~pment, 55 (1990) 199--206
199
nd Ltd.
CAL T R A I N I N G ON T H E FUN( ED BLOOD CELLS
.'HANGES
IRGER b and KRZYSZTOF SPODARYKL LESZEK BERGER a STEE AL °Academy of Physical Education, Dept. of Physiology, 31571 Krakow, K Biochemical Lab. of Clinical Hospital, Krakow and Clnstitute oof Sport, D, (Poland)
52A, ~Central 'ogy, Warsaw
(Received March 5th, 1990)
SUMMARY
Eleven male elite endurance-trained athletes and am 10 mab gth-trained athletes were compared to a non-trained group o f : men, to c Le effect of 3sc U 1 1 1 ¢ indic I I I g l I U t I L O L ~ / 131 I U g l I cell t ~ U l l mem11~ training on some haematological parameters and some e-fractionated by centrifugation in Percoll Pel brane properties. Erythrocytes were age-fractionat gradients. It has been found that in the reticuloc~.ytes and young erythrocyte rtes of endurance trained athletes activity of acetylcholine dcholinesterase (ACHE) and concerntration o f glutathione (GSH) were higher than in stren!gth-trained athletes and nd control. con The red cell osmotic fragility (RCOF) and glycerol lysis time (GLT) of young cells were similar in all investigated groups. The endurance endur~ training indicating chronic chr adaptation mechanisms in significant changes of red rec cell metabolism but non memti brane properties.
Key words: Red blood cells cells; Ageing; Physical training INTRODUCTION
Exercise physiologists are continually investigating possible mechanism for increased maximal oxyg ygen utilization (VO2m~) resulting from a vigorus training program. A factor considered tered recently is the difference in the age distribution of erytlrrocytes between sedentar rotary and well trained subjects. Edwards and Staub [1] fcrand that young blood cellss have an oxygen association rate 25°7o faster than older erythrocytes. In young erythroc rythrocytes higher concentrations of 2,3 DPG was found founc [2] and it was suggested that the decreased Hb-O 2 affinity after training car can be explained by the presence race of more young erythrocytes in the blood of trained subjects [3]. As demonstrated ,ated by many authors, training cause to increased of red cell 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
atologic changes of athletes are k and functional changes in erythJ
still little is veil-trained
~tudy was to compare the basal re haemoglobin concentration (Hb) mean cell haemoglobin concentrat indicators of red cell m e m b r a n e acetylcholinesterase (ACHE), level of glutathione in the 1 reduc osmotic fragility (RCOF) and glycerol lysis time (GLT), (GL both ance sports (cycling, rowing) and strength sports (judo, v training, to sedentary healthy controls. The bioct biochemical 1: mined both in unfractionated and fractionated to ag, ge groups
meters: red rit (PCV), ~), reticuloactivity of H), red cell rom endurJring basal ~ere detercells.
MATERIALS AND METHODS
Subjects healthy noJ nale volunControl group (C) was composed f r o m eleven he ~d in heavy teers, aged 19--23. All were moderately active but ~ut none i reg~ me Polish l-'OllSn t)lymplc team am - - Seul exercise. Group of athletes were composed from tt the sports - - male, 6 cyc'88 and was divided into two groups: group E - - endurance e~ 5 judo, 5 wrestlers. All athlists, 5 rowers and group S - - strength sports - - male, m~ ae Olympic OlyI letes were in a excellent state of fitness at the time of o the study; during the Games most of them won Olympic medals.
Blood sampling ect in a seated position from the The blood samples were drawn from each subje~ aft 3 antecubital vein after overnight fasting. The blood specimens were drawn after ;ubjects were free of acute exercise. days at which time all sub
iochemical analysis Haematological and bioch~ CV and M C H C were determined by standard haematolo~gical RBC, HB, PCV, MCV ctes wer were filrr of procedures. Reticulocytes ~ere performed by counting at least 1000 cells on films ~latelles lly with new methylene blue. Then, white cells and plat cells stained supravitally ler's method [7]. Red cells were washed three times in a p~hoswere removed by Beutle ~olution; ion: the packed red cells was divided into two parts: one phate buffered saline solution (1 part (0.2 ml) was keptt without further treatment as the uncentrifuged cells (uc), centrifuga~.5 ml) was used for separation by density-gradient centrif another part of cells (0.5 (G tion. Activity of acety'lcholinesterase (ACHE - - EC3.1.1.7) and glutathione (GSH) RCOF) levels were determined as described by Beutler [7]. Red cell osmotic fragility (RC w / v to was measured in H E P~ES E S buffered saline (pH 7.4) over a range of 0.90O7o w/ M to 17 m M NaCI by method of Dacie and Lewis [8] and w a s 0.10o7o w/v, i.e. 154 mM ttion of expressed as concentration c NaC1 causing 50°7o lysis. Glycerol lysis time (GLT) w a s
201 o f Gottfried and Robertson [9] an, sity to fall to half the initial value. ythrocytes, i.e., layer 1 + layer 2.
as the time G L T were
:-fractionated by centrifugation in bed by Vettore et ai. [10]. F o u r la'. studied further (Fig. 1). Each cell layer was separate( ~arated, diluted buffered saline and washed two times. The activity o f ACh] determined in each layer, as described above.
)harmacia, rosen to be phosphate level were
I i
1
young
2 ~i~!i~i~i:~i~ !ii!i
ii~!i!ii~i i!iii~ii!!iii i
?////iiiiiii!ii!
.,m
4
ii
oLd
Fig. 1. Typical Percoll densit:y gradient separation and the four layers that were sampled and studied.
U-test was used for comparison oJ The data shown are the mean val ance.
.~s from dif.; P < 0.05
Figure 2 shows the comparison of haematolotIgical pan whole blood of control and trained subjects. The haemoglol h was lower in endurance athletes than in control subj~jects and 15.5; 15.3 g" 100 ml -I, respectively). Red cell mean c o r p u s c (MCHC) was lower for endurance athletes (33.6 g • 100 ml-ll g- 100 m1-1) and strength (34.8 g" 100 m1-1) subjects. subjects Erythrc (MCV) measured in investigated subjects were: 81.3;; 87.1 anc
ermined in ration (Hb) detes (14.4; acentration mtrol (35.0 .'ell volume group C, E
Hb ( g . l O 0 r n ( q )
16-
B
1'4CV ( f l )
100 ,~,
15
,. • ,~,
90
° ol
,_~. 80
14
.,
70
13, -* S D
o~ C
0,3
0,4 E
-+ S D
33
S
C
D
b4CHC ( g ' 1 0 0 m [ 1 }
38.
3.s
4.3
E
S
R e t ( 1'1"I )
20.
.~" 36
;
-_**
t
34
*
:
15.
:
~*
10
•
.
.'. =e
s.
32 -+ SD
o,g C
io E
o,6 S
o
:
-. SD
2.4 C
2,s E
2,5 S
c( Fig. 2. The haematological parameters of peripheral blood in individual groups of subjects C - - control led subjects; S - - strength trained subjects; the median is indicated by aa horigroup; E - - endurance trained zontal line; C v s . E and C vs.:. S ; * P < 0 . 0 5 ; * * P < 0.01.
203 rly, only for endurance athletes re ount (Ret) :hletes (8.3 n for control group (5.9 1" 1"1) ant ytes in dirLt endurance athletes had more you ntrol) and strength-trained subject.' e, especially of old erythrocytes, :ulocytosis BC) after the training lead to a shi er erythrodimination al blood. Schmidt et al. [6] suggest of the oldest cells, which possess more useless haemoglobin haer For oxygen blood voltransport (e.g., methHb, HbA~c), the oxygen transpol transport capacit ume should be increased after training. Therefore, the haer changes in athletes take parts in two ways: (1) destruction and an elimin IC and (2) increase of erythropoiesis. Physical activity may not only cause direct red blood bit cell d [ntravascular haemolysis, but also accelerate red cell aging and an impair ity [11,12]. tBC resultbre~ The most likely explanation of haemolysis is mechanical mechan ing from trauma of the contracting muscle tissue. Ft Furthermo~ ical factors leads to negative changes in properties of red cells and a ell destructo an of lac tion. Mairbaurl et al. [13] showed a pronounced influx inf ruvate into nllUX o ofi lla¢ a c t a t e is l a C l l lited t e t a during ou the red cells during exhaustive exercise and this linflux infl the lactacidosis occurring in exercise. The acidication acidicatic o f the red cell cause a reducre~ glycolytic tion in the N A D / N A D H ratio and changes of the concentration of the glyco intermediates [14]. In this paper evidence for the stimulation of erytl •ythropoiesis and for the increased incre~ number of young erythrocytes in the blood of trained train~ subjects can be derived from f the data on Ret, MCHC, MCV and activity o f ACHE. AC~ It is known that the M C ( HC increases with increasing red cell age as a MCV decre :crease [15]; the activity offf AChE AC~ is useful indicator o f red cell age [16]. The main question is: are there any biochemical changes between young and old ~tary and well-trained endurance and strength athletes? Then, TI red blood cells in sedentar Acetylo we investigated AChE activity and level of GSH in red cells of different age. A mbrane-associated me-associated enzyme and take parts in ion exchan~ge; a cholinesterase is a membrane :e AChE activity with cell age has been reported by several se~ decrease in erythrocyte nzyme is one o f the most convenient markers of red blood cell investigators, so this enz' several age. The GSH plays ana important role in the protection of haemoglobin and se~ lzymatic proteins of RBC against the oxidative damage; it has other structural and enz' ',tivities of contents decreased with red cell aging. The AChE activitk ~nts decre~ been found that GSH content the four erythrocyte subpopulations and the unfractionated (Un) cells with the ical analysis are presented in Fig. 3A. Comparision of the results of their statistical aclusions: f ions: the AChE activities of Un cells and red cells from means leads to the conclusior locytes, young and middle-aged cells) were higher in RBC RB~ of layer 1, 2 and 3 reticuloc etes than sedentary and strength-trained subjects; the old cells endurance trained athletes roups had similar activity of ACHE. from all investigated grou! ation in the unfractionated (Un) to aged red blood cells and The GSH concentration .
.
.
.
.
.
,
.
•
•
•
E
¢c
LAYER 1
2
3
4
Un
OSH (~Jmot.mlRB(~1) 3,2 ** w ~ 2,8
~
+
bE ?c
2,4 2,0
LAYER 1
2
3
4
Un
Fig. 3. Mean values and S.D. for acetylcholinesterase (ACHE) activities (A) and levels of glutat lutathione (GSH) (B) of the four erythrocytic s u b p o p u l a t i o n s (layer l - - reti~ reticulocytes; layer 2 - - young erythrocytes; erythro layer 3 - - middle-aged erythrocytes; layer 4 - - old erythrocytes) and o f the unfractionated (Un) cells c in individual g r o u p s of subjects. s. C - - Control group; E - - endurance trained subjects; S - - strength th trained tr subjects; C v s . E and C v s . S;;; * P < 0.05; * * P < 0.01.
fractionated ones, withh the results o f their statistical analysis are presented on Fig. ncentration z-aged and old red cells (layer 3 and layer 4) the concentr~ 3B. In Un cells, middle-a ,'ntration of reduced glutathione¢'was similar for all groups, but in reticulocytes concentr, athletes. tly higher both in endurance- and strength-trained athletet of GSH was significantly H data for density-separated cells in general agreement with The AChE and GSH ,.ndurance:heless, in reticulocytes and young erythrocytes of endure others authors. Nevertheless than in tivity of AChE and GSH concentration were higher tha trained athletes the activit GSH es and control subjects. Muller et al. [171 reported that I strength-trained athletes than in aa was higher in trained men (long-distance runners) th~ concentration in plasma that untrained men. After the bicycle ergometer test, Ohno et al. [181 has found m acute physical exercise.' had some effects on red blood cell GSH. The hexose monoatenance of IMP) in red blood cells is related primarily to maintenan( phosphate pathway (HMP)
205
tate (GSH). Fornaini et al. [19] an I activity of the H M P in human
I. [20] have dls during
otic fragility (RCOF) and glycerol imilar in all investigated groups (F upport thesis about differences o )rane between trained and untrain, ity and lysis are affected by many factors, which are arc not quit mere tion of physico-biochemical changes in the red cell , training should await further study and uses more prq )recise metl blue show] Reticulocytes on films stained with methylene bl only from first class of Heilmeyer and Trachtenber['g scale, s( the reticulocytes in similar cell age. Endurance training trait as w, caused to changes the age of red blood cells (i.e., nmore y o u l but to biochemical changes of RBC due only the endurant AChE and level of GSH in old cells (layer 3 and lalyer 4) in were similar. Additional studies of influences of p])hysical tr poiesis function of bone marrow are needed to determine d lgll directly implicated in red cell biochemistry or are lmerely nl l ~ - r ~ l y ~sig
A
ROCF ( r a m NoCt)
) lysis time 3). Data of iochemical but fragilrhe defini;r physical cells were contained th training :irculation) activity of ted groups ae erythro:ercises are ~ l l l turnover. LIAIIIU
UI l¢U
(se k)
GLT
B
60
78
~o
77
50
•
40
76
75 •* S D
•
•
30
•
0.9 C
0.7 E
0.7
S
-+SD
9.7
C
g,6
E
10.0
S
Fig. 4. The red cell osmotic fragility (RCOF) and glycerol lysis time (GLT) of young cells (cells fron lay;ells from ers 1 and 2) in individual groups s oups of subjects. C - - Control group; E - - endurance-trained subjects; iects; S - strength-trained subjects; thee median is indicated by a horizontal line.
ring our work to clarify the effects ysical properties in human red bloc rance training, in haematological p aism: the increased erythrocyte tur :ant changes of the function of the
1 2 3 4
5 6
7 8 9
10 11 12 13
14 15 16 17
18
19 20
training on T, indicating
Lding higher
M.J. Edwards and N.C. Staub, Kinetics of 02 uptake by erythrocyte~ of cell age. J. Appl. Physiol., 21 (1966) 173--176. S. Haidas, D. Labie and J.C. Kaplan, 2,3-diphosphoglycer~ ,cerate content inity as a function of red cell age in normal individuals. Blood, 38 (1971 )t 463--467. 4 H. Mairbaurl, E. Humpeler and H. Passenhofer, Trainingg dependent c c ell density and erythrocytic oxygen transport. J. AppL Physiol., 55 (1983) 1403--1407 : U.A. Brodthagen, K.N. Knudsen, J. Hausen, R. Jordal, O. ( Kristens~ tulev, Red cell 2,3-DPG, A T P and mean cell volume in highly trained athletes. atl Eur. :iol., 53 (1985) 334--338. J.D. Robertson, R.J. M a u g h a n and R.L. Davidson, C h a n tges in red c~ related indices in response to distance running. Eur. J. Appl. Physiol., 57( 57(1988) 265-W. Schmidt, N. Maassen, F. Trost and D. Boning, Training Trai induc~ 91ood volume, erythrocyte turnover and haemoglobin oxygen binding proI ~erties. Eur ~iol., 57 (1988) 490--498. E. Beutler, Red cell metabolism: a manual o f biochemical rr biochemical methods. Grune and Stratton, New York, 1984. J.V. Dacie and S.M. Lewis, Practical Haematology, Churchill ChurcI Livingstone, London, 1984. E.L. Gottfried and N.A. Robertson, Glycerol lysis 'sis time as a screening test for erythrocyte 'te diso disorders. J. Lab. Clin. Med., 83 (1974) 323--333. L. Vettore, M.C. DeMatteis and P. Zampini, A new density densi gradient system for the separat ~aration of h u m a n red blood cells. Am. J. Hematol., 8 (1980) 291--297. 291--297 W.M. Reinhart and S. Chein, Stomatocytic transformation transformati¢ of red cells after m a r a t h o n running. rut Am. J. HematoL, 19 (1985) 201--204. G. Galen and R.J. Davidson, Haemorheology of m maratho~ a r a t h o n running. Int. J. Sports Med., 6 (1985) q 136--138. H. Mairbaurl, W. Schobersbel obersberger, W. Hasibeder, G. Schwaberger, G. Gaesser and K.R. Tanaka, Ta ~siol., 55 Regulation of red cell 2,1 ~,,3-DPG and Hb-O2-affinity during acute exercise. Eur. J. Appl. Physic (1986) 174--180. Yosh G. Jacobasch, S. Min~tkami and S.M. Rapaport, Glycolysis of the erythrocyte, In: H. Yoshikawa (ed.), Cellular and mol ecular biology oferythrocytes, U r b a n Schwarzenberg, Berlin, 1974. holm, M.G. Luthra and D.J. H a n a h a n , Biochemical characterizati cterization of N.S. Cohen, J.E. Ekholm density-separated h u m aann erythrocytes. Biochim. Biophys. Acta, 419 (1976) 229--242. LS. Agutter, Changes in the activities of some membrane-associated enzymes em M. K a d l u b o w s k i a n d P.S. during in vivo ageing off the normal h u m a n erythrocyte. Br. J. Haematol., 33 (1977) 111--116. exercise on D. Muller, M. Kretzsc h m a r and J. Hubscher, Influence of training and acute physical exerc eroxides. es. (abstract), Proc. X X X I Int. Congr. o f Physiol. Sci., Helsinki, 1989, glutathione and lipid peroxides. $3086. H. O h n o , Y. Sato, K. Yamashita and R. Dei, The effect of brief physical exercise on free rradical terns in h u m a n red blood cells. Can. J. Physiol. Pharmacol., 64 (1985) 1263-1 scavenging enzyme systems 1265. "ythrocytes G. Fornaini, M. Dacia,, A. Accorsi, A. Fazi and E. Piatti, Glucose utilization in h u m a n erythrc during physical exercisee, Med. Sci. Sports Med. , 5 (1981) 322--324. cal exercise exerc on H. O h n o , H. W a t a n a b ee, C. Kishihara, M. Hishino and M. Taniguchi, Effect of physical hediates in men" crossover plot analysis, In: J. P o o r t m a n s and J. Niset ((eds.), blood glycolytic intermediates sell/, Biochemistry o f Exercise 11; University Park Press, Baltimore, 1981, pp. 100--107.
199
nd Ltd.
CAL T R A I N I N G ON T H E FUN( ED BLOOD CELLS
.'HANGES
IRGER b and KRZYSZTOF SPODARYKL LESZEK BERGER a STEE AL °Academy of Physical Education, Dept. of Physiology, 31571 Krakow, K Biochemical Lab. of Clinical Hospital, Krakow and Clnstitute oof Sport, D, (Poland)
52A, ~Central 'ogy, Warsaw
(Received March 5th, 1990)
SUMMARY
Eleven male elite endurance-trained athletes and am 10 mab gth-trained athletes were compared to a non-trained group o f : men, to c Le effect of 3sc U 1 1 1 ¢ indic I I I g l I U t I L O L ~ / 131 I U g l I cell t ~ U l l mem11~ training on some haematological parameters and some e-fractionated by centrifugation in Percoll Pel brane properties. Erythrocytes were age-fractionat gradients. It has been found that in the reticuloc~.ytes and young erythrocyte rtes of endurance trained athletes activity of acetylcholine dcholinesterase (ACHE) and concerntration o f glutathione (GSH) were higher than in stren!gth-trained athletes and nd control. con The red cell osmotic fragility (RCOF) and glycerol lysis time (GLT) of young cells were similar in all investigated groups. The endurance endur~ training indicating chronic chr adaptation mechanisms in significant changes of red rec cell metabolism but non memti brane properties.
Key words: Red blood cells cells; Ageing; Physical training INTRODUCTION
Exercise physiologists are continually investigating possible mechanism for increased maximal oxyg ygen utilization (VO2m~) resulting from a vigorus training program. A factor considered tered recently is the difference in the age distribution of erytlrrocytes between sedentar rotary and well trained subjects. Edwards and Staub [1] fcrand that young blood cellss have an oxygen association rate 25°7o faster than older erythrocytes. In young erythroc rythrocytes higher concentrations of 2,3 DPG was found founc [2] and it was suggested that the decreased Hb-O 2 affinity after training car can be explained by the presence race of more young erythrocytes in the blood of trained subjects [3]. As demonstrated ,ated by many authors, training cause to increased of red cell 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
atologic changes of athletes are k and functional changes in erythJ
still little is veil-trained
~tudy was to compare the basal re haemoglobin concentration (Hb) mean cell haemoglobin concentrat indicators of red cell m e m b r a n e acetylcholinesterase (ACHE), level of glutathione in the 1 reduc osmotic fragility (RCOF) and glycerol lysis time (GLT), (GL both ance sports (cycling, rowing) and strength sports (judo, v training, to sedentary healthy controls. The bioct biochemical 1: mined both in unfractionated and fractionated to ag, ge groups
meters: red rit (PCV), ~), reticuloactivity of H), red cell rom endurJring basal ~ere detercells.
MATERIALS AND METHODS
Subjects healthy noJ nale volunControl group (C) was composed f r o m eleven he ~d in heavy teers, aged 19--23. All were moderately active but ~ut none i reg~ me Polish l-'OllSn t)lymplc team am - - Seul exercise. Group of athletes were composed from tt the sports - - male, 6 cyc'88 and was divided into two groups: group E - - endurance e~ 5 judo, 5 wrestlers. All athlists, 5 rowers and group S - - strength sports - - male, m~ ae Olympic OlyI letes were in a excellent state of fitness at the time of o the study; during the Games most of them won Olympic medals.
Blood sampling ect in a seated position from the The blood samples were drawn from each subje~ aft 3 antecubital vein after overnight fasting. The blood specimens were drawn after ;ubjects were free of acute exercise. days at which time all sub
iochemical analysis Haematological and bioch~ CV and M C H C were determined by standard haematolo~gical RBC, HB, PCV, MCV ctes wer were filrr of procedures. Reticulocytes ~ere performed by counting at least 1000 cells on films ~latelles lly with new methylene blue. Then, white cells and plat cells stained supravitally ler's method [7]. Red cells were washed three times in a p~hoswere removed by Beutle ~olution; ion: the packed red cells was divided into two parts: one phate buffered saline solution (1 part (0.2 ml) was keptt without further treatment as the uncentrifuged cells (uc), centrifuga~.5 ml) was used for separation by density-gradient centrif another part of cells (0.5 (G tion. Activity of acety'lcholinesterase (ACHE - - EC3.1.1.7) and glutathione (GSH) RCOF) levels were determined as described by Beutler [7]. Red cell osmotic fragility (RC w / v to was measured in H E P~ES E S buffered saline (pH 7.4) over a range of 0.90O7o w/ M to 17 m M NaCI by method of Dacie and Lewis [8] and w a s 0.10o7o w/v, i.e. 154 mM ttion of expressed as concentration c NaC1 causing 50°7o lysis. Glycerol lysis time (GLT) w a s
201 o f Gottfried and Robertson [9] an, sity to fall to half the initial value. ythrocytes, i.e., layer 1 + layer 2.
as the time G L T were
:-fractionated by centrifugation in bed by Vettore et ai. [10]. F o u r la'. studied further (Fig. 1). Each cell layer was separate( ~arated, diluted buffered saline and washed two times. The activity o f ACh] determined in each layer, as described above.
)harmacia, rosen to be phosphate level were
I i
1
young
2 ~i~!i~i~i:~i~ !ii!i
ii~!i!ii~i i!iii~ii!!iii i
?////iiiiiii!ii!
.,m
4
ii
oLd
Fig. 1. Typical Percoll densit:y gradient separation and the four layers that were sampled and studied.
U-test was used for comparison oJ The data shown are the mean val ance.
.~s from dif.; P < 0.05
Figure 2 shows the comparison of haematolotIgical pan whole blood of control and trained subjects. The haemoglol h was lower in endurance athletes than in control subj~jects and 15.5; 15.3 g" 100 ml -I, respectively). Red cell mean c o r p u s c (MCHC) was lower for endurance athletes (33.6 g • 100 ml-ll g- 100 m1-1) and strength (34.8 g" 100 m1-1) subjects. subjects Erythrc (MCV) measured in investigated subjects were: 81.3;; 87.1 anc
ermined in ration (Hb) detes (14.4; acentration mtrol (35.0 .'ell volume group C, E
Hb ( g . l O 0 r n ( q )
16-
B
1'4CV ( f l )
100 ,~,
15
,. • ,~,
90
° ol
,_~. 80
14
.,
70
13, -* S D
o~ C
0,3
0,4 E
-+ S D
33
S
C
D
b4CHC ( g ' 1 0 0 m [ 1 }
38.
3.s
4.3
E
S
R e t ( 1'1"I )
20.
.~" 36
;
-_**
t
34
*
:
15.
:
~*
10
•
.
.'. =e
s.
32 -+ SD
o,g C
io E
o,6 S
o
:
-. SD
2.4 C
2,s E
2,5 S
c( Fig. 2. The haematological parameters of peripheral blood in individual groups of subjects C - - control led subjects; S - - strength trained subjects; the median is indicated by aa horigroup; E - - endurance trained zontal line; C v s . E and C vs.:. S ; * P < 0 . 0 5 ; * * P < 0.01.
203 rly, only for endurance athletes re ount (Ret) :hletes (8.3 n for control group (5.9 1" 1"1) ant ytes in dirLt endurance athletes had more you ntrol) and strength-trained subject.' e, especially of old erythrocytes, :ulocytosis BC) after the training lead to a shi er erythrodimination al blood. Schmidt et al. [6] suggest of the oldest cells, which possess more useless haemoglobin haer For oxygen blood voltransport (e.g., methHb, HbA~c), the oxygen transpol transport capacit ume should be increased after training. Therefore, the haer changes in athletes take parts in two ways: (1) destruction and an elimin IC and (2) increase of erythropoiesis. Physical activity may not only cause direct red blood bit cell d [ntravascular haemolysis, but also accelerate red cell aging and an impair ity [11,12]. tBC resultbre~ The most likely explanation of haemolysis is mechanical mechan ing from trauma of the contracting muscle tissue. Ft Furthermo~ ical factors leads to negative changes in properties of red cells and a ell destructo an of lac tion. Mairbaurl et al. [13] showed a pronounced influx inf ruvate into nllUX o ofi lla¢ a c t a t e is l a C l l lited t e t a during ou the red cells during exhaustive exercise and this linflux infl the lactacidosis occurring in exercise. The acidication acidicatic o f the red cell cause a reducre~ glycolytic tion in the N A D / N A D H ratio and changes of the concentration of the glyco intermediates [14]. In this paper evidence for the stimulation of erytl •ythropoiesis and for the increased incre~ number of young erythrocytes in the blood of trained train~ subjects can be derived from f the data on Ret, MCHC, MCV and activity o f ACHE. AC~ It is known that the M C ( HC increases with increasing red cell age as a MCV decre :crease [15]; the activity offf AChE AC~ is useful indicator o f red cell age [16]. The main question is: are there any biochemical changes between young and old ~tary and well-trained endurance and strength athletes? Then, TI red blood cells in sedentar Acetylo we investigated AChE activity and level of GSH in red cells of different age. A mbrane-associated me-associated enzyme and take parts in ion exchan~ge; a cholinesterase is a membrane :e AChE activity with cell age has been reported by several se~ decrease in erythrocyte nzyme is one o f the most convenient markers of red blood cell investigators, so this enz' several age. The GSH plays ana important role in the protection of haemoglobin and se~ lzymatic proteins of RBC against the oxidative damage; it has other structural and enz' ',tivities of contents decreased with red cell aging. The AChE activitk ~nts decre~ been found that GSH content the four erythrocyte subpopulations and the unfractionated (Un) cells with the ical analysis are presented in Fig. 3A. Comparision of the results of their statistical aclusions: f ions: the AChE activities of Un cells and red cells from means leads to the conclusior locytes, young and middle-aged cells) were higher in RBC RB~ of layer 1, 2 and 3 reticuloc etes than sedentary and strength-trained subjects; the old cells endurance trained athletes roups had similar activity of ACHE. from all investigated grou! ation in the unfractionated (Un) to aged red blood cells and The GSH concentration .
.
.
.
.
.
,
.
•
•
•
E
¢c
LAYER 1
2
3
4
Un
OSH (~Jmot.mlRB(~1) 3,2 ** w ~ 2,8
~
+
bE ?c
2,4 2,0
LAYER 1
2
3
4
Un
Fig. 3. Mean values and S.D. for acetylcholinesterase (ACHE) activities (A) and levels of glutat lutathione (GSH) (B) of the four erythrocytic s u b p o p u l a t i o n s (layer l - - reti~ reticulocytes; layer 2 - - young erythrocytes; erythro layer 3 - - middle-aged erythrocytes; layer 4 - - old erythrocytes) and o f the unfractionated (Un) cells c in individual g r o u p s of subjects. s. C - - Control group; E - - endurance trained subjects; S - - strength th trained tr subjects; C v s . E and C v s . S;;; * P < 0.05; * * P < 0.01.
fractionated ones, withh the results o f their statistical analysis are presented on Fig. ncentration z-aged and old red cells (layer 3 and layer 4) the concentr~ 3B. In Un cells, middle-a ,'ntration of reduced glutathione¢'was similar for all groups, but in reticulocytes concentr, athletes. tly higher both in endurance- and strength-trained athletet of GSH was significantly H data for density-separated cells in general agreement with The AChE and GSH ,.ndurance:heless, in reticulocytes and young erythrocytes of endure others authors. Nevertheless than in tivity of AChE and GSH concentration were higher tha trained athletes the activit GSH es and control subjects. Muller et al. [171 reported that I strength-trained athletes than in aa was higher in trained men (long-distance runners) th~ concentration in plasma that untrained men. After the bicycle ergometer test, Ohno et al. [181 has found m acute physical exercise.' had some effects on red blood cell GSH. The hexose monoatenance of IMP) in red blood cells is related primarily to maintenan( phosphate pathway (HMP)
205
tate (GSH). Fornaini et al. [19] an I activity of the H M P in human
I. [20] have dls during
otic fragility (RCOF) and glycerol imilar in all investigated groups (F upport thesis about differences o )rane between trained and untrain, ity and lysis are affected by many factors, which are arc not quit mere tion of physico-biochemical changes in the red cell , training should await further study and uses more prq )recise metl blue show] Reticulocytes on films stained with methylene bl only from first class of Heilmeyer and Trachtenber['g scale, s( the reticulocytes in similar cell age. Endurance training trait as w, caused to changes the age of red blood cells (i.e., nmore y o u l but to biochemical changes of RBC due only the endurant AChE and level of GSH in old cells (layer 3 and lalyer 4) in were similar. Additional studies of influences of p])hysical tr poiesis function of bone marrow are needed to determine d lgll directly implicated in red cell biochemistry or are lmerely nl l ~ - r ~ l y ~sig
A
ROCF ( r a m NoCt)
) lysis time 3). Data of iochemical but fragilrhe defini;r physical cells were contained th training :irculation) activity of ted groups ae erythro:ercises are ~ l l l turnover. LIAIIIU
UI l¢U
(se k)
GLT
B
60
78
~o
77
50
•
40
76
75 •* S D
•
•
30
•
0.9 C
0.7 E
0.7
S
-+SD
9.7
C
g,6
E
10.0
S
Fig. 4. The red cell osmotic fragility (RCOF) and glycerol lysis time (GLT) of young cells (cells fron lay;ells from ers 1 and 2) in individual groups s oups of subjects. C - - Control group; E - - endurance-trained subjects; iects; S - strength-trained subjects; thee median is indicated by a horizontal line.
ring our work to clarify the effects ysical properties in human red bloc rance training, in haematological p aism: the increased erythrocyte tur :ant changes of the function of the
1 2 3 4
5 6
7 8 9
10 11 12 13
14 15 16 17
18
19 20
training on T, indicating
Lding higher
M.J. Edwards and N.C. Staub, Kinetics of 02 uptake by erythrocyte~ of cell age. J. Appl. Physiol., 21 (1966) 173--176. S. Haidas, D. Labie and J.C. Kaplan, 2,3-diphosphoglycer~ ,cerate content inity as a function of red cell age in normal individuals. Blood, 38 (1971 )t 463--467. 4 H. Mairbaurl, E. Humpeler and H. Passenhofer, Trainingg dependent c c ell density and erythrocytic oxygen transport. J. AppL Physiol., 55 (1983) 1403--1407 : U.A. Brodthagen, K.N. Knudsen, J. Hausen, R. Jordal, O. ( Kristens~ tulev, Red cell 2,3-DPG, A T P and mean cell volume in highly trained athletes. atl Eur. :iol., 53 (1985) 334--338. J.D. Robertson, R.J. M a u g h a n and R.L. Davidson, C h a n tges in red c~ related indices in response to distance running. Eur. J. Appl. Physiol., 57( 57(1988) 265-W. Schmidt, N. Maassen, F. Trost and D. Boning, Training Trai induc~ 91ood volume, erythrocyte turnover and haemoglobin oxygen binding proI ~erties. Eur ~iol., 57 (1988) 490--498. E. Beutler, Red cell metabolism: a manual o f biochemical rr biochemical methods. Grune and Stratton, New York, 1984. J.V. Dacie and S.M. Lewis, Practical Haematology, Churchill ChurcI Livingstone, London, 1984. E.L. Gottfried and N.A. Robertson, Glycerol lysis 'sis time as a screening test for erythrocyte 'te diso disorders. J. Lab. Clin. Med., 83 (1974) 323--333. L. Vettore, M.C. DeMatteis and P. Zampini, A new density densi gradient system for the separat ~aration of h u m a n red blood cells. Am. J. Hematol., 8 (1980) 291--297. 291--297 W.M. Reinhart and S. Chein, Stomatocytic transformation transformati¢ of red cells after m a r a t h o n running. rut Am. J. HematoL, 19 (1985) 201--204. G. Galen and R.J. Davidson, Haemorheology of m maratho~ a r a t h o n running. Int. J. Sports Med., 6 (1985) q 136--138. H. Mairbaurl, W. Schobersbel obersberger, W. Hasibeder, G. Schwaberger, G. Gaesser and K.R. Tanaka, Ta ~siol., 55 Regulation of red cell 2,1 ~,,3-DPG and Hb-O2-affinity during acute exercise. Eur. J. Appl. Physic (1986) 174--180. Yosh G. Jacobasch, S. Min~tkami and S.M. Rapaport, Glycolysis of the erythrocyte, In: H. Yoshikawa (ed.), Cellular and mol ecular biology oferythrocytes, U r b a n Schwarzenberg, Berlin, 1974. holm, M.G. Luthra and D.J. H a n a h a n , Biochemical characterizati cterization of N.S. Cohen, J.E. Ekholm density-separated h u m aann erythrocytes. Biochim. Biophys. Acta, 419 (1976) 229--242. LS. Agutter, Changes in the activities of some membrane-associated enzymes em M. K a d l u b o w s k i a n d P.S. during in vivo ageing off the normal h u m a n erythrocyte. Br. J. Haematol., 33 (1977) 111--116. exercise on D. Muller, M. Kretzsc h m a r and J. Hubscher, Influence of training and acute physical exerc eroxides. es. (abstract), Proc. X X X I Int. Congr. o f Physiol. Sci., Helsinki, 1989, glutathione and lipid peroxides. $3086. H. O h n o , Y. Sato, K. Yamashita and R. Dei, The effect of brief physical exercise on free rradical terns in h u m a n red blood cells. Can. J. Physiol. Pharmacol., 64 (1985) 1263-1 scavenging enzyme systems 1265. "ythrocytes G. Fornaini, M. Dacia,, A. Accorsi, A. Fazi and E. Piatti, Glucose utilization in h u m a n erythrc during physical exercisee, Med. Sci. Sports Med. , 5 (1981) 322--324. cal exercise exerc on H. O h n o , H. W a t a n a b ee, C. Kishihara, M. Hishino and M. Taniguchi, Effect of physical hediates in men" crossover plot analysis, In: J. P o o r t m a n s and J. Niset ((eds.), blood glycolytic intermediates sell/, Biochemistry o f Exercise 11; University Park Press, Baltimore, 1981, pp. 100--107.
