Acta physiol. scand. 1975. 93. 150-156 From the Department of Physiology, Gymnastik- och idrottshogskolan, Stockholm, Sweden

LDH Isozymes in Skeletal Muscles of Endurance and Strength Trained Athletes BY

J. KARLSSON, B. SJODIN,A. THORSTENSSON, B. HULTENand K. FRITH Received 3 1 May 1974

Abstract KARLSSON, J., B. SJODIN,A. THORSTENSSON, B. HULTBNand K. FRITH. LDH isozymes in skeletal muscles of endurance and strength trained athletes. Acta physiol. scand. 1975. 93. 150-156. Muscle biopsy samples were obtained from arm and leg muscles of endurance and strength trained athletes, respectively. Total LDH activity as well as occurrence and activity of LDH isozymes were determined. Comparing the results from the athletes with those from non-trained subjects with corresponding fibre compositions, it was found that the endurance athletes had a lower total LDH activity, a higher relative activity of the most heart-specific isozymes, LDH ( I I 2), and, on electrophoretic separation, a complete absence of LDH (4 I- 5 ) in both arm and leg muscles. As compared to the untrained material the strength trained athletes tended to have a higher total LDH activity, a similar distribution of relative isozyme activities, and, in the leg muscles, a strong electrophoretic band corresponding to LDH 5 , the most skeletal muscle specific isozyme.

Human skeletal muscle has been shown to consist of 2 major fibre types-slow twitch fibres (ST) with alkaline labile myofibrillar ATPase, and fast twitch fibres (FT) with alkaline stabile myofibrillar ATPase (Gollnick et al. 1972). Recently it was demonstrated (Karlsson et a/. 1974) that LDH (E.C. 1.1.1.27) activity (Vmax) increased with increased percentage of fast twitch fibres in humans. Along with the higher LDH activity there was a decline in the relative contribution of the more heart specific isozymes, LDH-I and LDH-2, thus indicating that the fast twitch fibres contained in greater concentration the more skeletal muscle specific LDH isozymes. As the group examined did not consist of athletes, it was felt of interest to study endurance as well as strength trained subjects according to a similar protocol.

Materials and Methods Needle muscle biopsies (Bergstrom 1962) for histological classification of muscle fibre types (Gollnick C I ul. 1972) and LDH isozyme examination were obtained from the vastus lateralis (weight lifters and long distance runners), gastrocnemius (long distance runners and cross country skiers), and deltoid muscles

150

LDH ISOZYMES IN TRAINED MUSCLES

151

TABLEI. Mean values and standard error (S.E.) for the percentage of slow twitch fibres in different muscles of long distance runners, cross country skiers and weight lifters. ~~

Fibre types

Weight lifters n=7 Long distance runners n 7 Cross country skiers n = I3 2

ST

Vastus lateralis

Gastrocnemius

Deltoideus

49k3

-

57+ 12

71 + 5

60k3

-

-

79&4

76+5

(weight lifters and cross country skiers). The athletes were aged 26 years (range 19-34 years). All of them trained and competed regularly, although not all of them o n the top elite level. The biopsy samples for L D H isozyme studies were immediately frozen in liquid nitrogen and stored at 80°C until further analyzed. After thawing, the specimens were sonificated in 0. I M Tris buffer, both in the direction pH 7.5. The homogenate was immediately analyzed for total LDH activity (iz.V,,) of lactate and pyruvate production, using methods based on Lowry and Passoneau (1964). and modified by Karlsson, Diamant and Saltin (1968). Homogenates were then stored frozen (below - 80°C) until subsequently analyzed for L D H activity using different substrates (lactate and ketobutyrate) to distinguish the fraction of the total activity attributable to L D H 1 plus L D H 2 (Rosalki and Wilkinson 1960). Thernioinactivation at 65°C for 30 min was then applied to distinguish the activity of L D H I (Strandjord and Clayson 1961, Wroblewski and Gregory 1961, and Meurman pi a/. 1964). The enzyme activity determinations were performed with an LKB 8600 Reaction Rate analyzer (LKB-Produkter AB, S-161 25 Stockholm, Sweden) operating at 340 nm and 37°C (LKB-Produkter AB, application note MLC/an-4, Smith, Brown and Taylor 1970). The LDH isozymes were also identified by means of qualitative disc-electrophoresis and subsequent staining as described by Dietz and Lubrano (1967). For this purpose 50,uI of the homogenate was placed onto columns of 7.5 percent polyacrylamide gel in 5 60 mm tubes. The electrophoresis was run for 2 h at 3 mA a tube. A second portion of the biopsy sample was prepared for histochemical analysis. Cross-sections 10 p m thick were stained for myofibrillar adenosine triphosphatase activity at p H 9.4 after preincubation a t pH 10.3 (Padykula and Herman 1955). The fibres in each sample were then identified a s slow twitch (ST) o r fast twitch (FT) as described by Gollnick e r a / . (1972). ~

I

Results The skeletal muscle fibre distribution patterns in the examined athletes were similar to those reported by Gollnick et a/. (1972), i.e. a high percentage of fast twitch fibres in the strength and a high percentage of slow twitch fibres in the endurance trained muscles, respectively (Table I). In the examined leg muscles of the weight lifters total LDH activity averaged 1.44 < and in the endurance trained 0.65 (long distance runners), and 0.56 y mol Y g-' y min-l (cross country skiers), respectively. In relation to fibre type distribution a similar pattern was observed for total LDH activity as previously described (Karlsson et al. 1974), i.e. a lower total LDH activity corresponded to a relatively higher percentage slow twitch fibres (Fig. I). The differences between the weight lifters and the endurance trained athletes were, however, larger than could be expected from only the difference in fibre composition. Compared to an unselected population (Karlsson et a/. 1974) the total LDH activity of the

152

J. KARLSSON,

B. SJODIN, A. THORSTENSSON, B.

HULTBN

AND K . FRITH

LACTATEEHYDROGENASE ACTIVITY IN MUSCLE roles. g-1. fin-’

% SLOW TWITCH FIBER Fig. I . Means 5 I S.E. of total lactate dehydrogenase (LDH)activity in skeletal muscles of the different athlete groups in relation to fibre types. The untrained subjects are identical to those presented by Gollnick c r ul. 1974, where vastus lateralis and soleus muscles were compared.

weight lifters was almost significantly higher and that of the endurance athletes significantly lower for the corresponding fibre type composition (p ~ 0 . 0 5and p ~0.01,respectively). The qualitative electrophoretic analysis of LDH isozyme patterns demonstrated bands corresponding to isozymes LDH-1, LDH-2, and LDH-3 in all the muscles investigated from the endurance trained athletes and the deltoid muscles of the weight lifters. The vastus lateralis in the weight lifters was the only muscle that appeared with a distinct band corresponding to the most skeletal muscle specific isozyme, LDH-5 (Fig. 2). This is in contrast to what was previously found in untrained healthy subjects (Karlsson et al. 1974). where LDH-5 was always present in skeletal muscle when the percentage slow twitch fibres was in the order of 40-50 percent. Not until the percentage slow twitch fibres approached 90-100 percent did the band corresponding to LDH-5disappear. This is exemplified with a sample from the soleus muscle (Fig. 3), which generally has a high percentage of slow twitch fibres (Gollnick et al. 1974b). Although there is a great variation in total LDH activity for a certain fibre composition, it has been demonstrated that the activity of certain isozyrnes, expressed as percent of total LDH activity, is linearly related to the percentage of slow twitch fibres (Karlsson et af. 1974). To examine whether a shift of the relationship between isozyme activity and fibre distribution was present in endurance and strength trained subjects, as indicated by the qualitative electrophoretic analysis (Fig. 2), the activity of LDH-(I i 2) in percent of total

153

LDH ISOZYMES IN TRAINED MUSCLES

Fig. 2. Electrophoretic separation and staining of LDH isozymes in different muscles of 4 individual athletes. ( I is a long distance runner, 2 is a cross country skier, 3 and 4 are weight lifters). The percentages denote the portion of slow twitch (ST) fibres and the figures 1-5 the position of the different LDH isozymes (LDH-Iis the most heart muscle specific and LDH-5 the most skeletal muscle specific isozyme, respectively).

activity was related to fibre type composition (Fig. 4). The leg muscle of long distance runners and the arm and leg muscles of the cross country skiers demonstrated a significantly higher relative activity of LDH-(I + 2 ) for a certain percentage of slow twitch fibres cornpared to non-conditioned subjects. The corresponding values for the arm and leg muscles of the weight lifters were similar to those of the untrained group. In absolute terms the activity of LDH-(I + 2 ) in the long distance runners averaged approximately 0.39 (leg muscles), the cross country skiers 0.46 4 mol g-' (arm muscles), and 0.44 min-I (leg muscles), respectively. The corresponding values for arm and leg muscles in the weight lifters were 0.65 and 0.63 mol g-' min-l, respectively. A

I

y

Fig. 3. L D H isozyme patterns in the deltoid, soleus and gastrocnemius muscles of an untrained subject. The percentages denote the portion of slow twitch fibres.

)I

154

1 .I I

J. KARLSSON, B. SJODIN, A . THORSTENSSON, B.

HULTCN

AND K . FRITH

v.1. gastr. &It.

;

v

70

t?

crossc. skiers

v

lo@. runners weight lifters

++ '/

t /

50-

/X/ /

-

-

-

KJ

301

y * .36X*27;

T

4

i

+lo%

/

T

t

J

40

60

80

100

% ST Fig. 4. The activity of LDH-(I - t 2 ) in percent of total LDH activity versus fibre type composition i n different muscles of the different athlete groups. The regression line (i10%) represents corresponding values from an untrained material (Karlsson et af. 1974).

The most heart specific isozyme, LDH-I, appeared in a similar pattern as LDH-(I + 2), i.e. the long distance runners and cross country skiers had higher relative activity of this isozyme. The relative contribution of LDH-1 averaged in the long distance runners 40 k6.0 (S.E.) and 45 k7.3 (S.E.) percent in the vastus lateralis and gastrocnemius muscles, respectively. The deltoid and gastrocnemius muscles of the cross country skiers averaged in this respect 71 k2.7 (S.E.) and 58 k3.9 (S.E.) percent, respectively. In the weight lifters the contribution from LDH-1 averaged 4.5 k3.9 (S.E.) (vastus lateralis) and-6.3 f2.O(S.E.) (deltoid) percent, respectively, i.e. a contribution similar to that found in untrained subjects. In absolute figures, however, the activity of LDH-I was greater in the weight lifters as compared to the normals due to the fact that the weight lifters had a higher total LDH activity.

Discussion Total L D H activity was found to decrease with an increase in percentage of slow twitch fibres, thus confirming the results of Karlsson et a/. (1974). However, the endurance trained athletes had a lower total L D H activity than an unselected population with the corresponding fibre type composition. Simultaneously the relative activity of LDH-(I +2) in percent of total LDH activity, was higher in the endurance trained subjects, suggesting that the lower total LDH activity in these athletes might be caused by a decrease in activity of the more skeletal muscle specific

LDH ISOZYMES IN TRAINED MUSCLES

155

isozymes (LDH-3, 4, and 5). This suggestion is supported by the results from the qualitative electrophoretic separations and subsequent staining of LDH isozymes. The isozyme band corresponding to LDH-5, normally present in muscles of untrained subjects with the same fibre composition, was completely lost in the endurance trained muscles. These findings are in agreement with the hypothesis that endurance training shifts the relationship between LDH isozymes to a more heart specific pattern, thus affecting and depressing total LDH activity. The rationale for such an hypothesis would be that (i) a linear relationship exists between relative area and percentage of slow twitch fibres (Gollnick e f a / . 1972), and that (ii) 5 months of endurance training increased the relative area of the slow twitch fibres (Gollnick et al. 1973). If the more heart specific isozymes LDH-1 and LDH-2 are the predominating isozymes in the slow twitch fibres as suggested by Karlsson et a / . (1974) and the relative volume of the slow twitch fibres would increase while the volume of the fast twitch fibres would decrease with endurance training, a similar LDH isozyme pattern as that deinonstrated for endurance athletes in this study should be obtained. Total L D H activity in the muscle samples from the weight lifters was higher than for a group of untrained subjects with a corresponding fibre composition. The relative activity of LDH isozymes 1 and 2 was not significantly different from that of the untrained. The electrophoretic separation of leg muscle samples showed a strong band corresponding to LDH-5, but bands were also present for the more heart specific isozymes; i.e. the isozyme picture was similar to the one for nonconditioned subjects. As basic knowledge of the effect of strength training on human skeletal muscle fibres at present is scarce, as compared to what is known about endurance training, it is more difficult to interpret the results obtained for the weight lifters. Gollnick e t a / . (1972) concluded from their study that strength training was the reason for the enlarged area of the fast twitch fibres in weight lifters. This would suggest a corresponding increase in the relative activity of the more skeletal muscle specific isozymes (e.g. LDH-5) in the strength trained muscles. This suggestion is not fully supported by the present findings. If this is due to an insufficient degree of strength training of the weight lifters or that such an expectation is basically wrong cannot be evaluated at present. In conclusion it is suggested from the present results that the lower total LDH activity in endurance athletes compared to untrained subjects is caused by a relative loss of muscle specific isozymes and a subsequent shift in isozymes to a more heart specific pattern. This in turn might be due to a specific effect of the endurance training program on the slow twitch muscle fibres. The higher total LDH activity in the strength trained athletes was not accompanied by a comparable shift in isozymes. This study was supported by grants from the Swedish Medical Research Council (875-04-4251-02) and the Research Council of the Swedish Sports Federation.

References BERGSTROM, J.. Muscle electrolytes in man. Scand. J. d i n . Lab. Inoesf. 1962. Suppl. 68. DIETZ,A. A. and T. LUBRANO, Separation and Quantitation of Lactic Dehydrogenase Isoenzymes by Disc Electrophoresis. Analyf. Biochem. 1967. 20. 246-257.

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HULTBN

AND K . FRITH

GOLLNICK, P. D., R. B. ARMSTRONG. C. W. SAUBERT IV, K. PIEHLand B. SALTIN, Enzyme activityand fiber composition in skeletal muscle of untrained and trained men. J. appl. Physiol. 1972. 33. 312-319. GOLLNICK, P. D., R. B. ARMSTRONG, B. SALTIN, C. W. SAUEERT IV, W. L.SEMBROWICH and R. E. SHEPHERD. Effect of training o n enzyme activity and fiber cornposition of human skeletal muscle. J . appl. Physiul. 1973a. 34. 107-111.

GOLLNICK, P. D., B. SJODIN,J. KAKLSSON, E. JANSSONand B. SALTIN,Human Soleus Muscle: A Comparison of Fiber Composition and Enzyme Activities with Other Leg Muscles. P/liiger.r Arch. g ~ r . Physiul. 1974. 348. 247-255.

KARLSSON, J., B. DIAMANT and B. SALTIN,Lactate dehydrogenase activity in muscle after prolonged severe exercise in man. J. appl. Physiol. 1968. 25. 88-91. KARLSSON, J., K. FRITH,B. SJODIN,P. D. GOLLNICK and B. SALTIN,Distribution of LDH isozymes in human skeletal muscle. Scand. J. d i n . Lab. Ini;esr. 1974. 33. 307-3 12. LOWRY,0. H. and J. V. PASSONEAU, The relationship between substrates and enzymes of glycolysis in brain. J. biol. Chcni. 1964. 239. 3 1-42, MEURMAN. L.. G.-R. HAGQVIST,0. NORDENFELT and R. OKDELL,Thermostable lactate dehydrogenase (LD,,) in serum in acute myocardial infarction. Norrl. Med. 1964. 7 / . 129. PADYKULA, H. and E. HERMAN, The specificity of the histochemical method of adenosine triphosphatase. J. Histocherti. Cytochcni. 1955. 3. 170-195. ROSALKI, S. and J. H. WILKINSON, Reduction of a-ketobutyrate by human serum. Naturc, (Lond.) 1960. 188. 1110-1111.

SMITH, A. F.. S. S. BROWNand R. TAYLOR, Assessment of an automatic enzyme reaction rate monitor. Clin. d i m . Acta 1970. 30. 105-1 13. STRANUJORD, P. E. and K. 1. CLAYSON, The diagnosis of acute myocardial infarction on the basis of heatstable serum lactic dehydrogenase. J . Lab. d i n . M d . 1961. 58. 962. F.I ,and K. F. GREGORY, Lactic dehydrogenase isozymes and their distribution in normal WR~ELEWSK tissue and plasma and in disease states. Ann. N . Y . Arad. Sci. 1961. 94. 912-932.

LDH isozymes in skeletal muscles of endurance and strength trained athletes.

Muscle biopsy samples were obtained from arm and leg muscles of endurance and strength trained athletes, respectively. Total LDH activity as well as o...
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