Acta physiol. scand. 1978. 103. 40-46 F r o m the L a b o r a t o r y for Human Performance, National Defence Research Institute, Stockholm,
Sweden
Relationship between lactate accumulation, LDH activity, LDH isozyme and fibre type distribution in human skeletal muscle BY
PERTESCH, BERTIL SJODINand JAN KARLSSON Received 31 October 1977
Abstract TESCH,P.. B. SJODINand .I. KARLSSON.Relaiioriship betweerr lactate accumulniiori, L D H crciii~it,v.L D H isoqrne arid fibre t j p e disrribrrtioti itr hiirnati skeletal muscle. Acta physiol. scand. 1978. 103. 40-46. Lactate concentration, total L D H activity a n d muscle-specific L D H isozymes were determined in pools of the ~ M Oniain types of human skeletal muscle fibres. Analyses were made from biopsy specimens obtained after intense d ) namic exercise lasting approximately 30 s. Lactate Concentration, total L D H activity and muxlc-specific L D H activity displayed higher average values for FT (fast twitch) fihres than for ST (slow tibitch) fibres. In addition. positive correlations were found both between the individual percentage of FT fibres and muscle lactate concentration a n d between lactate concentration and total L D H activity and muicle-specific L D H actib ity respecti\ely.
High lactate levels have been demonstrated both after exhaustive isometric exercise (Ahlborg ei ul. 1972, Karlsson and Ollander 1972) and maximal dynamic exercise (Karlsson 1971,
Knuttgen and Saltin 1972) of brief duration and have been suggested as one possible factor contributing to muscle fatigue in maximal exercise of brief duration. When the lactate concentration in different parts of a muscle sample were analysed after maximal exercise, values in the contracting elements were found to be 3-5 times greater than in elements supposed to be non-contracting (Karlsson 1971). Therefore, it may be assumed that t h e concentration of metabolites found in whole muscle biopsy specimens does not always reflect the true state of lactate accumulation in exercised muscle. Essen and Hiiggrnark (1975) reported a greater accuinulation of lactate in type 11 (fast twitch, FT) muscle fibres than in type I (slow twitch. ST) muscle fibres after exhaustive isometric exercise. Recent studies of lactate accumulation in different single muscle fibres have indicated that the fibre type distribution of a muscle is important to lactate accumulation in single muscle fibres (Tesch and Karlsson 1977). In other words, a muscle rich in FT fibres displals a greater potential for accumulating lactate than a muscle rich in ST fibres. This difference may be due to differences in the activity of the glycogenolytic system’s regulating
40
LACTATE AND LDH IN HUMAN SKELETAL MUSCLE
41
enzymes, i.e. phosphofructokinase (E.C.2.7.1.11.), glycogen phosphorylase (E.C.2.4.1. I .) and lactate dehydrogenase ((LDH) E.C.1.1.1.27.), in the two main muscle fibre types (Essen ef a / . 1975, Sjodin 1976, Thorstensson 1976). Thus, both LDH activity and the LDH isozyme distribution pattern in an untrained or moderately trained human population were found to be related to the percentage of FT fibres in the muscle (Karlsson et al. 1974). A muscle rich in F T fibres displayed greater LDH activity and a larger contribution of muscle-specific LDH (M-LDH) than a muscle rich in ST fibres in which the heart-specific type of LDH (H-LDH) was predominant. The intracellular distribution of these two main LDH isozyrnes and their kinetic properties differ (for ref. see Sjodin 1976). In normal conditions, the M-LDH isozyrnes are mainly associated with membranes, such as the sarcoplasmic reticulum. On the other hand, H-LDH fractions have been found in the inner membrane of the mitochondria. The aim of the present investigation was to study lactate accumulation in-the muscles of individuals differing with respect to certain relevant muscle qualities. Therefore, muscle lactate concentration was determined after intense, standardized exercise of brief duration and related to individual muscle fibre type distribution and LDH characteristics.
Subjects, Procedure and Methods 10 male physical education students served as subjects. Their mean ( k S . E . ) age, height and weight were 2 4 i 1 yrs, 1 7 9 k 1 cm and 71.312.1 kg respectively. Muscle biopsies (Bergstrom 1962) were taken from the vastus lateralis of the left leg at rest. Histochemical staining for myofibrillar ATPase was undertaken after preincubation at p H 10.3, 4.6 and 4.3 according to Brooke and Kaiser (1970) in order to classify muscle fibres as fast twitch (FT) and slow twitch (ST) fibres respectively and in order to separate the F T fibres into F T a and F T b subgroups. Muscle fibre area was determined according to Thorstensson (1976) from transverse muscle sections stained for NADH diaphorase activity according to Novikoff ef af. (1961). The subjects performed 25 repeated maxinial isokinetic leg extensions a t a velocity corresponding to 3.14 rad.s-' so as to induce substantial local muscle fatigue (Thorstensson 1976). The exercise was performed with the subject seated in a fired position with his leg attached to the lever arm of an isokinetic dynamometer (Cybex 11, Lumex Inc., New York) as described elsewhere (Thorstensson 1976). During this type of exercise, EMG recordings have shown that both medial and lateral parts of the vastus muscle are heavily activated (Merrifield and Dostal 1977). In order to ensure that subjects initially exerted maximal force, 3 single leg extensions were performed a few minutes prior to the experiment. The mean force exerted when a single extension was performed amounted to 161.6 Newtonmeter (Nm) compared to 160.4 N m in the experimental situation. Immediately after (within 3-4 s) the 25th leg extension, a muscle biopsy was taken from the vastus lateralis, frozen in liquid nitrogen and stored at - 80°C until analysed. Muscle biopsies were then freeze-dried, and approximately 100 fibres were dissected out (21°C and 30% humidity) and separated into FT and ST fibres on the basis of myofibrillar ATPase staining following preincubation at p H 10.3 according to Essen et a / . (1975). Pooled S T and F T fibres respectively consisting of 5-10 fibre fragments per pool were weighed on a Cahn electro-balance (Karlsson 1971). The samples ranged 0.01-0.03 mg in weight. The lactate concentration was determined by fluorometric means (Karlsson 1971). Each value was then calculated as a mean value for the lactate concentration in 2-7 pools of the respective fibre type. All values were converted into wet weight values. A water content of 77% was assumed for muscle biopsy specimens (Karlsson 1971). Additional dissected pools of F T and ST fibres consisting of about 100 fibres each were homogenized in 100pl of 0.5 M KCI. Total L D H activity in the forward reaction (pyruvate- lactate, LDHt,,) i n the homogenate, diluted 1 :4 with 0. I M Tris-HCI buffer p H 7.5, was determined by fluorometric means according to Lowry and Passonneau (1972). Discelectrophoresis for separating the L D H isorymes was performed according to Dietz and Lubrano (1967) on 25 ,uI of the homogenate. The relative contribution of musclespecific L D H monomeres (?& M-LDH) was determined using a densiometric scanning technique on the separated and stained L D H isozyme bands in the gels according to Sjodin (1976). The activity corresponding to the musclespecific L D H (M-LDH) was then calculated (x M-LDH ; LDH,,,).
42
PER TESCH. BERTIL SJODIN AND JAN KARLSSON
TmLE I . Mean values and range for lactate concentration, total LDH activity and M-LDH activity in
different muscle fibre types and whole muscle respectively.
Lactate concentration mmol . kg-' wet weight LDH,,, activity: mmol min-I kg-I wet weight lo1 M-LDH activity inniol min-' ' kg-' \ret weight ' 10'
FT fibres
ST fibres
Mean Range
Mean Range
11.7
(4.0-31.3)
15.0
1.12
(0.52-1.82)
0.82
(0.37-1.76)
Whole muscle Difference
Mean Range
(3.4-30.5)
p < 0.005
183
(3.5-31.1)
0.61
(0.33-1.11)
p-'0.001
0.90
(0.38-1.65)
0.28
(0.10-0.81)
p~:O.OOI
0.60
(0.18-134)
,'
1
The following formula was used in order to express lactate concentration for the entiremuscle investigated: Lactate concentration in FT fibres FT area -lactate concentration in ST fibres? "/ST I area. L D H activity or M-LDH activity were calculated in the corresponding manner by substituting lactate content for LDH activity in the t u o main fibre types.
Results The average percentage of FT fibres in the studied subjects was 46.506 FT fibres (range 29-74",). When the FT fibres were subdivided into FTa and F T b fibre groups, the mean values were found to be 28 and 1 5 O , respectively of the total number of fibres. 3 per cent of the fibres were classified as unidentified. The mean relative area taken up by FT fibres amounted to 62.0°,, (range 28-7600). Lactate concentration after exercise averaged 21.7 and 15.0 mmol kg-l wet muscle in FT fibres and ST fibres respectively. There was a considerable inter-individual spread in terms of the lactate concentration in both FT (4.0-31.3 mmol kg-') and ST fibres (3.4-30.5 minol kg-I) (Table I). The lowest lactate concentration, irrespective of muscle fibre type, was observed in the subject with a muscle richest in ST fibres, whereas the highest lactate concentration observed was found in a muscle consisting of predominantly FT fibres. The whole muscle values calculated for lactate concentration, LDH,,, and M-LDH activity
.
Fig. 1. The relationship between muscle lactate concentration (lactate concentration in FT fibres%96 F T area + lactate concentration in ST fibres ST area) and individual muscle fibre tvue o t:
43
LACTATE AND LDH IN HUMAN SKELETAL MUSCLE
TABLE 11. Correlation coefficients and levels of significance for some variables investigated.
Lactate concentration in muscle-muscle fibre type distribution Lactate content in FT fibres-muscle fibre type distribution Lactate content in ST fibres-muscle fibre type distribution Lactate concentration in muscle-LDH,,, activity Lactate concentration in muscle-M-LDH activity LDH,,, activity-muscle fibre type distribution M-LDH activity-muscle fibre type distribution 9; M-LDH in F T fibres-muscle fibre type distribution 7; M-LDH in ST fibres-muscle fibre type distribution
Correlation coefficient
Level of significance
r=0.74 r=0.91
p.r.o.01 p
Sweden
Relationship between lactate accumulation, LDH activity, LDH isozyme and fibre type distribution in human skeletal muscle BY
PERTESCH, BERTIL SJODINand JAN KARLSSON Received 31 October 1977
Abstract TESCH,P.. B. SJODINand .I. KARLSSON.Relaiioriship betweerr lactate accumulniiori, L D H crciii~it,v.L D H isoqrne arid fibre t j p e disrribrrtioti itr hiirnati skeletal muscle. Acta physiol. scand. 1978. 103. 40-46. Lactate concentration, total L D H activity a n d muscle-specific L D H isozymes were determined in pools of the ~ M Oniain types of human skeletal muscle fibres. Analyses were made from biopsy specimens obtained after intense d ) namic exercise lasting approximately 30 s. Lactate Concentration, total L D H activity and muxlc-specific L D H activity displayed higher average values for FT (fast twitch) fihres than for ST (slow tibitch) fibres. In addition. positive correlations were found both between the individual percentage of FT fibres and muscle lactate concentration a n d between lactate concentration and total L D H activity and muicle-specific L D H actib ity respecti\ely.
High lactate levels have been demonstrated both after exhaustive isometric exercise (Ahlborg ei ul. 1972, Karlsson and Ollander 1972) and maximal dynamic exercise (Karlsson 1971,
Knuttgen and Saltin 1972) of brief duration and have been suggested as one possible factor contributing to muscle fatigue in maximal exercise of brief duration. When the lactate concentration in different parts of a muscle sample were analysed after maximal exercise, values in the contracting elements were found to be 3-5 times greater than in elements supposed to be non-contracting (Karlsson 1971). Therefore, it may be assumed that t h e concentration of metabolites found in whole muscle biopsy specimens does not always reflect the true state of lactate accumulation in exercised muscle. Essen and Hiiggrnark (1975) reported a greater accuinulation of lactate in type 11 (fast twitch, FT) muscle fibres than in type I (slow twitch. ST) muscle fibres after exhaustive isometric exercise. Recent studies of lactate accumulation in different single muscle fibres have indicated that the fibre type distribution of a muscle is important to lactate accumulation in single muscle fibres (Tesch and Karlsson 1977). In other words, a muscle rich in FT fibres displals a greater potential for accumulating lactate than a muscle rich in ST fibres. This difference may be due to differences in the activity of the glycogenolytic system’s regulating
40
LACTATE AND LDH IN HUMAN SKELETAL MUSCLE
41
enzymes, i.e. phosphofructokinase (E.C.2.7.1.11.), glycogen phosphorylase (E.C.2.4.1. I .) and lactate dehydrogenase ((LDH) E.C.1.1.1.27.), in the two main muscle fibre types (Essen ef a / . 1975, Sjodin 1976, Thorstensson 1976). Thus, both LDH activity and the LDH isozyme distribution pattern in an untrained or moderately trained human population were found to be related to the percentage of FT fibres in the muscle (Karlsson et al. 1974). A muscle rich in F T fibres displayed greater LDH activity and a larger contribution of muscle-specific LDH (M-LDH) than a muscle rich in ST fibres in which the heart-specific type of LDH (H-LDH) was predominant. The intracellular distribution of these two main LDH isozyrnes and their kinetic properties differ (for ref. see Sjodin 1976). In normal conditions, the M-LDH isozyrnes are mainly associated with membranes, such as the sarcoplasmic reticulum. On the other hand, H-LDH fractions have been found in the inner membrane of the mitochondria. The aim of the present investigation was to study lactate accumulation in-the muscles of individuals differing with respect to certain relevant muscle qualities. Therefore, muscle lactate concentration was determined after intense, standardized exercise of brief duration and related to individual muscle fibre type distribution and LDH characteristics.
Subjects, Procedure and Methods 10 male physical education students served as subjects. Their mean ( k S . E . ) age, height and weight were 2 4 i 1 yrs, 1 7 9 k 1 cm and 71.312.1 kg respectively. Muscle biopsies (Bergstrom 1962) were taken from the vastus lateralis of the left leg at rest. Histochemical staining for myofibrillar ATPase was undertaken after preincubation at p H 10.3, 4.6 and 4.3 according to Brooke and Kaiser (1970) in order to classify muscle fibres as fast twitch (FT) and slow twitch (ST) fibres respectively and in order to separate the F T fibres into F T a and F T b subgroups. Muscle fibre area was determined according to Thorstensson (1976) from transverse muscle sections stained for NADH diaphorase activity according to Novikoff ef af. (1961). The subjects performed 25 repeated maxinial isokinetic leg extensions a t a velocity corresponding to 3.14 rad.s-' so as to induce substantial local muscle fatigue (Thorstensson 1976). The exercise was performed with the subject seated in a fired position with his leg attached to the lever arm of an isokinetic dynamometer (Cybex 11, Lumex Inc., New York) as described elsewhere (Thorstensson 1976). During this type of exercise, EMG recordings have shown that both medial and lateral parts of the vastus muscle are heavily activated (Merrifield and Dostal 1977). In order to ensure that subjects initially exerted maximal force, 3 single leg extensions were performed a few minutes prior to the experiment. The mean force exerted when a single extension was performed amounted to 161.6 Newtonmeter (Nm) compared to 160.4 N m in the experimental situation. Immediately after (within 3-4 s) the 25th leg extension, a muscle biopsy was taken from the vastus lateralis, frozen in liquid nitrogen and stored at - 80°C until analysed. Muscle biopsies were then freeze-dried, and approximately 100 fibres were dissected out (21°C and 30% humidity) and separated into FT and ST fibres on the basis of myofibrillar ATPase staining following preincubation at p H 10.3 according to Essen et a / . (1975). Pooled S T and F T fibres respectively consisting of 5-10 fibre fragments per pool were weighed on a Cahn electro-balance (Karlsson 1971). The samples ranged 0.01-0.03 mg in weight. The lactate concentration was determined by fluorometric means (Karlsson 1971). Each value was then calculated as a mean value for the lactate concentration in 2-7 pools of the respective fibre type. All values were converted into wet weight values. A water content of 77% was assumed for muscle biopsy specimens (Karlsson 1971). Additional dissected pools of F T and ST fibres consisting of about 100 fibres each were homogenized in 100pl of 0.5 M KCI. Total L D H activity in the forward reaction (pyruvate- lactate, LDHt,,) i n the homogenate, diluted 1 :4 with 0. I M Tris-HCI buffer p H 7.5, was determined by fluorometric means according to Lowry and Passonneau (1972). Discelectrophoresis for separating the L D H isorymes was performed according to Dietz and Lubrano (1967) on 25 ,uI of the homogenate. The relative contribution of musclespecific L D H monomeres (?& M-LDH) was determined using a densiometric scanning technique on the separated and stained L D H isozyme bands in the gels according to Sjodin (1976). The activity corresponding to the musclespecific L D H (M-LDH) was then calculated (x M-LDH ; LDH,,,).
42
PER TESCH. BERTIL SJODIN AND JAN KARLSSON
TmLE I . Mean values and range for lactate concentration, total LDH activity and M-LDH activity in
different muscle fibre types and whole muscle respectively.
Lactate concentration mmol . kg-' wet weight LDH,,, activity: mmol min-I kg-I wet weight lo1 M-LDH activity inniol min-' ' kg-' \ret weight ' 10'
FT fibres
ST fibres
Mean Range
Mean Range
11.7
(4.0-31.3)
15.0
1.12
(0.52-1.82)
0.82
(0.37-1.76)
Whole muscle Difference
Mean Range
(3.4-30.5)
p < 0.005
183
(3.5-31.1)
0.61
(0.33-1.11)
p-'0.001
0.90
(0.38-1.65)
0.28
(0.10-0.81)
p~:O.OOI
0.60
(0.18-134)
,'
1
The following formula was used in order to express lactate concentration for the entiremuscle investigated: Lactate concentration in FT fibres FT area -lactate concentration in ST fibres? "/ST I area. L D H activity or M-LDH activity were calculated in the corresponding manner by substituting lactate content for LDH activity in the t u o main fibre types.
Results The average percentage of FT fibres in the studied subjects was 46.506 FT fibres (range 29-74",). When the FT fibres were subdivided into FTa and F T b fibre groups, the mean values were found to be 28 and 1 5 O , respectively of the total number of fibres. 3 per cent of the fibres were classified as unidentified. The mean relative area taken up by FT fibres amounted to 62.0°,, (range 28-7600). Lactate concentration after exercise averaged 21.7 and 15.0 mmol kg-l wet muscle in FT fibres and ST fibres respectively. There was a considerable inter-individual spread in terms of the lactate concentration in both FT (4.0-31.3 mmol kg-') and ST fibres (3.4-30.5 minol kg-I) (Table I). The lowest lactate concentration, irrespective of muscle fibre type, was observed in the subject with a muscle richest in ST fibres, whereas the highest lactate concentration observed was found in a muscle consisting of predominantly FT fibres. The whole muscle values calculated for lactate concentration, LDH,,, and M-LDH activity
.
Fig. 1. The relationship between muscle lactate concentration (lactate concentration in FT fibres%96 F T area + lactate concentration in ST fibres ST area) and individual muscle fibre tvue o t:
43
LACTATE AND LDH IN HUMAN SKELETAL MUSCLE
TABLE 11. Correlation coefficients and levels of significance for some variables investigated.
Lactate concentration in muscle-muscle fibre type distribution Lactate content in FT fibres-muscle fibre type distribution Lactate content in ST fibres-muscle fibre type distribution Lactate concentration in muscle-LDH,,, activity Lactate concentration in muscle-M-LDH activity LDH,,, activity-muscle fibre type distribution M-LDH activity-muscle fibre type distribution 9; M-LDH in F T fibres-muscle fibre type distribution 7; M-LDH in ST fibres-muscle fibre type distribution
Correlation coefficient
Level of significance
r=0.74 r=0.91
p.r.o.01 p
