Europ. J. appl. Physiol. 36, 267--273 (1977) European Journal of

Applied Physiology and Occupational Physiology c by Springer Veriag 1977

Muscle Amino Acid Arylpeptidases and Their Serum Changes with Exercise 1

A. Berg and J. Keul

Lehrstuhl f'fir Leistungs- und Sportmedizin (Direktor: Prof. Dr. J. Keul), Medizinische Universitgtsklinik, D-7800 Freiburg i. Br., Federal Republic of Germany

Summary. Activities of serum amino acid arylpeptidases were determined in five groups of healthy male adults at rest and after various exercise types using the following substrates: Ala-, Leu-, Phe-, Tyr-, Gly- and Pro-4-Nitroanilide. The exercise-induced changes were compared to the activities of the respective en- zymes in resting leg muscles. A regression function was found, showing a close correlation between the mentioned parameters in all groups. The influence of haemoconcentration and intravascular hemolysis on the postexercise measured activities was of no consequence.

Key words: Aminopepfidases - Muscle -- Serum - Exercise.

In a previous paper from this laboratory, activities of amino acid arylamidases in skeletal muscle and serum of healthy male subjects have been investigated [5]. A relationship has been found between the increase of the serum activities with various substrates (Ala-, Leu-, Phe-4-nitroanilides) after physical exercise and their respec- tive activities in the resting leg muscle. It was the purpose of this study to gain more information on the hypothesis that AAP are increasingly released from working muscle. To verify this, investigations were performed:

a) on AAP-activities in human muscle with the substrates already described, as well as with other amino acid paranitroanilides (Tyr-, Gly- and Pro-4-nitroanilide) as substrates;

b) on changes of serum activities after physical strain using the same sub- strates.

The possibility of intravascular (or in vitro) hemolysis influencing the results was investigated by measuring serum hemoglobin before and after exertion. Eventually the changes of total serum protein were determined, purporting to seek information as to the amount of haemoconeentration.

1 The authors express their thanks to G. Haralambie for his encouragement during this work

268 A. Berg and J. Keul

Subjects and Methods

Muscle samples were obtained surgically from male subjects, aged 18--36 years, under conditions described in [5]. In no case there were any symptoms of muscular disease. After removing fat and connective tissue, the samples weight was between 80 and 360 mg. The preparation of a muscle extract in triethanolamine buffer (0.3 M, pH 7.6, containing 150 mM KC1) is detailed in [5]. After centrifuga- tion at 30,000 g for 20 min, the clear supernatant (final dilution 1 :21 w/v) was analyzed immedi- ately.

Determinations of the AAP-activities were performed as indicated by Nagel et al. [13]. Activities in muscle extracts were measured at 37 ~ , serum activities at 25 ~ . For comparison with the activities in muscle, serum activities were multiplied by the coefficient 2.4 (see ref. [5]). The following substrates were used:

L-alanine-4-nitroanilide hydrochloride (Merck, Darmstadt), L-leucine-4-nitroanilide (Boehringer, Mannheim), L-Phenylalanine-4-nitroanilide (Schuchardt, Mtinchen), L-Tyrosine-4-nitroanilide (Merck, Darmstadt), L-glycine-4-nitroanilide (Serva, Heidelberg), L-proline-4-nitroanilide (Sehuchardt, Mfinchen). The final concentration of all substrates used was 0.8 mM with the exception of L-proline-4-

nitroanilide. Serum analyses with this latter substrate were performed with a final concentration of 15.6 mM. Measurements in muscle extract showed, that there was no difference in the extent of split- ting of L-proline-4-nitroanilide, when concentration was raised above 0.8 mM.

Serum hemoglobin was determined spectrophotometrically according to Harboe [7]. Serum total protein was measured with the biuret reaction (reaction kit of the Fa. Merck, Darmstadt, Art. No. 3327).

Five groups of male subjects, all trained students (groups B, C, E), or trained athletes (groups A, D) aged 16-41 years were examined before and after their respective races (Table 1). All subjects were clinically healthy, i.e. there was no muscular, connective tissue or systemic disease. Blood was drawn from the cubital vein before and 5 - 8 min after the end of the exercise, avoiding stasis and without anticoagulant. After clotting serum was centrifugated twice and analyzed immediately.

Mean values and S.D. were calculated and the significance of difference was tested with the Student's t-test for paired data. Correlations were computed as linear regression functions.

Table 1. Data on subjects and type of exercise

Group n Mean age Type of exercise Mean duration of subject (min) (years)

A 8 17.5 10 km skiing 25.1 B 13 29.6 20 km cross running 80.0 C 18 28.5 42 km marathon race 186.0 D 10 29.0 50 km skiing 135.0 E 10 32.0 60 km skiing 340.0

Results

Values o f s e r u m - A A P - a c t i v i t i e s at res t a n d af ter the di f ferent exerc ise types are

g iven in T a b l e 2.

T a b l e 3 p r e sen t s the A A P - a c t i v i t i e s wi th the desc r ibed s u b s t r a t e s a t 37 ~ in var -

ious leg- a n d a rm-m us c l e s . W h e n a lso the va lues o f the p receed ing d e t e r m i n a t i o n [5]

Tab

le 2

. S

eru

m A

AP

-act

ivit

ies

at 2

5 ~

C i

n tr

aine

d m

ale

subj

ects

bef

ore

and

aft

er v

ario

us*

exer

cise

du

rati

on

(m

ean

val

ue •

st

and

ard

dev

iati

on,

acti

vity

in

mU

/ml)

Ala

-AP

L

eu-A

P

Ph

e-A

P

Ty

r-A

P

GIy

-AP

P

ro-A

P

befo

re

afte

r be

fore

af

ter

befo

re

afte

r be

fore

af

ter

befo

re

afte

r be

fore

af

ter

>

3"

O

A

18.6

0 19

.60

..

..

5.

04

5.48

1.

21

1.35

+

1.

98

• 2.

71

+

1.28

+

1.

28

• 0.

33

• 0.

43

P <

0.

02

0.02

0.

05

B

16.6

0 18

.70

15.4

0 16

.80

5.20

6.

50

5.65

6.

55

1.42

1.

85

• 2.

28

• 2.

85

• 1.

84

• 2.

01

• 1.

59

• 1.

47

• 0.

92

• 1.

18

• 0.

23

• 0.

37

P <

0.

001

0.00

5 0.

001

0.00

1 0.

005

C

17.3

0 19

.60

13.4

0 14

.80

6.95

8.

25

3.85

4.

54

1.02

1.

24

• 2.

90

• 4.

10

• 2.

01

• 2.

08

++ 2

.57

• 3.

09

• 0.

63

• 0.

72

• 0.

11

• 0.

12

P <

0.

001

0.00

1 0.

001

0.00

1 0.

001

D

15.6

0 16

.50

..

..

5.

13

5.36

1.

02

1.16

•

2.98

•

2.89

•

0.97

•

0.96

•

0.35

•

0.30

P

<

0.05

0.

05

0.05

E

15.3

0 17

.60

..

..

3.

92

4.50

1.

60

1.91

•

3.04

+

3.

32

• 0.

83

• 0.

82

• 0.

34

• 0.

49

P <

0.

001

0.00

1 0.

01

1.55

1.

60

• 0.

65

+ 0

.87

n.s

. n

.s.

1.60

1.

91

_+ 0

.63

_+ 0

.92

n.s.

n.

s.

n.s

. n

.s.

* S

ee S

ubje

cts

and

Met

ho

ds

270

Table 3. AAP activities at 37 ~ C in muscle extracts of male subjects (n = 15) (mean value + S.D., activity in mU/g wet weight)

A. Berg and J. Keul

Ala-AP Leu-AP Phe-AP Tyr-AP GIy-AP Pro-AP

M. vastus lat. quadr, fern. 2211 1039 983 277 112 96 n = 7 , 2 5 . 0 + _ 6.5 yrs. _+ 245 _+ 181 + 269 + 60 + 24 + 24

M. vastus med. quadr, fem. 1918 1015 1103 219 98 65 19 yrs.

M. rectus fem. quadr. 1366 798 789 102 44 61 20 yrs.

M. gluteus maximus 1911 794 1025 200 80 95 21 yrs.

M. gluteus maximus 2501 981 1249 292 102 123 20 yrs.

M. biceps bracch, cap. lg. 2172 1432 1205 322 59 83 18 yrs.

M. biceps bracch, cap. reed. 1781 747 659 185 83 92 36 yrs.

M. deltoideus 1295 555 535 146 68 57 18 yrs.

M. deltoideus 1650 685 660 220 85 65 29 yrs.

Table 4. Correlation between AAP-muscle-activities (mU/g wet weight, 37 ~ C) and the respective in- creases of their activities in serum (mU/ml, calculated for 37 ~ C) after different athletic races

Muscle activities Ala-AP Leu-AP Phe-AP Tyr-AP Gly-AP (x) 2086 1136 952 277 112

Increase of A + 3.24 - - + 1.06 + 0.34 serum activities C + 5.57 + 3.36 + 3.12 + 1.63 + 0.53 (y) D + 2.16 - - + 0.55 + 0.34

E + 5.52 -- -- + 1.39 + 0.74

Muscle activities Pro-AP R P Regression (x) 96

Increase of A + 0.12 0.9796 < 0.01 y = 0.0014 x + 0.26 serum activities C -- 0.9899 < 0.001 y = 0.0024 x + 0.65 (y) D -- 0.9995 < 0.001 y = 0.0009 x + 0.27

E -- 0.9987 < 0.005 y = 0.0024 x + 0.60

a re c o n s i d e r e d , t he f o l l o w i n g

ca l cu l a t ed :

A l a - A P 2 0 8 6 + 517 m U / g ,

L e u - A P 1136 + 233 m U / g ,

P h e - A P 952 + 278 m U / g .

mean activities in M. vast. lat. (n = 17) w e r e

Muscle Aminopeptidases 271

mt

5.0-

4.0 �9

3 .0"

2.0"

1.O"

0.5:

/rnl J

C .... m O I g

. . . . 5bo d o o ' 2doo

Fig. 1. Postexercise variations of AAP-activities in serum (mU/ml, calculated for 37 ~ C) in relation to their respective muscle activities (mU/g wet weight) in group B; y = 0.0020 x + 1.01, R = 0.9734, P < 0.001

Table 5. Changes in serum total protein (g/100 ml) and serum free hemoglobin (mg/100 ml) in the investigated groups

Group Changes in serum total protein Changes in serum free hemoglobin

before after p < + A% before after P <

A 7.58 • 0.53 7.52 • 0.52 n.s. @ 6.3 • 3.4 4.8 • 1.4 n.s. B 7,43 • 0.52 7.43 • 0.50 n.s. @ 3.1 • 2.5 9.6 • 5.4 0.01 C 7.60 • 0.30 7.86 • 0.20 n.s. 3.4 3.8 • 2.6 8.3 • 4.1 0.005 D 7.64 • 0.34 7.41 _+ 0.31 0.05 @ 4.2 • 1.2 3.3 • 1.5 n.s. E 7.52 • 0.45 7.78 • 0.48 0.02 3.5 3.1 • 0.9 4.1 • 2.4 n.s.

These da t a and the m e a n activities o f T y r - A P , G l y - A P and P r o - A P in the seven

samples o f M. vast. lat. show a s ignif icant cor re la t ion to their respect ive increases in

se rum activit ies after the var ious exercise types (groups A, C, D, E: Tab le 4). The

var iab le increase o f the se rum act ivi ty (y) is descr ibed here as a l inear funct ion (y =

bx + A) of the rest ing musc le act ivi ty (x). F igure 1 shows this func t ion for g roup B,

because in this case da ta with all substra tes used in this w o r k are available.

Based on the m e a n activites for all M m . vast . lat. and the average rest ing se rum

values o f all groups , the fol lowing musc le to se rum enzyme- ra t ios were obta ined:

A l a - A P 5 1 . 1 , L e u - A P 3 3 . 2 , P h e - A P 63.82, T y r - A P 24.9, G l y - A P 3 7 , 9 , Pro-

A P 25.3.

The pos texerc ise changes in se rum free hemoglob in and in se rum total prote in

are shown in Tab le 5.

z In re• [5] the figure of 16.5 instead of the real value 72.8, was erroneously given

272 A. Berg and J. Keul

Discussion

In spite of numerous publications on this subject, there are still discussions on the mechanism by which serum enzyme activities increase during and after physical strain. On the one side it is assumed that this increase has its origin in the enzyme release from active cells [2, 16]. Meanwhile, other authors have pointed out the importance of secondary effects of exercise, such as haemoconcentration in conse- quence of fluid redistribution and intravascular haemolysis [4, 8]. Since enzymes, as macromolecules, are not freely diffusible between intracellular and intracapillar space, it is obvious that an increase of their activity may be measured in serum as a consequence just of hypohydration, without effective release from tissue. However, when quantitative aspects are considered, it appears that a reduction in plasma volume:

a) is present only after intensive, short-lasting exercise (e.g. up to 30 min); b) does not exceed a maximum of ca. -15%, generally being less; c) is transient, e.g. preexercise values are reestablished within 30 rain of recovery

or sooner [11, 17]. Several facts of the present investigation strongly suggest that haemoconcentra-

tion alone cannot explain the observed changes in serum AAP-activities: a) Qualitatively, there is no parallelism between the changes of AAP-activities

and that of serum total protein concentrations. b) Quantitatively, the postexercise changes observed in serum proteins are in

none of the studied groups higher than +3.5%, whereas increases in AAP-activities are frequently 15-25%.

In addition, in group E blood hemoglobin has been determined as an indicator of haemoconcentration [1, 3], with the result that a 2% decrease was obtained after exercise.

Since it is known that AAP-activities in red blood cells are considerably higher than in plasma [12], it was important to gain information on the amount of haemoly- sis in our samples; this was done by determing the serum hemoglobin, before and after exertion. Increases of this parameter were found only after the running-races, in contrast to the ski-races. When the obtained figures are computed with the data from Lorentz et al. [12] on AAP-activities in erythrocytes (expressed in U/g hemo- globin), the highest possible increase attributable to intra- (or extra-) vasal haemoly- sis are about 0.12 mU/ml, and can thus be neglected. Moreover, there is no relation- ship between exercise-induced haemotysis and increase of the AAP-activities in the various groups.

On the other hand, there is a strong correlation of postexercise increase in AAP- activities with various substrates and the respective activities in resting M. vastus lateralis quadr, fern. This muscle has been here given preference since:

a) It is involved biochemically in both running and skiing; b) it has been shown, that a significant correlation exists between relative mito-

chondrial volume of this muscle and maximal oxygen uptake during exercise in man [9]. This suggests its character as a "reference" muscle with respect to exercise investigations.

An interesting fact is the lack of change in serum gamma-GT-activity with exercise in man [6], an enzyme which could not be found in muscle tissue [10, 15].

Muscle Aminopeptidases 273

The gamma-GT-ac t iv i ty in erythrocytes is about 1000 mU/g hemoglobin [12]; this again would suggest the lack of any noticeable influence on serum enzyme levels by int ravascular haemolysis during exercise.

In spite of all evidence speaking for a muscular origin of serum A A P increases, the variable magnitude of the activity changes in the different groups, expressed by the slope "b" in Table 4 and in Figure I, is still difficult to interprete. I t is however noticeable that groups A and D as compared to groups B, C and E have the lowest slopes of increase after exercise. The former two groups consist of highly trained athletes having a very good physical fitness status and an optimal run technique. It is possible that these factors m a y reduce the loss of enzymes from the muscle. This would express an adapta t ion of the organism and of a given organ (here: muscle) to the physical strain, like that described by Nut ta l and Jones [14] for CPK-increase after weight lifting exercise.

References

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(Evansblau) und radioaktivem Chromat. Cardiologia 47, 25-44 (1965) 4. Friedel, R., Mattenheimer, H., Trautschold, I., Forster, G.: Der vorget~uschte Enzymaustritt. J.

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arylpeptidase activity in man. Clin. chim. Acta 69, 433-439 (1976) 6. Haralambie, G.: Serum gamma-glutamyl-transpeptidase and physical exercise. Clin. chim. Acta

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human skeletal muscle. A morphometric analysis on untrained men, women and well-trained orienteers. Pflfigers Arch. 344, 217-232 (1973)

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13. Nagel, W., Willig, F., Schmidt, F.: Ober die Aminos~mrearylamidase- (sog. Leucinaminopepti- dase-)Aktivit/it im menschlichen Serum. Kiln. Wschr. 42, 447-449 (1964)

14. Nuttal, F., Jones, B.: Creatine kinase and glutamic oxalacetic transaminase activity in serum: kinetics of change with exercise and effect of physical conditioning. J. Lab. clin. Med. 71, 847- 854 (1968)

15. Rosalki, S. B.; Gamma-glutamyltranspeptidase, In: Adv. Clin. Chem., Vol. 17 (O. Bodansky, A. L. Lather, eds.), pp. 53--107. New York: Academic Press 1975

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17. Van Beaumont, W., Strand, J. C., Petrofski, J. S., Hipskind, S., Greenleaf, J.: Changes in total plasma content of electrolytes and plasma proteins with maximal exercise. J. appl. Physiol. 34, 102--106 (1973)

Received August 23, 1976