Changes in serum lysozyme activity with physical exercise

217

Clinica Chimica Acta, 55 (1974) 217-224 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

CGA 6565

CHANGES IN SERUM LYSOZYME

G. HARALAMBIE

WITH PHYSICAL EXERCISE

and F.J. CERNY*

Lehrstuhl fiir Leistungsmedizin Freiburg/Breisgau (G.F.R.) (Received

ACTIVITY

und Kreislaufforschung,

Medizinische Klinik der Universittit

April 11,1974)

Summary

Serum lysozyme activity significantly increased up to a maximum of 70% above the rest value after various types of physical exertion. During recovery, values rapidly decreased towards initial levels. When measured 24 h after exertion, pre-exercise levels had been reached. Three possible mechanisms for these changes are discussed: lysosomal disruption, leucocyte destruction, and leakage from non-lysosomal sources. The mechanism may vary with the type of exercise. It is concluded that previous physical activity history has little importance, if any, when serum lysozyme levels are considered for clinical purposes.

Introduction

In recent years, much attention has been given to the changes in serum (plasma) enzyme activity as a result of various forms of physical exercise [ 1,2] . It has been shown that, in the case of several enzymes, there can be an immediate and/or a delayed increase in activity after effort. This may often lead to misinterpretations in clinical examination of patients. These delayed changes, in fact, may be the result of very variable types of exertion [3-71. Immediate history of activity must therefore be an important point in the patient’s examination. Because determinations of enzyme activities for diagnostic purposes are constantly being extended and more and more enzymes - for many of which the effects of exercise have not been, or only poorly been studied - are being introduced, we undertook the examination of the effects of various types of exercise on these “diagnostic” enzymes. In a previous paper this problem was discussed concerning amino acidarylpeptidase activity in serum [ 51. This

* Recipient of Alexander

“on

Humboldt Foundation Research Fellow Grant.

218

report presents data obtained on serum mucopeptide glucohydrolase (lysozyme, EC 3.2.1.17) activity; the possible significance of the observed changes is also discussed. Subjects

and Methods

All subjects involved in these examinations were adult (18-38 years) males. Subjects in groups A, C and D were university students, physically active, but not involved in a regular training program. Groups B, E, F and G were highly trained athletes, participating in training programs of 12 to 16 h/week (Table I). Multiple laboratory examinations were done on all subjects (serum enzymes, urea, uric acid, serum iron and copper etc.); those having clearly pathological values - excepting the characteristic changes described in athletes in training [ 7,8] - were not considered in this study. In groups A, C and E-G, blood was drawn, without stasis and anticoagulant, from the cubital vein into plastic disposable syringes, allowed to clot, and serum separated by centrifuging twice (at 4000 and 12 000 rev./min, respectively). Samples in group B were drawn through a catheter inserted in the pulmonary artery. In group D, a plastic catheter was placed in the cubital vein for the duration of the experiment. To determine the simple effect of catheterisation on serum lysozyme activity, blood was drawn in eight subjects resting in the supine position, immediately and 40 to 90 min after insertion; no difference has been observed. The method for lysozyme activity determinations followed, in principle, that described by Daniels et al. [9] . Successively pipetted into a disposable plastic cuvette were: (a) 2.75 ml phosphate buffer 0.066 M, pH 6.3-6.4; (b) 0.2 ml of freshly prepared suspension of killed Micrococcus Zuteus cells, 2 mg/ml in buffer (Boehringer, Mannheim); (c) after temperature equilibration (25”), 50 ,ul of serum.

TABLE GROUPS

I EXAMINED

IN THE PRESENT

STUDY

Group

N

Activity

Blood sampled

A

37 40

Normal daily activity Athletes in training

Morning, Morning,

B

5

Maximal Vo2 test on bicycle ergometer

Before, during (at 200 and 300 W) and 3 min after exercise

10 14

G

1 h ergometer to exhaustion

exercise (x 64% max.

2 h ergometer exercise (65% max. work load)

9

42kmrun

8

50 km competitive

11

Bobsled

competition

walk

GO,)

at rest at rest

Before, immediately, after exercise

1 h and 24 h

Before, 10. 40, ‘IO and 100 min during and 10. and 20 min after exercise Before

and 3-S

min after

Before

and 3-S

min after

Before and after runs on 2 consecutive days

219

The content of the cuvette was mixed and the change in absorbance recorded for 4 min, using an Eppendorf Hg-lamp photometer at 623 nm. During the first 4 min, the decrease in absorbance was linear; in many samples linearity was observed for longer periods. A calibration curve was constructed using a lyophilized human serum standard (Behringwerke, Marburg/Lahn). The relationship between decrease in absorbance/min and the amount of lysozyme added to the analysis was linear up to a calculated serum concentration of at least 300 pg human lysozyme/lOO ml. Using various amounts of fresh human serum, a linear relationship to the decrease in absorbance was also observed. The results in this paper are expressed in pg human lysozyme/lOO ml serum* and differ from the usual expression based on egg-white lysozyme by a factor of l/5.37. This factor was taken into consideration when the present results were compared to those of other authors. 01~-Acid glycoprotein was determined with the radial immunodiffusion technique of Mancini et al. [lo] using M-Partigen immunoplates and a standard serum (Behringwerke) . Determination of serum hemoglobin was done spectrophotometrically according to Harboe [ 111. Results

Group A Reference values (Fig. 1). A mean serum lysozyme activity of 128.3 f 43.6 pg/lOO ml (range 40-216) was found in the randomly selected subjects. Athletes in training showed a mean value of 126.3 5 64 pug/100 ml (range 45-318). Group B In this group, serum lysozyme maximal V. z test from 132 f. 47.3 (P < 0.01) at 300 W after 18 min 3 min of recovery the value was still

activity increased pg/lOO ml at rest of total exercise 161 f 64.2 pg/lOO

progressively during the to 160 it 57.7 c(g/lOO ml duration (Fig. 2). After ml (P < 0.01).

Group C After 1 h of severe exercise, lysozyme increased from 140.4 + 60 at rest to 170.9 f 57 pug/100 ml (P< 0.005); 1 h after exercise, the level had already decreased to 153.7 + 49 (P < 0.05); and 24 h later it had returned to the resting level (142 + 41 pg/lOO ml). Group D During 2 h of moderate exercise (Fig. 3) serum lysozyme progressively increased to a peak value of 156.8 + 43.5 (P < 0.025) at 70 min and thereafter decreased. This decrease continued during 20 min of recovery to 144.6 + 38.4 pg/lOO ml (P > 0.1).

* It should be emphasized that, although results are given as concentrations, ments of activities and merely represent the amount of “active” lysozyme

they are in fact measurein serum.

220

Athletes n=40

6-

n=

5

180

16

in ”

140

CCWWOI subjects nz37

12

4

~~

2

0

50

100

LysOZYme

150

200

250

0

p9IlOOml

300

200

100 I 6

I 12

watt

, 16

,

ml”

Fig. 1. Frequency distribution of lysozyme activity in serum from a group of athletes in tmining, measured at rest (above), and a group of control subjects (below). Fig. 2. Increase in serum lysozyme activity during and after a maximal Vo2 test on the bicycle ergometer. Bars indicate S.E. (See text for details.)

Group E

A 42 km (marathon) race resulted in an increase in lysozyme activity from 133 f 32 to 191 f 47 ILg/lOOml (P< 0.001). Group F After a 50 km competitive walk values increased from 124 + 29 at rest to 196 + 41.3 pg/lOO ml immediately after (P < 0.001). Group G Resting lysozyme values on day 1 were, in this group, 191.8 rt 55.4 and

I

125 I

Watt I 30

1 60

1 90

I I 120

mm

Fig. 3. Time Course of changes in serum lysozyme during and after 2 h of moderate ergometer exercise. Bars indicate S.E. (See text for details.)

221

increased increased

to 198.9 + 56 pg/lOO ml after four bobsled runs. On day 2 levels from 185.4 f 67 to 202 + 75 (P < 0.01) after another four runs.

Discussion The reference values described here agree well with those reported in adult subjects using either an egg-white lysozyme standard [ 12,131 or a standardized human serum [14], with the turbidimetric procedure. Higher values are reported using the lysoplate technique [ 15-181; this may be explained in part by the higher incubation temperature (37” ). Increases in lysozyme activity of up to 60% were found as a result of very different types of exertion; these relative increases are much lower than those reported [ 19,201 or found in this laboratory for serum lactate dehydrogenase, creatine phosphate kinase, isocitrate dehydrogenase, malate dehydrogenase, aldolase, etc. after similar physical stress. The rise in lysozyme activity due to exertion is followed by a rapid decline, during recovery, towards pre-exercise levels (group C); if exercise is moderate, this decrease may occur during the activity itself (Fig. 3). The day after intense, long-lasting (group C) or short, violent exercise (group G) lysozyme activity was not significantly different from the pre-exercise level. After short, severe exercise (6 min, 8 male subjects), Poortmans [13] found a significant increase of 18% in serum lysozyme; after 30 min recovery, values decreased to 112% of the resting level. The distribution of lysozyme activities is also much the same in control subjects and in athletes, measured at rest, during training, which is not the case, for example, for CPK activity [7] . For these reasons, the determination of serum lysozyme for clinical diagnostic purposes can be made with less concern over exercise (physical activity) history, which is apparently not the case with enzymes such as CPK and LDH [4,7,21] . A possible exception may be the activities involving severe physical contact (e.g. boxing, wrestling, bob-sleding) which may result in tissue damage. This is suggested by the high (191 f 55.4 E.cg/lOOml) resting values in group G. Three main causes for the observed increases in serum lysozyme activity after physical exercise may be considered, as follow. (a) Lysosomal rupture During exercise this may occur either as a consequence of tissue destruction, or as a result of unfavorable conditions within the tissue (e.g. severe acidosis, lowered synthesis of energy-rich compounds etc.). In the present study blood pH changes have been measured only for group B. The mean pH value, recorded in the third minute of recovery, was 7.28 + 0.063 for arterial and 7.198 f 0.051 for mixed venous blood from the pulmonary artery; peak venous lactate values ranged from 7 to 15 mmoles/liter. In group G, blood lactate data are available, showing that immediately after the bob runs the subjects had arterial lactate levels between 2.8 and 8 mmoles/liter, which may also indicate a certain degree of tissue acidosis; this may, however, be less than in group B. De Duve [ 221 suggests that the release of lysosomal enzymes is pH dependent, with greater release at lower pH. Thus it is possible that some

222

TABLE

II

CHANGES

IN

SEVERAL Results

&I-ACID

OF

THE

expressed

GLYCOPROTEIN

GROUPS in

AND

SERUM

HEMOGLOBIN

DUE

TO

EXERCISE

IN

STUDIED

mg/lOO

ml

serum

+

S.D.

Significance

calculated

using

Student’s

t test

for

paired

differences.

Group

N

Exercise

5

B

Max.

eGz

al-Acid

glycoprotein

Serum

Before

After

Before

test

5.8

Hb After

f

14

D

2 h ergometer

3.6

f

9

Marathon

run

54.4

f

61.1

6.4 F

8

50 km

walk

86.5

+

27 G

11

Bobsled

(19.2)

80.9

(20.2)

88.2 21.7

101

+

85.1

6.0

&

+***

11.0 1.0

6.5

+ *

10.7

+

5.35

4.7

25

24.2 Bobsled

& ***

7.0

f **

2.1

2.2 E

12.5 4.6

2.1

*

18.4

f ***

6.0

+ ***

26.0 f

94.5

f ***

27.0

* P KO.02. ** ***

P co.005.

P
lysosomal disruption occurred as a consequence of exercise acidosis in these groups of subjects. This would, however, not be the case in the other groups, since long-lasting exercise is not usually associated with a fall in arterialized or even venous blood pH lower than 7.30-7.35. (b) Leakage of extralysosomal lysozyme from tissues; It has recently been shown that human cartilage has a lysozyme activity of non-lysosomal origin of about 10 3 times that of serum [ 231, thereby being a possible source of considerable amounts of lysozyme if leakage were to occur here. From the present investigation (Table II) as well as from other papers [24,25] it is known that long-lasting exercise is accompanied by a significant increase in the serum “acute phase” inflammatory cyl -acid glycoprotein. It has been shown in animal experiments that during the acute phase response, inflamed connective tissue significantly contributes to the increase in acid-soluble serum glycoproteins; this may occur rapidly, since maximal activity. of injected radioactive glucosamine was found in serum 30 min later [ 261. Although it is not clear whether the increase in a! 1-acid glycoprotein observed here represents a true “inflammatory reaction” during exercise, it may indicate some metabolic involvement of connective tissue in certain types of exertion (see also ref. 27). (c) Lysis of lysozyme-containing leucocytes The lysozyme activity in plasma represents

only 3.7% of the total per unit

223

volume of whole blood [ 281. Lysozyme values in leucocytes have been measured to be 1.0 to 2.8 ,ug/106 cells [16]. Thus, a lysis of less than 4% of the leucocytes normally present in blood would result in an increase of about 70 pg lysozyme/lOO ml serum, e.g. the highest increase in the present experiments. It must be remembered that a pronounced leucocytosis has been observed after long-lasting exercise [29,30] . In this investigation, determinations of serum hemoglobin levels have been made, purporting to obtain some information on the hemolysis during exercise. Although the results (Table II) seem to show a very moderate hemolysis, the strong decrease in serum haptoglobin after endurance events 1311 which is correlated with the serum hemoglobin increase after the 50 km walk (G. Haralambie, in prep~ation), strongly suggests that the me~urement of serum hemoglobin underestima~s the true blood cell destruction occurring during certain types of exercise. It seems then, that leucocyte lysis could be a factor responsible for a part of the changes in lysozyme activity reported here. This is further substantiated by the fact that the greatest changes both in lysozyme activity and in serum hemoglobin occur in the groups performing “impact” types of exercise, known to cause hemolysis substantially more than ergometer exercise (Table II); the latter being associated with more moderate increase in lysozyme. It appears from the present data that exercise of very high intensity and/ or long duration does not cause an increase in serum lysozyme activity above the range reported for reference values. It is also apparent that, if exercise is moderate and long enough, a rise in activity may be compensa~d for during the stress itself (Fig. 3). Hansen et al. 1321 have reported a lysozyme turnover rate of 76% per h. It thus seems probable that a very effective inactivationelimination mechanism for lysozyme exists in the human body. Acknowledgements Appreciation is extended to Prof. Dr J. Keul and members of the sport medicine staff for their cooperation in obtaining some of the blood samples needed for this study. Supported by the Bundesinstitut ftii’ ~po~~ssens~h~~n. References 1 P.O. Schmidt and F.W. Schmidt, Klin. Wochenschr., 42 (1964) 75 2 G. Siest and M.M. Galteau. in: Proc. 2nd Int. Colloquium Automatiiation and Prospective Biology, S. Karger. Basel, 1973, p. 223 3 B. AhIborg and J. Brohult. Fdrsvarsmed. (Stockholm), 2 (1966) 35 4 P.D. Griffiths, Clin. Chim. Acta, 13 (1966) 413 5 G. HaraIambie and J. Keul, Med. Sci. Sport, 3 (1971) 79 6 W. I&f. B. Klein and E. MLUIer. Med. Klin., 67 (1972) 195 7 G. Haralambie, in: Proc. 2nd Int. Colloquium Automatisation and Prospective Biology, S. Karger, Basel, 1973, p. 243 8 G. Hawdambie and F.J. Cemy, in press 9 J.C. Daniels, M. Fukushima, D. Larson, S. Abston and S. Ritzmann, Texas Rep. Biol. Med., 29 (1971) 13 10 G. Mancini, A. Carbonara and J. Heremans. Immunochem., 2 (1965) 235 11 M. Harboe, Stand. J. Clin. Lab. Invest., 11 (1959) 66 12 DJ. Prockop and W.D. Davidson, New En& J. Med., 270 (1964) 269 13 J.R. Poortmans. GIin. Sci., 43 (1972) 115

224 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Miiller, 5th Int. Symp. Clinical Enzymology, Venice, 1973, in press J. Hays&t, P. Perillie and S. Finch, New En& J. Med., 279 (1968) 506 H. Asamer, F. Schmalzl and H. Braunsteiner, K&n. Wochenschr., 49 (1971) 587 H. Haupt and N. Heimburger, Hoppe Seiler’s Z. Physioi. Chem., 353 (1972) 1125 F. Tischendorf, M. Tischendorf and G. Ledderose, in: A. Engelhard and H. Lommel (eds), Serumproteine, Verl. Chemie. Weinheim, 1974, p. 147 J. McKechnie, W. Leary and S. Joubert, Sth. Afr. Med. J., 41 (1967) 722 L. Rose, J. Bousser and K. Cooper, J. Appl. F’hysiol.. 29 (1970) 355 F. Nuttal and B. Jones, J. Lab. Clin. Med., 71 (1968) 847 Chr. De Duve, in A. de Reuck and M. Cameron (eds), Lysosomes, CIBA Found. Symp., Churchii, London, 1963, p. 1 R.A. Greenwald, AS. Josephson, H.S. Diamond and A. Tsang, J. Clin. Invest., 51 (1972) 2264 G. Haralambie, Clin. Chim. Acta, 27 (1970) 475 G. Haralambie and J. Keul, Xrztl. Forschg., 24 (1970) 112 G. Bole and J. Leutz. J. Lab. Clin. Med., 70 (1967) 880 H. Cleve and G. Strohmeyer, Klin. Wochenschr., 45 (1967) 1051 P. Flanagan and F. Lionetti, Blood, 10 (1955) 497 F. Leibetseder, M. Angerer and H. Schrijcksnadel, Sportmed. Ergebnisse der IX. Olymp. Winterspiele. Universitlt Innsburek. 1966, p. 127 K. Jung, Med. Welt, 23 (1972) 1105 K. Bichler. E. Lachmann and F. Porzsoit, Sportarzt Sportmed., 23 (1972) 9 N.E. Hansen, H. Karle, V. Andexsen and K. (plgaard, J. Clin. Invest., 51 (1972) 1146