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The effect of lactic acid on erythropoietin production and the rate of disappearance of erythropoietin from rat plasma during exercise
Life Sciences Yol, 10, Part II, pp, i1-17, 1971 . Printed in Great Britain
Pergamon Press
THA TFFACT OF LACTIC ACID OA ARYTHROPOI$TIN PRODDCTION A~ TH>s RATA OF DISAPPEARANCE OF ERYTHROPOIATIN FROlL ßAT PLASi(A DURING S%SRCISE Jaroslav Ziv~r, Jan Neuwirt and Tomsé TrdvniL'ek Department of Pathological Physiology, Faculty of
General Medicine, Charles University, U neaocnice 5, Praha 2, Czechoslovakia (Received ß October 1970; in final form 18 November 1970)
RÉYNAFARJE (1) has found an increase in hemoglobin and hematocrit in volunteers performing ezercise at sea level and at an altitude corresponding to 4,540 m. Heßias explained these findings due to alterations in hemoconcentration and blood distribution. However, on the other hand, it has recently been shown (2) that erythropoietin (AP) activity in the plasma of rats performing ezercise depends on the duration of ezercise and on the tension of ozygen in the inspired air. The ltiP activity in plasma was increased in rats performing work is mild hypozia. The exposure of rata to exercise of long duration at higher altitudes had a reverse effect and EP activity was decreased. An explanation for these findings was sought . The level of lactic acid after both hypozia and ezercise of long duration at the higher altitude was significantly increased. The effect of lactic acid on $P activity was determined . However, SP activity was not altered after incubation in vitro with lactic acid . In the present study an attempt was made to find out if lactic acid affected AP formation and if the rate of disappearance of ezogenous AP from plasma was altered in rats performing 11
12
Erythropoietin Producüon
Vol. 10, No. 1
exercise . Materials and Methods lisle Wistar rats, weighing from 250 - 280 g, acclimatized to laboratory conditions for at least two Weeks were used. Lactic acid and
roduction of EP . FP production in rats was
stimulated by reduction of circulating red cell mesa . The right carotid artery was cannulated under ether an9sthesia . Beven to eight mililiters of blood Were removed
3 hours after cannulation,
and immediately replaced by transfusion into the tail vein using the same amount of heparinized plasma obtained from donor rats . Ho significant bleeding occurred during or following the operation . The anemic rats Were divided into two groups . each control rat Was given 2 ml saline into the tail vein immediately after transfusion and 1 ml of saline 2 and 4 hours later. The rats from the second group sere given the same amount of a molar solution of lactic acid at the same time intervals . The blood Was withdrawn from the abdominal aorta into heparinized syringes 6 hours after the transfusion . The blood was centrifuged 30 minutes at 2,200 G. The plasma from 5 - 6 rata was pooled, stored at -15°C until EP assay (3) . The hematocrit was determined by the macrohematocrit method . Lactic acid concentration in the rat arterial blood was determined by enaymatic test - Lactate Biochemica Test Combination, C . F . Boehringer and Schone G .M .B .H . Mannheim, Germany, Biochemical Department . The rate of disappearance of EP from rat plasma . The SP isolated from the urine of anemic patients at our laboratory was used (4) . Sia International Standard Units of FP were given into the tail vein . A narrow cylindrical revolving cage on a horizontal aid s whose turning speed Was 4 meters e minute was used to induce exercise in rata (2) . Rate were subjected to exercise in
Erythropoietin Production
Vol. 10, No. 1
13
the cage for periods of 2, 4 and 8 hours . Plasma from 5 - ~ rats was pooled, stored at -15°0 until EP assay. EP assay was carried out on polycythemic, white, H mice (3) " Polycythemia was induced by two injections of 0 .8 ml of ?0 ~ erythrocyte suspension . On the sixth and seventh day after the second erythrocyte injection, 0 .5 ml of assayed plasma was injected inzraperitoneally and 24 hours later, 0.5 ~uC of 59Fe (citrate) in 0.4 ml of saline was injected intravenously . Forty eight hour incorporation into circulating red cell mass was determined . Reticulocyte counts were made from blood films stained with brilliant cresyl blue . Each plasma sample was assayed on a group of 6 - 8 mice . The statistical significance of the results was compared using the Student's t - test . Differences significant at the level were considered as significant . Results Fig. 1
shows EP activity in the plasma of the anemic rata
after the administration of saline or lactic acid . No significant difference between plasma EP activity was found in the two groups of rats (p ~ 0 .05) . Table I
illustrates results of hematocrit and lactic acid
concentration in the rat arterial blood 15 and 360 minutes after the administration of saline or lactic acid (or 15 and 360 minu tes after the induction of anemia) . There were no significant differences between the hematocrite in the rats (p > 0.05) . Lactic acid concentration was significantly increased 15 and 360 minutes after lactic acid injection (p ~ 0 .05) . The rate of disappearance of EP from the plasma of rats is depicted in Fig . 2. The EP activity is expressed as a percentage of the control value . EP activity in plasma 5 minutes after the
14
Vol, 10, No . 1
Erythropoietin Production
0
9.ß~INCORPORATION
® RTC
. 0
PLASMA " LACTIC ACIO
PLASMA " SALINE
FIG . 1
The EP activity in the plasma of anemic rats after administration of saline or lactic acid . Bars indicate + one standard deviation; n = 7; aTC = the number oP reticulocyteâ per 1,000~red blood cells. PLASMA EP - NORMAL PLASMA EP - EXERCISE _____ y. 100:
R25
50_
.01" 5'
2
4
0.15 ,12 8 hours
FIG. 2 The rate oß disappearance of erogenous EP Brom rat plasma . The numbers situated above curves represent EP activity of rat plasma ezpressed as percentage of radioiron incorporation into circulating red cell mass in polycythemic mice ; n = 7.
Erythropoietin Production
Vol . 10, No . 1
15
administration of exogenous BP Was as the control value . No significant difference was found (p y o .05) . TABLE I Hematocrit and lactic acid concentration in the arteriaY blood of the anemic rata
Saline
AN~üA 15 min. Lactic acid
AI~.1~A 360 min. Saline Lactic acid
Hematocrit (9~)
20 .7 + 1 .3 -
21 .5 + _ 1.1
20 .4 ± 1 .5
20 .5 ± 0 .9
Lactic acid (~)
19 .5 + 2 .2 -
47 .8 + 4 .1
20 .2 + 2 .0
35 " 1 ± 3.3
ues are means +
ne s
ar
e
a
oni n=
Discussion The changes in plasma EP activity depend on the rate of EP production and on the rate of EP disanpearaace from the plasma (5) . It . has been shown previously (2) that ~P activity increases in the plasma of rats performing ezercise at low altitudes and decreases in the plasma of rats performing strenuous ezercise at high altitudes for a long period. It is generally known that lactic acid concentration increases in the plasma during ezercise (6) . Since plasma EP concentration, however, was not changed after incubation in vitro with increased concentration of lactic acid (2), it could not be ezcluded that lactic acid has an effect on EP formation. In this ezperiment EP production was stimulated by induction of anemia and lactic acid concentration in plasma was increased 2 - 3 times above the normal level . The activity of plasma EP was not altered
It would indicate that
EP production is independent of lactic acid concentration in plasma, although it cannot be ezcluded that lactic acid concent-
16
Erythropoietin Producüon
Vol. 10, No. 1
ration at the site s) of EP production in individuals performing e:ercise is mush higher than in the plasma and that only a high concentration of lactic acid impairs EP production . Altland et al .(7) have reported that serum glutamic-oxalacetic and glutamic-pyruvic transaminase, serum aldolase, serum lactic debydrogenase and blood urea nitrogen were increased and fatty changes sad necrosis were found in the renal tubular epithelium and in the liver in rats performing exercise for periods of 16 hours at sea level . Exercise at altitude corresponding to 21,500 ft . produced greater changes in serum enzymes and in tissues than in either at sea level (8) . The changes in the kidney tissue /kidney is a main site of SP production (9)/ or elevated level of serum enzymes in plasma may be important in the observed decrease of EP production . On the other hand the hypothesis that greater amount of EP disappears from the plasma during exercise was tested . It was suggested that EP may escape more quickly from plasma into eztra vascular spaces, may be more rapidly inactivated or the renal threshold for EP may decrease during exercise . This hypothesis, however, was not confirmed. Also the explanation for changes of EP activity in the plasma during exercise at altitude are not known. This complicated question can only be solved by further research . Summary Lactic acid does not influence EP formation in anemic rata . There are no differences in the disappearance of exogenous EP from plasma between normal rats and rata performing exercise at normal atmospheric pressure . Acknowledgement - The authors are grateful to Dr . A. Hides who corrected the English language .
Vol. 10, No. 1
Erythropoietin Production
17
References l.
C . RBIIUI~ABJB, In:Proceedinge from Symposium Interlaken
2.
J . LIVNg, T. TR1PNfCEg and J . NEUYPIRT, Amer . J . Physiol . (in Press) .
3.
J. LIYN~, J . NEUWIRT and T. TRAYN2CES, Endocrinol . ewer . 4, 143 (1970) "
4.
J. ZIVN~, J . NEUWIRT, T . TRIYNtCEB and R. N&UWIRTOPI,
5.
P " 73, ed . W .H . Weihe, Pergamon Preen, London (1964) .
Pi~ysiol . bohemoslov . 18, 379 (1969) "
S.B . SRANTZ and L.O . JACOBSON, Erythropoietin and the Regulation of Erythropoiesis, p" 56, The University of Chicago Press , Chicago and London (1970) .
6.
E .J . Van LIERE and J .C . STICSNEY, Hypozia, p.64, The
7.
P.D . ALTLAND and B . HIGHIIA,N, Amer . J . Physiol. 201, 393 j1961) .
8.
P.D . ALTLAND, B . HIGHILAN and B.D . NEISON, Amer . J . Physiol. 214, 28 (1969) .
9.
L.O . JACOSSON, E . GOLDWASSER, W . FRIED and L. PLZAS, Nature 633 (1957) "
University of Chicago Press , Chicago and London (1963) .
Pergamon Press
THA TFFACT OF LACTIC ACID OA ARYTHROPOI$TIN PRODDCTION A~ TH>s RATA OF DISAPPEARANCE OF ERYTHROPOIATIN FROlL ßAT PLASi(A DURING S%SRCISE Jaroslav Ziv~r, Jan Neuwirt and Tomsé TrdvniL'ek Department of Pathological Physiology, Faculty of
General Medicine, Charles University, U neaocnice 5, Praha 2, Czechoslovakia (Received ß October 1970; in final form 18 November 1970)
RÉYNAFARJE (1) has found an increase in hemoglobin and hematocrit in volunteers performing ezercise at sea level and at an altitude corresponding to 4,540 m. Heßias explained these findings due to alterations in hemoconcentration and blood distribution. However, on the other hand, it has recently been shown (2) that erythropoietin (AP) activity in the plasma of rats performing ezercise depends on the duration of ezercise and on the tension of ozygen in the inspired air. The ltiP activity in plasma was increased in rats performing work is mild hypozia. The exposure of rata to exercise of long duration at higher altitudes had a reverse effect and EP activity was decreased. An explanation for these findings was sought . The level of lactic acid after both hypozia and ezercise of long duration at the higher altitude was significantly increased. The effect of lactic acid on $P activity was determined . However, SP activity was not altered after incubation in vitro with lactic acid . In the present study an attempt was made to find out if lactic acid affected AP formation and if the rate of disappearance of ezogenous AP from plasma was altered in rats performing 11
12
Erythropoietin Producüon
Vol. 10, No. 1
exercise . Materials and Methods lisle Wistar rats, weighing from 250 - 280 g, acclimatized to laboratory conditions for at least two Weeks were used. Lactic acid and
roduction of EP . FP production in rats was
stimulated by reduction of circulating red cell mesa . The right carotid artery was cannulated under ether an9sthesia . Beven to eight mililiters of blood Were removed
3 hours after cannulation,
and immediately replaced by transfusion into the tail vein using the same amount of heparinized plasma obtained from donor rats . Ho significant bleeding occurred during or following the operation . The anemic rats Were divided into two groups . each control rat Was given 2 ml saline into the tail vein immediately after transfusion and 1 ml of saline 2 and 4 hours later. The rats from the second group sere given the same amount of a molar solution of lactic acid at the same time intervals . The blood Was withdrawn from the abdominal aorta into heparinized syringes 6 hours after the transfusion . The blood was centrifuged 30 minutes at 2,200 G. The plasma from 5 - 6 rata was pooled, stored at -15°C until EP assay (3) . The hematocrit was determined by the macrohematocrit method . Lactic acid concentration in the rat arterial blood was determined by enaymatic test - Lactate Biochemica Test Combination, C . F . Boehringer and Schone G .M .B .H . Mannheim, Germany, Biochemical Department . The rate of disappearance of EP from rat plasma . The SP isolated from the urine of anemic patients at our laboratory was used (4) . Sia International Standard Units of FP were given into the tail vein . A narrow cylindrical revolving cage on a horizontal aid s whose turning speed Was 4 meters e minute was used to induce exercise in rata (2) . Rate were subjected to exercise in
Erythropoietin Production
Vol. 10, No. 1
13
the cage for periods of 2, 4 and 8 hours . Plasma from 5 - ~ rats was pooled, stored at -15°0 until EP assay. EP assay was carried out on polycythemic, white, H mice (3) " Polycythemia was induced by two injections of 0 .8 ml of ?0 ~ erythrocyte suspension . On the sixth and seventh day after the second erythrocyte injection, 0 .5 ml of assayed plasma was injected inzraperitoneally and 24 hours later, 0.5 ~uC of 59Fe (citrate) in 0.4 ml of saline was injected intravenously . Forty eight hour incorporation into circulating red cell mass was determined . Reticulocyte counts were made from blood films stained with brilliant cresyl blue . Each plasma sample was assayed on a group of 6 - 8 mice . The statistical significance of the results was compared using the Student's t - test . Differences significant at the level were considered as significant . Results Fig. 1
shows EP activity in the plasma of the anemic rata
after the administration of saline or lactic acid . No significant difference between plasma EP activity was found in the two groups of rats (p ~ 0 .05) . Table I
illustrates results of hematocrit and lactic acid
concentration in the rat arterial blood 15 and 360 minutes after the administration of saline or lactic acid (or 15 and 360 minu tes after the induction of anemia) . There were no significant differences between the hematocrite in the rats (p > 0.05) . Lactic acid concentration was significantly increased 15 and 360 minutes after lactic acid injection (p ~ 0 .05) . The rate of disappearance of EP from the plasma of rats is depicted in Fig . 2. The EP activity is expressed as a percentage of the control value . EP activity in plasma 5 minutes after the
14
Vol, 10, No . 1
Erythropoietin Production
0
9.ß~INCORPORATION
® RTC
. 0
PLASMA " LACTIC ACIO
PLASMA " SALINE
FIG . 1
The EP activity in the plasma of anemic rats after administration of saline or lactic acid . Bars indicate + one standard deviation; n = 7; aTC = the number oP reticulocyteâ per 1,000~red blood cells. PLASMA EP - NORMAL PLASMA EP - EXERCISE _____ y. 100:
R25
50_
.01" 5'
2
4
0.15 ,12 8 hours
FIG. 2 The rate oß disappearance of erogenous EP Brom rat plasma . The numbers situated above curves represent EP activity of rat plasma ezpressed as percentage of radioiron incorporation into circulating red cell mass in polycythemic mice ; n = 7.
Erythropoietin Production
Vol . 10, No . 1
15
administration of exogenous BP Was as the control value . No significant difference was found (p y o .05) . TABLE I Hematocrit and lactic acid concentration in the arteriaY blood of the anemic rata
Saline
AN~üA 15 min. Lactic acid
AI~.1~A 360 min. Saline Lactic acid
Hematocrit (9~)
20 .7 + 1 .3 -
21 .5 + _ 1.1
20 .4 ± 1 .5
20 .5 ± 0 .9
Lactic acid (~)
19 .5 + 2 .2 -
47 .8 + 4 .1
20 .2 + 2 .0
35 " 1 ± 3.3
ues are means +
ne s
ar
e
a
oni n=
Discussion The changes in plasma EP activity depend on the rate of EP production and on the rate of EP disanpearaace from the plasma (5) . It . has been shown previously (2) that ~P activity increases in the plasma of rats performing ezercise at low altitudes and decreases in the plasma of rats performing strenuous ezercise at high altitudes for a long period. It is generally known that lactic acid concentration increases in the plasma during ezercise (6) . Since plasma EP concentration, however, was not changed after incubation in vitro with increased concentration of lactic acid (2), it could not be ezcluded that lactic acid has an effect on EP formation. In this ezperiment EP production was stimulated by induction of anemia and lactic acid concentration in plasma was increased 2 - 3 times above the normal level . The activity of plasma EP was not altered
It would indicate that
EP production is independent of lactic acid concentration in plasma, although it cannot be ezcluded that lactic acid concent-
16
Erythropoietin Producüon
Vol. 10, No. 1
ration at the site s) of EP production in individuals performing e:ercise is mush higher than in the plasma and that only a high concentration of lactic acid impairs EP production . Altland et al .(7) have reported that serum glutamic-oxalacetic and glutamic-pyruvic transaminase, serum aldolase, serum lactic debydrogenase and blood urea nitrogen were increased and fatty changes sad necrosis were found in the renal tubular epithelium and in the liver in rats performing exercise for periods of 16 hours at sea level . Exercise at altitude corresponding to 21,500 ft . produced greater changes in serum enzymes and in tissues than in either at sea level (8) . The changes in the kidney tissue /kidney is a main site of SP production (9)/ or elevated level of serum enzymes in plasma may be important in the observed decrease of EP production . On the other hand the hypothesis that greater amount of EP disappears from the plasma during exercise was tested . It was suggested that EP may escape more quickly from plasma into eztra vascular spaces, may be more rapidly inactivated or the renal threshold for EP may decrease during exercise . This hypothesis, however, was not confirmed. Also the explanation for changes of EP activity in the plasma during exercise at altitude are not known. This complicated question can only be solved by further research . Summary Lactic acid does not influence EP formation in anemic rata . There are no differences in the disappearance of exogenous EP from plasma between normal rats and rata performing exercise at normal atmospheric pressure . Acknowledgement - The authors are grateful to Dr . A. Hides who corrected the English language .
Vol. 10, No. 1
Erythropoietin Production
17
References l.
C . RBIIUI~ABJB, In:Proceedinge from Symposium Interlaken
2.
J . LIVNg, T. TR1PNfCEg and J . NEUYPIRT, Amer . J . Physiol . (in Press) .
3.
J. LIYN~, J . NEUWIRT and T. TRAYN2CES, Endocrinol . ewer . 4, 143 (1970) "
4.
J. ZIVN~, J . NEUWIRT, T . TRIYNtCEB and R. N&UWIRTOPI,
5.
P " 73, ed . W .H . Weihe, Pergamon Preen, London (1964) .
Pi~ysiol . bohemoslov . 18, 379 (1969) "
S.B . SRANTZ and L.O . JACOBSON, Erythropoietin and the Regulation of Erythropoiesis, p" 56, The University of Chicago Press , Chicago and London (1970) .
6.
E .J . Van LIERE and J .C . STICSNEY, Hypozia, p.64, The
7.
P.D . ALTLAND and B . HIGHIIA,N, Amer . J . Physiol. 201, 393 j1961) .
8.
P.D . ALTLAND, B . HIGHILAN and B.D . NEISON, Amer . J . Physiol. 214, 28 (1969) .
9.
L.O . JACOSSON, E . GOLDWASSER, W . FRIED and L. PLZAS, Nature 633 (1957) "
University of Chicago Press , Chicago and London (1963) .