensity of the muscle samples of the two groups were similar.
Human Muscle Fiber Glycogen Depletion during Work
11
The FT fibers of human skeletal muscle generally have higher glycolytic and lower oxidative capacity as judged histochemically than ST fibers [9,12]. However, a spectrum of oxidative capacities exists within each fiber type and a third fiber type has been identified on the basis of oxidative capacity [21]. To determine whether differences in glycogen depletion existed for the FT fibers with relatively high oxidative capacity they were identified and examined after the various work times. I n one of the ten subjects the high-oxidative FT fibers became PAS negative before those with lower oxidative capacity. I n another subject the highoxidative FT fibers had the highest PAS staining intensity at the termination of exercise. Both subjects were trained and had completed 3 h of exercise. Thus, it does not seem possible to distinguish these fibers from their glycogen depletion rate during exercise. I n summary, the present data suggest that a preferential pattern of fiber use exists in human muscle during prolonged exercise. However, this is based on the PAS staining technique for glycogen disappearance. This method is indirect and additional experiments are needed to fully establish whether or not fibers are active under different exercise conditions. The present data do, however, firmly establish that differential rates of glycogen depletion occur in the fibers of human skeletal muscle during exercise.
References 1. Bagby, G. B., Sembrowich, W. L., Gollnick, P. D.: Myosin ATPase and fiber composition from trained and untrained rat skeletal muscle. Amer. J. Physiol. 223, 1415--1417 (1972) 2. Baldwin, K. M., Klinkerfuss, G. tt., Terjung, R. L., Mol~, P. A., Holloszy, J. O. : Respiratory capacity of white, red, and intermediate muscle: adaptive response to exercise. Amer. J. Physiol. 222, 373--378 (1972) 3. Barnard, 1%.J., Edgerton, V. R., Furukawa, T., Peter, J. B. : Histochemicu], biochemical, and contractile properties of red, white, and intermediate fibers. Amer. J. Physiol. 220, 410--415 (1971) 4. Bergstrhm, J.: Muscle electrolytes in man. Scand. J. clin. Lab. Invest. 14, Suppl. 68 (1962) 5. Bergstrhm, J., Hermansen, L., Hultman, E., Saltin, B. : Diet, muscle glycogen and physical performance. Acta physiol, scand. 71, 140--150 (1967) 6. Buchthal, F., Dahl, K., Rosenfalck, P. : Rise time of the spike in fast and slowly contracting muscle of man. Acta physiol, scand. 87, 261--269 (1973) 7. Corcoran, P. J., Brengelmann, G. L. : Oxygen uptake in normal and handicapped
subjects, in relation to speed of walking beside velocity-controlled cart. Arch. Phys. Med. 1%ehab.51, 78--87 (1970) 8. Dole, V. F., Meinertz, It.: IViicrodetermination of long-chain fatty acids in plasma and tissues. J. biol. Chem. 235, 2595--2599 (1960) 9. Dubowitz, V., Pearse, A. G. E. : A comparative histochemica] study of oxidative enzymes and phosphorylase activity in skeletal muscle. Histochemie 2, 105--117 (1960)
12
P.D. GoIlnick et al.
10. Edgerton, V. R., Barnard, R. J., Peter, J. B., Gillespie, C. A., Simpson, D. R. : Overloaded skeletal muscles of a nonhuman primate (Galago senegalensis). Exp. Neurol. 37, 322--339 (1972) 11. Goldspink, G., Larson, R. E., Davies, R. E. : The immediate energy supply and the cost of maintenance of isometric tension for different muscles in the hamster. Z. vergl. Physiol. 66, 389--397 (1970) 12. Gollnick, P. D., Armstrong, 1~. B., Saltin, B., Saubert, C. W., IV, Sembrowich, W. L., ShePherd, R. E. : Effect of training on enzyme activity and fiber composition of human skeletal muscle. J. appl. Physiol. 34, 107--111 (1973) 13. Gollnick, P. D., Piehl, K., Sanbert, C. W., IV, Armstrong, R. B., Saltin, B.: Diet, exercise, and glycogen changes in human muscle fibers. J. appl. Physiol. 33, 421--425 (1972) 14. Hermansen, L., Hultman, E., Sa]tin, B.: Muscle glycogen during prolonged severe exercise. Acta physiol, seand. 71, 129--139 (1967) 15. Hoes, H. J. A. J.M., Binkhorst, R.A., Smeekes-Kuyl, A. E. M.C., Vissers, A. C. A. : Measurements of forces exerted on pedal and crank during work on a bicycle ergometer at different loads. Intern. Z. angew. Physiol. 20, 33--42 (1968) 16. Hultman, E. : Muscle glycogen in man determined in needle biopsy specimens. Methods and normal values. Scand. J. elin. Lab. Invest. 19, 209--217 (1967) 17. Hultman, E., BergstrSm, J., l~och-Norhnd, A. E. : Glycogen storage in human skeletal muscle. In: Muscle Metabolism during Exercise, pp. 273--288. New York: Plenum Press 1971 18. Karlsson, J., Diamant, B., Saltin, B. : Muscle metabolites during submaximal and maximal exercise in man. Scand. J. clin. Lab. Invest. 26, 385--394 (1971) 19. Kugelberg, E., EdstrSm, L.: Differential effects of muscle contractions on phosphorylase and glycogen in various types of fibers: relation to fatigue. J. Ncurol. Neurosurg. Psyehiat. 31, 415--423 (1968) 20. Novikoff, A. B., Shin, W., Drueker, J. : Mitoehondrial localization of oxidation enzymes: staining results with two tetrazolinm salts. J. biophys, bioehem. Cyto]. 9, 47--61 (1961) 21. Ogata, T., Murata, F.: Cytological features of three fiber types in human striated muscle. Tohoku J. exp. Med. 99, 225--245 (1969) 22. Padyku]a, H. A., Herman, E. : The specificity of the histochemieal method of adenosine triphosphatase. J. Histochem. Cytoehem. 3, 170--195 (1955) 23. Pearse, A. G. E. : Histoehemistry--Theoretical and Applied. Boston: Little Brown Comp., appendix 9, p. 832 (1961) 24. Romanul, C. A. F. : Capillary supply and metabolism of muscle fibers. Arch. ~eUrol. (Chic.) 12, 497--509 (1965) 25. Staneloni, 1%., Piras, R.: Changes in glycogen synthetase and phosphorylase during muscular contraction. Biochem. biophys. Res. Commun. 36, 1032--1038 (1969) 26. Stein, J. M., Padykula, H. A.: ttistochemical classification of individual skeletal muscle fibers of the rat. Amer. J. Anat. ll0, 103--123 (1962) 27. StrSm, G. : The influence of anoxia on lactate utilization in man after prolonged muscular work. Acta physiol, scand. 17, 440--451 (1949) Philip D. Gollnick, Ph.D. Department of Physiology Gymnastik- och idrottshSgskolan Liding5v~gen 1 114 33 Stockholm/Sweden
