Eur J Appl Physiol (1984) 52:219-224 European Journal of

Applied Physiology and Occupational Physiology �9 Springer-Verlag 1984

Serum enzyme variations in men during an exhaustive "square-wave" endurance exercise test*

Manuel Gimenez and Michel Florentz

Laboratory of Exercise Physiology, Unit6 14 of the Institut National de la Sant6 et de la Recherche M6dicale (INSERM), Centre Hospitalier Universitaire de Brabois, Case Officielle 10, F-54500 Vandoeuvre-16s-Nancy, France

Summary. The present study was designed to test if both the intensity and duration of the 45-rain Square-Wave Endurance Exercise Test (SWEET) would produce changes in serum enzyme activities. Nine men, four sedentary (S) and five athletes (A), p e r f o r m e d VO2 max and SWEET, at their Maximal Intensity of Endurance (MIE45) as defined by maximal heart rate and the impossibility of main- taining MIE 45 + 5% for 45 rain. Arterial blood was sampled at rest (R), exercise (Ex) (45th rain) and during recovery (15th rain) for measurements of levels of Haemoglobin (Hb), Haematocrit (Hct), pH and seven serum enzymes: Creatine kinase (CPK), Hexose-phosphate isomerase (PHI), Aldolase (ALD), Lactate dehydrogenase (LDH), Malate dehydrogenase (MDH), Aspartate amino-transferase (ASAT or GOT), and Alanine aminotransferase (ALAT or GPT). Five enzymes increased signifi- cantly during exercise (MIE4.5), the A % (Ex- R/R) increases were as follows: PHI (72%), MDH (28%), LDH (21%), CPK (17%), and GOT (13.5%), whilst only a 10% increase was observed for Hct and Hb and there was no significant change in the arterial pH. There was no correlation between the A% of Hb, Hct, pH, and the results for the enzymes. Thus, it does not seem that haemoconcentration and arterial blood acidosis which occur during exercise are only at the origin of the observed increases in enzymes. A difference between "sedentary" and "athletes" sub- jects was found at rest and exercise (A% = A - S/S) for CPK (R = 222%; Ex = 235%), GOT (R = 90%; Ex -- 75%) and ALD (R = 99%; Ex -- 54%). These results suggest that the MIE45, by measured increases in enzymatic activity, seems to require great muscular effort.

Offprint requests to: M. Gimenez at the above address * This investigation was in part supported by the European

Economic Community (EEC), Luxembourg

Key words: Serum enzymes (CPK, PHI, MDH, LDH, GOT, GPT, ALD) - Arterial pH - Hae- moglobin - Cycle-ergometer - Sedentary men - Athletes

Introduction

The quantitative aspects of enzymatic activity in skeletal muscle of animals have been studied at rest, during exercise and after training (Wagner and Critz 1970; Mole et al. 1973; Benzi et al. 1975; Thomson et al. 1975; Garcia 1977). The evolution of circulating enzymes in the course of exercise has been less studied in men, and most work has been concerned with exercises of variable duration (Critz and Merrick 1962; Fowler et al. 1962, Tesch et al. 1973; Costill et al. 1976; Garcia 1977; Berg and Haralambie 1978; Komi and Karlsson 1978; Kelly et al. 1979). There is no extensive information about serum enzymatic variations during prolonged intense exercise and recovery (Sanders and Bloor 1975; Berg and Haral- ambie 1978; Stromme et al. 1978; Haralambie and Senser 1980). The mode of exercise (race, treadmill, cycle ergometer, etc.), the number of enzymes studied, and the degree of training of the subjects, vary from study to study.

We have recently applied a 45 rain exhaustive "square-wave" endurance exercise test (SWEET) for training purposes (Gimenez et al. 1982). This exhaustive exercise requires a considerable caloric output in men, depending on the fitness of the subjects (Gimenez et al. 1982).

The aim of this work is to measure, in normal sedentary and trained subjects, seven circulating enzymes chosen to represent several energy metab- olisms in order to verify: 1) whether an exhaustive exercise (SWEET) produces an increase in some of

220 M. Gimenez and M. Florentz: Serum enzyme responses during SWEET

these enzymes, and 2) whether among these seven enzymes, there is a difference between trained and untrained subjects.

Subjects and methods

Nine healthy subjects, four sedentary and five athletes, volun- teered for this study after having been informed of the protocol involved.

Constant power exercises and triangular exercises were used for the direct measurement of maximal oxygen consumption (1>o2 max) (Gimenez et al. 1982). In the latter exercise, from an initial 30 W the load was increased every 3 rain; the highest level of this progressive test maintained for at least 2 min is referred to as "maximal tolerated power" (MTP).

The level of reference for the SWEET is the MTP. The SWEET includes an intense submaximal exercise at a % of MTP for 45 rain, with periodic maximal surcharges (MTP) of 1 min, imposed every 5 min. The Maximal Intensity of Endurance for the 45 min (MIE45) is defined as a % of MTP by the maximal cardiac frequency at the end of the test and by the impossibility of maintaining the MIE45 + 5% for 45 rain (Gimenez et al. 1982).

The athletes did not take part in any endurance competition during the week preceding the examination, nor in any training 2 or 3 days before the fixed date of the biological test.

The subjects arrived at the laboratory at about 8.00 a.m., having eaten a light non-fat breakfast 11/z h before. They rested for 30 min in a chair and a catheter was placed in a radial artery. The first blood sample and measurements were taken after 45 rain of rest (R), then the subject performed the MIE45 exercise on a cycle ergometer at the predetermined intensity of maximal endurance. A second blood sample (Ex) was taken at the final peak of the MIE45 (45th min of exercise) and a third blood sample 15 min after recovery (Rec) in a chair, pH, (BSM3 Radiometer), haemoglobin (Hb) (OSM2 Radiometer) and haematocrit (Hct) (capillary technique) were immediately measured in duplicate.

The blood for enzymal~analysis was taken in an heparinized tube, kept in ice and then, within 5 min after withdrawal, was

centrifuged for 15 min at 3,000 g. Analyses were carried out on the plasma at 37 ~ C, with a Beckman spectrophotometer (Beckman, Mfinchen, FRG), Model 25K, with the techniques in Table 1. Enzyme activities are expressed in micromole of modified substrate per minute, and per litre of serum (U/l). Statistical analysis was performed with the Student's t test in order to note paired data, and differences between the values of athletic and sedentary subjects.

Results

Table2 shows the mean values (x + SEM) of physical characteristics, the maximal value of MTP and heart frequency (fH), the maximal oxygen consumption and the observed MIE45 of the two groups of untrained and trained subjects. With similar physical characteristics and maximal fH, significant differences exist between the two groups for MTP, 1>o2 max and MIE45.

Judging the two measurements of 1>o2 max and by the accuracy of the measured MIE45 , all the subjects, both trained and sedentary, reached their maximal level of SWEET. Exhaustion occured by the end of the test (Gimenez et al. 1982).

The uncorrected haematocrit (R = 41 + 1.4; Ex = 44.9 + 1.4; and Rec = 43.7 + 1.5 Vol%) increased significantly during exercise (P < 0.05), and haemo- globin showed the same evolution (R = 13.8 + 0.5; Ex = 15.2 + 0.5, P < 0.01 Rec = 14.6 + 0.5 g �9 dl-~), but the difference between rest and recovery was more sensitive for Hb (P < 0.001) than for Hct (P < 0.05). pH (7.451 + 0.014) did not vary during exercise (7.410 + 0.003) nor during recovery (7.420 +_ 0.007).

Table 1. Methods used for the seven enzyme activity determinations at 37~

Enzymes E . C . Abbreviation Test-Kit No. Method ref.

1 Creatine kinase (Creatine phosphokinase)

2 Hexose phosphate isomerase

3 Lactate dehydrogenase

4 Malade dehydrogenase

5 Aldolase

6 Asparate aminotransferase (Glutamate-oxaloacetate transaminase)

7 Alanine aminotransferase (Glutamate pyruvate transaminase)

2.7.3.2. CK Boehringer, 126322 Szasz et al. 1976 (CPK) Mannheim, FRG

5.3.1.9 PHI Behring, OTL 10/11 Schwartz et al. 1971 Marburg, FRG

1.1.1.27 LDH Boehringer, 124885 Bergmeyer 1974 Mannheim, FRG

1.1.1.37 MDH Boehringer, 124940 Bergmeyer 1974 Mannheim, FRG

4.1.2.13 ALD Merck, Haralambie and Darmstadt, FRG Senser 1980

2.6.1.1. ASAT Merck, 3342 Bergmeyer 1974 (GOT) Darmstadt, FRG

2.6.1.2. ALAT Merck, 3372 Bergmeyer 1974 (GPT) Darmstadt, FRG

In parenthesis, old terminology

M. Gimenez and M. Florentz: Serum enzyme responses during SWEET 221

Figure 1 represents the variations of the mean values of the seven enzymes at exercise and during recovery. There was a difference between rest and exercise for PHI, LDH, MDH, GOT, and CPK; there was no difference for GPT and Aldolase. The individual variations were rather diverse for GPT, but for Aldolase, six of the nine subjects increased the values at exercise. If we consider the difference between exercise and rest, expressed as a percentage (A%), the most sensitive variation corresponds to PHI (+72%); followed in descending order by MDH (+28%), LDH (+20.5%), CPK (+17.3%) and

finally GOT (+13.5%). There was no difference in these percentages between the sedentary and trained subjects. At the 15th min of recovery, the activity of PHI, LDH, and GOT decreased significantly. A difference was found between sedentary (S) and athletes (A) subjects (A% = A - S/S) at rest and during exercise for CPK (R = 222%; Ex = 235%), GOT (R = 90%; Ex 75%) and ALD (R = 99%; Ex = 54%).

Discussion

300_

200

100

CHANGES IN SERUM ENZYMES DURING AND AFTER EXERCISE

U.L -1 ~ n=9

R+_SEM T

~ " ' " ' - ~ MDH (n =7)

25

20 t oot . . . . . . I oPT

{n=8)

6] I - - - - ' - - ~ - ' - ' - - - ' - - - - - ~ . . . . . . --I ALD 3 (n=S)

REST EX -R.. R&O- elSE VERY (45 min) (15 min)

Fig. 1. The enzymatic activity of five enzymes increases signifi- cantly during exercise (MIE45). The most sensitive variation corresponds to PHI (+ 72 p. 100). PHI, LDH and GOT decrease their activity at recovery. * P < 0.05; ** P < 0.01; �9 ** P < 0.001

The significant increase in five enzymes during exercise and the difference in enzymatic activities between the trained and sedentary subjects were the two most striking results of this study.

It is known that serum enzyme response to exercise depends on the intensity and duration of the test, and also on the training of the subjects. Table 3 shows the variations of serum enzymes that were observed after exercise and/or training. In men, it seems that the most frequently studied enzymes that increase during exercise are CPK, LDH, and GOT, always coinciding with high level of exercise. On the other hand, whereas the increase in these enzymes is often noted, it is the increase in the PHI during exercise which is the most significant in this study (72%).

In the course of exercise, haemoconcentration and acid-base balance in blood can influence the stages of enzymatic activity in the plasma.

The loss of weight in marathons or in endurance races can excede 2 kg, which coincides with a net and significant increase in the activity of some enzymes (Magazanik et al, 1974; Sanders and Bloor 1975). On the other hand, after intense muscular exercise of short duration (10-20 rain), where neither loss of weight nor dehydration seem to play a role, sensitive enzymatic variations have been described (Fowler et al. 1962; Kelly et al. 1979). It does not seem that haemoconcentration, secondary to dehydration, is

Table 2. Physical characteristics, maximal levels of exercise, and Vo2 max in sedentary and trained subjects

Groups of Age Height Weight MTP fH 1)o2 max. MIE45 subjects (years) (cm) (kg) (W) (b . rain -1) (ml- kg -1- min -1) % MTP

Sedentary x 29 178 73.3 210 192 40.7 45 (n = 4) SEM 2.5 1.5 3.8 0 6.4 2.0 1.4

** *** **

Trained x 26.6 174 67 282 192 58.5 57.2 (n = 5) SEM 1.4 3.8 4.2 15 4.4 2.4 2.8

MTP Maximal Tolerated Power of the progressive exercise (+ 30 W/3 min); f H heart frequency; V o 2 m a x . maximal oxygen consumption; MIE45 = Maximal Intensity of Endurance maintained during the 45 rain of the "square-wave" exercise endurance-test (SWEET) ** P < 0.01; *** P < 0.001

222 M. Gimenez and M. Florentz: Serum enzyme responses during SWEET

Table 3. Changes in serum enzymes observed in men after exercise and/or training

Reference Subjects Enzymes Experimental conditions Conclusions

Fowler et al. 1962 28 M (T, U) LDH, GOT, GPT Treadmill: max. sub. re GOT/*GPT/*T = U 11 F MDH, ALD

Rose et al. 1970 6 M CPK, LDH Marathon CPK, LDH, LDH-3, LDH-isoenzymes 4 and 5/*

Hunter and Gritz 12 M CPK, LDH, GOT E max. sub before and 1971 after training

Maqazanik et al. 6 M (T) CPK, GOT, GPT Marathon 1974 SDH, ALD

Wyndhan et al. 30 M CPK, LDH E sub room temperature 1974 15 M (T) GOT, GPT and at 32~

Raimondi et al. 4 M CPK, LDH, GOT After 12 weeks training 1975 MDH (cycle-ergometer)

Kaman et al. 1977 5 M (T) CPK, LDH Runs 6 to 19 miles iso CPK, LDH

Francesconi et al. 8 M CPK, LDH, GOT, GTP Treadmill sub 90 rain 1977

Berg and Haralambie 166 M (T) CPK, PHI Cycle-ergometer 1978 (4 to 100 min),

ski-running, running-skating

Pate et al. 1978 4 M (T) CPK, LDH Treadmill: 3 E of varying GOT, GPT duration and intensity

Stromme et al. 16 M (T) CPK, GOT, GPT Run: 90 and 70 km 1978 OCT, LDH-iso

Haralambie and 16 M (T) CPK, PHI, MDH, ALD Senser 1980 3-PGK, TPDH

Haralambie 1981 21 M (T) ALD iso A - B 21 M (U)

Davies et al. 1982 11 M (T) CPK, LDH, iso 1 - 2 CPK iso MB

This study 5 M (T) CPK, LDH, GOT, GPT 4 M (U) PHI, MDH, ALD

LDH-t,2 =

CPK, LDH, G O T S a t max CPK, G O T S a d sub L D H S a t R in T

CPK, GOT, A L D S GPT, SDH =

CPK, GOT = at room temp. CPK, GOT/*at 32 ~ C

CPK, LDH/* at R

Swimming 5 km (90 min)

Runs of varying duration and intensities

Treadmill and 8 km race

Cycle-ergometer SWEET

CPK, LDH~ LDH-17/* CPK-MM/*/*

CPKS

CPK, P H I S

CPK/*LDH/*GOT/*

CPK, GOT, LDH, LDH 4 - 5 7

Except TPDH, all five enzymes increase

ALD and iso-A are higher in T than in U

CPKSonly after race

CPK, LDH, MDH, PHI, GOT,.," CPK, GOT, ALD in T > U at R and E

M males; F females; T trained; U untrained; R rest; E exercise; RE recovery; M A X maximal; SUB submaximal; ALD aldolase; = do not vary; A acclimatized; ISO isoenzymes; .,*, S / increases

only at the origin of the observed increases in enzymes during exercise. If this were the case, all plasma enzymes studied would have increased. In fact two of them (GPI and ALD) did not change. Furthermore in this study there was no correlation between rest-exercise differences of each of the studied enzymes which were always higher than 13 %, and those of haemoglobin and haematocrit which varied by of 10%. Kelly et al. (1979) showed that these mentioned changes in the dehydrated state do not occur. The enzymes considered as regulators of glycolysis are sensitive to variations in pH. The constancy of arterial pH, at the end of MIE45, suggests that the observed enzymatic variations are

not influenced by an arterial pH effect. In fact, there is no correlation between the sensitive variations of the enzymes at exercise, and the minor one of arterial pH.

Finally, it has been suggested that intravascular haemolysis, temperature increases and increases in lymph in the blood may influence the increased level in serum enzymes (Young 1974). However, it seems that intravascular haemolysis is less after a race of 100 km, and that consequently its influence on the enzymatic variations, which are increased further, only plays a minor role (Berg and Haralambie 1978; Haralambie 1981). Moreover, the acclimation effects of heat over circulating enzymatic activities were

M. Gimenez and M. Florentz: Serum enzyme responses during SWEET 223

analyzed by two groups with controversial results (Wyndham et al. 1974; Francesconi et al. 1977).

The origins of enzymes studied are the cardiac and skeletal muscle for CPK and PHI, the liver for GPT and the three types of tissues for ALD, GOT and LDH (Young 1974; Haralambie et al. 1976; Stromme et al. 1978; Berg and Haralambie 1978). Since GPT, which comes from the liver (Young 1974) does not vary during exercise, it is reasonable to think that the striated muscle could be the principal source of the enzymatic discharge. However, if the results of Arnold and Pette (1968) indicate that the liaison of glycolytic enzymes with structural proteins could be affected by the variations of metabolic concentrations (Poortmans 1975), there is no experimental informa- tion concerning the mechanism of enzymatic dis- charge from skeletal muscle into the bloodstream during intense exercise (Young 1974). After 10 min of stimulation in situ of skeletal muscle in the rat we observed an increase in the activity of oxydative enzymes (citrate syntase) and a simultaneous decrease in Phosphohexoisomerase (PHI) and 3-phosphoglycerate kinase (3PGK), variations which were not evident at the non-stimulated extremity (Gimenez and Florentz 1979). All the increases in serum enzymes observed in this work with men agree with these experimental facts, and with the enzymatic activity observed at the muscular level (Gollnick et al. 1973; Raimondi et al. 1975; Haralambie et al. 1976).

More information can be obtained by a simulta- neous analysis of total enzyme activity together with isoenzymes distribution. The increase in CPK (Gar- cia 1977; Davies et al. 1982), and LDH (Rose et al. 1970) was due to the rise in the respective specific muscular isoenzymes. The absence of a variation in LDH-1, -2 isoenzymes (Rose et al. 1970) suggests that exhaustive exercise does not increase the loss of liver or heart isoenzymes. Consequently, it could not substantially affect the increase in the total LDH enzyme activity. Moreover, athletes in train- ing have considerably higher levels of serum aldo- lase activity at rest in comparison with non-athletes. This is due to the higher level of aldolase isoenzyme A, predominant in the muscle (Haralambie 1981).

Sanders and Bloor (1970) think that CPK is the enzyme most sensitive to muscular "stress" exercise, whereas Berg and Haralambie (1978) consider that the increase in both CPK and PHI are "indicators of stress". For the CPK, this was recently confirmed by Davies et al. (1982). If the increase of the CPK, PHI, and LDH enzymes is considered as evidence of a certain degree of muscular stress (Sanders and Bloor 1970; Hunter and Critz 1971:; Berg and Haralambie 1978), our results confirm that the MIE45, through simultaneous elevation of CPK, PHI, and LDH can produce muscular "stress" at the end of exercise.

v 2OO t,, (.J

C O M P A R I S O N BETWEEN SEDENTARYr -~n=~ U.L-1 A N D T R A I N E D Ez~ n=S

300 ~ + S E M

100

T

O 20 ~

REST EXERCISE RECOVERY (A5 min) (15 rnin)

Fig. 2. Three enzymes (CPK, GOT and ALD) were different between sedentary and trained subjects not only during exercise, but also at rest, and for CPK and GOT at recovery. * P < 0.05; �9 * P < 0.01

The second point of this discussion concerns the behaviour of CPK, GOT and ALD at rest and during exercise, between trained and untrained subjects (Fig. 2). Simultaneously with our results, Haralambie (1981) has found similar differences at rest. More- over, it is known that human muscle has rather high GOT activity (Raimondi et al. 1975; Stromme et al. 1978). Also, after training, Raimondi et al. (1975), observed an increase of muscular enzymatic activity of GOT and MDH of greater than 100% at rest in men's skeletal muscle. Moreover, the increase of several enzyme activities in the serum tends, to be maintained for several hours or even several days after the exercise, particularly in the case of ALD (Haralambie 1981), GOT (Sanders and Bloor 1971, Raimondi et al. 1975; Pate et al. 1978) and CPK (Fowler et al. 1962; Rose et al. 1970; Haralambie et al. 1976). This may contribute to the significant "chronically" different values observed between trained and sedentary subjects at rest; the enzyme level in the serum could well be an index reached during training (Raimondi et al. 1975; Berg and Haralambie 1978). However, further studies are still necessary for a better understanding of the adapta- tion of the muscle to training and its relation with the observed enzyme responses at the blood level.

From the results presented here one can con- clude: 1) that the MIE45 causes a significant increase

224 M. Gimenez and M. Florentz: Serum enzyme responses during SWEET

of five enzymes, of which at least three (CPK, PHI, and LDH) are considered as representative of "intense muscular exercise", and 2) that among the seven studied enzymes, three of them, CPK, GOT, and Aldolase, clearly distinguish trained from untrained subjects, both at rest and during exercise. It appears very likely from the present study that the activity of these three enzymes levels may well enable one to obtain reliable information on physical fitness and might well be an index for attainment during training in man. Acknowledgements. We are greatly indebted to Doctor B. Dull and Miss Sallyann Horlick for their assistance in preparing the English manuscript. We also wish to thank Mrs D. Harteman for technical assistance and the secretaries Ms M. C. Rohrer for illustration and Ms J. Bara and Ms B. Clement for typing the manuscript.

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Accepted October, 1983