purine metabolism involves synthesis and degradation of purine nucleotides , and determines the adenylate and guanylate pool value . in this way , it is responsible for the absolute value of intracellular atp and gtp concentrations .
the unique erythrocyte structure and metabolism is due to , among others , a deficiency of glutamine 5-phosphoribosyl 1-pyrophosphate ( prpp ) amidotransferase ( ec 2.4.2.14 ) and its inability to synthesize purine nucleotides in the de novo pathway .
thus , in erythrocytes these compounds are formed in the reactions of reutilization , i.e. the reutilization of free purine bases ( adenine , ade ; hypoxanthine , hyp ; guanine , gua ) and nucleosides ( adenosine , ado ; inosine , ino ; guanosine , guo ) .
the metabolic aim of these reactions is the introduction of these compounds into the high energy purine nucleotide synthesis pathways ( atp , adp , gtp and gdp ) ( dudzinska et al .
1.fig .  1overview of purine metabolism in human erythrocytes ( straight line ) and out of them ( dotted line ) .
ada adenosine deaminase ( ec 3.5.4.4 ) , ak adenosine kinase ( ec 2.7.1.20 ) , ampd amp deaminase ( ec 3.5.4.6 ) , aprt adenine phosphoribosyltranferase ( ec 2.4.2.7 ) , cn - i cytosolic amp - specific 5-nucleotidase ( ec 3.2.3.5 ) , cn - ii cytosolic imp and gmp - specific 5-nucleotidase ( ec 3.2.3.5 ) , gk guanosine kinase ( ec 2.7.1.73 ) , hgprt hypoxanthine - guanine phosphorybosyltransferase ( ec 2.4.2.8 ) , ik inosine kinase ( ec 2.7.1.73 ) , mtado methylothioadenosine , 5-nt-5-nucleotidase ( ec 3.1.3.5 ) , pnp purine nucleoside phosphorylase ( ec 2.4.2.1 ) , prpp 5-phosphoribosyl 1-pyrophosphate , sah s - adenosylhomocysteine , sahh s - adenosylhomocysteine hydrolase ( ec 3.3.1.1 ) , sam s - adenosylmethionine overview of purine metabolism in human erythrocytes ( straight line ) and out of them ( dotted line ) . based on simmonds et al .
ada adenosine deaminase ( ec 3.5.4.4 ) , ak adenosine kinase ( ec 2.7.1.20 ) , ampd amp deaminase ( ec 3.5.4.6 ) , aprt adenine phosphoribosyltranferase ( ec 2.4.2.7 ) , cn - i cytosolic amp - specific 5-nucleotidase ( ec 3.2.3.5 ) , cn - ii cytosolic imp and gmp - specific 5-nucleotidase ( ec 3.2.3.5 ) , gk guanosine kinase ( ec 2.7.1.73 ) , hgprt hypoxanthine - guanine phosphorybosyltransferase ( ec 2.4.2.8 ) , ik inosine kinase ( ec 2.7.1.73 ) , mtado methylothioadenosine , 5-nt-5-nucleotidase ( ec 3.1.3.5 ) , pnp purine nucleoside phosphorylase ( ec 2.4.2.1 ) , prpp 5-phosphoribosyl 1-pyrophosphate , sah s - adenosylhomocysteine , sahh s - adenosylhomocysteine hydrolase ( ec 3.3.1.1 ) , sam s - adenosylmethionine physical exercise causes an oxygen deficit in the working muscles .
the evolving hypoxia impairs oxidative atp resynthesis , which increases atp degradation , accompanied by the accumulation of imp ( stathis et al .
most of the imp is very quickly resynthesized to atp during restitution , but part of the imp is dephosphorylated which results in the production of ino and hyp ( stathis et al .
the products of purine catabolism , not recovered intramuscularly via purine salvage , efflux the muscle and are collected in the plasma ( bangsbo et al .
thanks to membrane transporters , nucleosides and purine bases ( mainly hypoxanthine ) are in equilibrium between plasma and red blood cells where they constitute the substrate in the salvage reactions .
these processes involve prpp , adenine phosphoribosyltransferase ( aprt ) , hypoxanthine - guanine phosphoribosyltransferase ( hgprt ) and nucleoside kinases ( dudzinska et al .
many in vitro studies have shown that nucleosides and purine bases may participate in the resynthesis of adenine nucleotides in red blood cells ( bontemps et al .
kim ( 1990 ) and komarova et al . ( 1999 ) demonstrated the participation of adenosine in the resynthesis of the adenine nucleotide pool , especially under conditions of high pi concentrations .
the results of those experiments suggest the participation of pi in the stimulation of adenosine kinase .
in contrast , van der berghe and bontemps ( 1990 ) and bontemps et al .
( 1986 ) reported an pi - induced inhibition of the activity of 5-nucleotidase and amp deaminase .
thus , the increase in erythrocyte and plasma pi accompanying physical effort ( yamamoto et al .
1994 ) may lead to changes in the activities of enzymes involved in purine metabolism .
in addition , one of the phenomena regularly associated with intense physical effort is metabolic acidosis .
the increase in the concentration of hydrogen ions in body fluids is greater , the greater the intensity of effort .
a fall in ph in red blood cells results in a decrease in adp and 2.3 dpg ( allosteric inhibitors of prpp synthetase , ec 2.7.6.1 ) with a concomitant increase in intracellular pi ( activator of prpp synthetase ) and atp ( berman et al .
thus , intense physical activity is likely to encourage increased synthesis of prpp  a co - substrate in reactions catalyzed by aprt and hgprt .
berman et al . ( 1988 ) reported that the uptake of hyp and accumulation of imp in red blood cells are significantly increased at an acid ph , high external phosphate concentrations , and low \documentclass[12pt]{minimal } 
				 \usepackage{amsmath } 
				 \usepackage{wasysym } 
				 \usepackage{amsfonts } 
				 \usepackage{amssymb } 
				 \usepackage{amsbsy } 
				 \usepackage{mathrsfs } 
				 \usepackage{upgreek } 
				 \setlength{\oddsidemargin}{-69pt } 
				 \begin{document}$$ { \text{p}}_{{{\text{o}}_{2 } } } $ $ \end{document}. in addition , they suggested that erythrocytes could play a role in the removal of hyp from anoxic tissue .
so far , there has been few reports of post - exercise changes in the erythrocyte concentration of adenine , guanine , and pyridine nucleotides .
therefore , we decided to measure the concentrations of adenine ( atp , adp , amp ) inosine ( imp ) , guanine ( gtp , gdp , gmp ) , as well as pyridine ( nad , nadp ) nucleotides in red blood cells immediately after standardized physical effort with increasing intensity , and at the 30th min of rest .
we examined the effect of muscular exercise on the adenylate ( guanylate ) energy charge 
aec ( gec ) , and on the concentration of nucleosides ( guanosine , inosine , adenosine ) and hypoxanthine .
their age , height , weight , and peak oxygen consumption were 21.9    2.33  years , 179.8    8.33  cm , 75.6    7.89  kg and 45.8    4.11  ml  kg  min , respectively .
the subjects were fully informed of any risks and discomfort associated with the experimental procedures before giving their consent to participate .
the examined individuals were subjected to a continuous effort test with progressively increasing intensity ( up to a refusal ) on a cycloergometer ( kettler x-7 , germany ) .
the test was preceded by a 5-min warm - up on the cycloergometer ( 25  w ) .
the test proper began at a resistance of 70  w while maintaining 70 revolutions per min .
the effort continued with an increasing load ( 20  w every 3  min ) until refusal , or until the tested individual was not able to maintain the required frequency of rotation . in the last 15  s of each 3-min effort at a given load , arterialized blood was collected to assess lactate concentration .
lactate levels were determined during the exercise and were used to establish an individual lactic threshold .
lactate concentration was determined using a dr lange lp-20 kit ( lange , germany ) .
a heart rate monitor ( polar s610 , finland ) was used to record resting heart rate and changes during exercise .
the uptake of oxygen during exercise was examined using an oxycon gas analyzer ( jaeger , germany ) .
based on the heart rate increase curve and the lactic acid concentrations at subsequent loads , we wanted to determine the load that was followed by a significant increase in blood lactate concentration .
individual lactate threshold was calculated using the plot of log la linear regression and log of the effort intensity .
the selection of loads was aligned with the power achieved at corresponding lactic thresholds ( ppa ) .
table  1 shows the anthropometric and physiological parameter characteristics of the study.table  1anthropometrical and physiological characteristics of subjectsparametersmean    sdbody height ( cm)179.8    8.33body mass ( kg)75.6    7.89hrrest ( bpm)72.1    8.03hrmax ( bpm)195.6    6.60ppa ( w)134.5    26.39mpo ( w)218    40.77vo2max ( ml / min / kg)45.8    4.11values are given as mean    sd ; n  =  22hr heart rate , ppa anaerobic threshold , mpo maximum power output , vo2max maximum of oxygen uptake anthropometrical and physiological characteristics of subjects values are given as mean    sd ; n  =  22 hr heart rate , ppa anaerobic threshold , mpo maximum power output , vo2max maximum of oxygen uptake concentrations of purine and pyridine nucleotides , guanosine ( guo ) , inosine ( ino ) , adenosine ( ado ) and hypoxanthine ( hyp ) were determined in whole blood using high - performance liquid chromatography .
designations were made of the blood taken from the antecubital vein immediately before and after exercise , and 30  min after exercise testing .
the samples ( 500  l ) of heparinized blood were deproteinized with an equal volume of 1.3  m hclo4 , mixed , and then centrifuged at 20,000 g for 5  min at 4c . the supernatant ( 400  l ) was neutralized with 130160  l of 1  m k3po4 ( to ph 57 ) .
the neutralized extract was again centrifuged as above , and the supernatant was stored at 80c until analysis .
chromatographic analysis was performed using a hewlett - packard series 1100 chromatograph according to the method used by smolenski et al .
the concentrations of nucleotides ( atp , adp , amp , imp , gtp , gdp , gmp ) , guo , ino , ado and hyp were determined in the whole blood .
taking into account , the hematocrit values ( hematocrit values were determined in duplicate by the standard microhematocrit method and expressed in percentage ) , we were able to calculate the intraerythrocyte nucleotide concentrations ( mol / l rbc ) with the assumption that there were no significant concentrations in plasma due to the action of the nucleotidases .
the concentrations of nucleosides ( ino , ado , guo ) and oxypurine ( hyp ) , which are present in both erythrocytes and plasma , are expressed as mol / l whole blood . they are not directly comparable to concentrations measured in separated erythrocytes or plasma .
inorganic phosphorous in plasma was determined using a randox laboratory diagnostic kit ref ph1016 ( randox laboratories ltd , co. antrim , uk ) .
when the anova was significant ( rir ) tukey s post hoc tests were used to localize the difference . the accepted level of significance was defined as p  <  0.05 . in order to demonstrate whether the observed correlations were statistically significant , spearman s rank correlation coefficient was applied .
their age , height , weight , and peak oxygen consumption were 21.9    2.33  years , 179.8    8.33  cm , 75.6    7.89  kg and 45.8    4.11  ml  kg  min , respectively .
the subjects were fully informed of any risks and discomfort associated with the experimental procedures before giving their consent to participate .
the examined individuals were subjected to a continuous effort test with progressively increasing intensity ( up to a refusal ) on a cycloergometer ( kettler x-7 , germany ) .
the test was preceded by a 5-min warm - up on the cycloergometer ( 25  w ) .
the test proper began at a resistance of 70  w while maintaining 70 revolutions per min .
the effort continued with an increasing load ( 20  w every 3  min ) until refusal , or until the tested individual was not able to maintain the required frequency of rotation . in the last 15  s of each 3-min effort at a given load , arterialized blood was collected to assess lactate concentration .
lactate levels were determined during the exercise and were used to establish an individual lactic threshold .
lactate concentration was determined using a dr lange lp-20 kit ( lange , germany ) .
a heart rate monitor ( polar s610 , finland ) was used to record resting heart rate and changes during exercise .
the uptake of oxygen during exercise was examined using an oxycon gas analyzer ( jaeger , germany ) .
based on the heart rate increase curve and the lactic acid concentrations at subsequent loads , we wanted to determine the load that was followed by a significant increase in blood lactate concentration .
individual lactate threshold was calculated using the plot of log la linear regression and log of the effort intensity .
the selection of loads was aligned with the power achieved at corresponding lactic thresholds ( ppa ) .
table  1 shows the anthropometric and physiological parameter characteristics of the study.table  1anthropometrical and physiological characteristics of subjectsparametersmean    sdbody height ( cm)179.8    8.33body mass ( kg)75.6    7.89hrrest ( bpm)72.1    8.03hrmax ( bpm)195.6    6.60ppa ( w)134.5    26.39mpo ( w)218    40.77vo2max
( ml / min / kg)45.8    4.11values are given as mean    sd ; n  =  22hr heart rate , ppa anaerobic threshold , mpo maximum power output , vo2max maximum of oxygen uptake anthropometrical and physiological characteristics of subjects values are given as mean    sd ; n  =  22 hr heart rate , ppa anaerobic threshold , mpo maximum power output , vo2max maximum of oxygen uptake
concentrations of purine and pyridine nucleotides , guanosine ( guo ) , inosine ( ino ) , adenosine ( ado ) and hypoxanthine ( hyp ) were determined in whole blood using high - performance liquid chromatography .
designations were made of the blood taken from the antecubital vein immediately before and after exercise , and 30  min after exercise testing .
the samples ( 500  l ) of heparinized blood were deproteinized with an equal volume of 1.3  m hclo4 , mixed , and then centrifuged at 20,000 g for 5  min at 4c . the supernatant ( 400  l ) was neutralized with 130160  l of 1  m k3po4 ( to ph 57 ) .
the neutralized extract was again centrifuged as above , and the supernatant was stored at 80c until analysis .
chromatographic analysis was performed using a hewlett - packard series 1100 chromatograph according to the method used by smolenski et al .
( 2003 ) . the concentrations of nucleotides ( atp , adp , amp , imp , gtp , gdp , gmp ) , guo , ino , ado and hyp were determined in the whole blood .
taking into account , the hematocrit values ( hematocrit values were determined in duplicate by the standard microhematocrit method and expressed in percentage ) , we were able to calculate the intraerythrocyte nucleotide concentrations ( mol / l rbc ) with the assumption that there were no significant concentrations in plasma due to the action of the nucleotidases
. the concentrations of nucleosides ( ino , ado , guo ) and oxypurine ( hyp ) , which are present in both erythrocytes and plasma , are expressed as mol / l whole blood . they are not directly comparable to concentrations measured in separated erythrocytes or plasma .
inorganic phosphorous in plasma was determined using a randox laboratory diagnostic kit ref ph1016 ( randox laboratories ltd , co. antrim , uk ) .
when the anova was significant ( rir ) tukey s post hoc tests were used to localize the difference . the accepted level of significance was defined as p  <  0.05 . in order to demonstrate
whether the observed correlations were statistically significant , spearman s rank correlation coefficient was applied .
the characteristics of some anthropometrical and physiological parameters are shown in table  1 . in the group of subjects ,
the testing time was 24.0    5.97  min , hr recorded before the exercise was 72.1    8.03  beats / min , while the maximum was 195.6    6.60  beats / min .
the maximum power value was 218    40.77  w , while the anaerobic threshold was set at 134.5    26.39  w. in the examined group , the oxygen consumption at maximum load was 45.8    4.11  ml / min / kg , which indicates that the subjects had a good aerobic endurance for this age group in comparison with reference data ( astrand and rodhal 1986 ) .
the mean and standard deviation of guanine and pyridine nucleotides concentrations are presented in table  2 there were no significant changes in the concentrations of gtp , gdp , gmp and tgn in red blood cells immediately after exercise , and at the 30th min of recovery .
no significant changes were observed for gec nor the concentrations of nad and nadp.table  2concentration of guanine nucleotides
( gtp , gdp , gmp ) and pyridine nucleotides ( nad , nadp ) in red blood cells before exercise , after exercise and 30  min of recoverybefore exerciseafter exercise30  min of recoverygtp ( mol / l rbc)66.80    8.2565.27    7.4163.01    8.02gdp ( mol / l rbc)28.02    4.5529.04    6.2328.42    6.67gmp ( mol / l rbc)41.46    6.2939.53    6.2341.08    6.31tgn ( mol / l rbc)133.84    12.26133.66    10.07132.49    12.76gec0.58    0.030.59    0.030.58    0.03nad ( mol / l rbc)72.84    7.1372.88    9.5571.77    8.36nadp ( mol / l rbc)45.80    4.5345.18    3.4044.89    3.80concentrations of gtp , gdp , gmp , tgn , nad , nadp are expressed as mol / l rbcvalues are given as mean    sd ;
=  22tgn ( total guanine nucleotides )  =  [ gtp ]  +  [ gdp ]  +  [ gmp]gec ( guanylate energy charge ) was evaluated according to the formula by atkinsongec  =  ( [ gtp ]  +  0.5[gdp])/([gtp ]  +  [ gdp ]  +  [ gmp ] ) concentration of guanine nucleotides ( gtp , gdp , gmp ) and pyridine nucleotides ( nad , nadp ) in red blood cells before exercise , after exercise and 30  min of recovery concentrations of gtp , gdp , gmp , tgn , nad , nadp are expressed as mol / l rbc values are given as mean    sd ; n  =  22 tgn ( total guanine nucleotides )  =  [ gtp ]  +  [ gdp ]  +  [ gmp ] gec ( guanylate energy charge ) was evaluated according to the formula by atkinson gec  =  ( [ gtp ]  +  0.5[gdp])/([gtp ]  +  [ gdp ]  +  [ gmp ] ) atp , adp , and amp stoichiometrically couple all of the metabolic sequences of a living cell .
the amount of metabolically available energy that is momentarily stored in the adenylate system is linearly related to the mole fraction of atp plus half the mole fraction of adp ; this parameter has been termed the aec of the adenylate pool . in this study ,
physical exercise with increasing intensity did not contribute to significant changes in atp concentration in red blood cells immediately after exercise and at the 30th min of recovery ( table  3 ) .
we demonstrated a significantly lower ( p  <  0.01 ) concentration of adp in red blood cells at the 30th min of recovery in comparison to concentrations observed immediately before the test .
similarly , the concentration of amp in red blood cells was significantly lower on the 30th min of recovery .
at the same time , the decrease observed in the concentration of amp was significant both with respect to the levels observed immediately before exercise ( p  <  0.0001 ) and immediately after exercise ( p  <  0.001 ) .
physical exercise with increasing intensity contributed to a significant increase in imp concentration in red blood cells compared to resting levels ( p  <  0.01 ) . at the 30th min of rest
, we observed a significant decrease in the concentration of imp in comparison to its level immediately after the exercise ( p  <  0.01 ) .
aec was significantly higher at the 30th min of recovery , both in comparison to the value observed before ( p  <  0.006 ) and immediately after exercise ( p  <  0.01 ) ( table  3).table  3concentrations of adenine nucleotides ( atp , adp , amp ) and inosine nucleotides ( imp ) in red blood cells before exercise , after exercise and 30  min of recoverybefore exerciseafter exercise30  min of recoveryatp ( mol / l rbc)1,797.00    158.831,777.80    130.521,808.522    155.26adp ( mol / l rbc)227.42    40.49220.13    42.30189.18    39.57*amp ( mol / l rbc)14.25    2.9413.21    3.099.77    2.43***tan ( mol / l rbc)2,038.59    172.872,010.73    154.922,006.79    171.29aec0.93    0.010.93    0.010.94    0.009**imp ( mol / l rbc)7.16    1.358.43    1.86 * 6.82    2.00atp / adp ( mol / l rbc)8.13    1.548.41    1.539.91    1.96*adp / amp ( mol / l rbc)16.06    1.1316.95    1.8419.56    1.67***concentrations of atp , adp , amp , tan and imp are expressed as mol / l rbcvalues are given as mean    sd ; n  =  22tan ( total adenine nucleotides )  =  [ atp ]  +  [ adp ] )  +  [ amp]aec ( adenylate energy charge ) was evaluated according to the formula by atkinsonaec  =  ( [ atp ]  +  0.5[adp])/([atp ]  +  [ adp ]  +  [ amp ] ) *  p  <  0.01 ; * * p  <  0.006 ; * * * p  <  0.0001 , significantly different from before exercisep  <  0.01 ; p  < 
0.001 ; p  <  0.0001 , significantly different from after exercise concentrations of adenine nucleotides ( atp , adp , amp ) and inosine nucleotides ( imp ) in red blood cells before exercise , after exercise and 30  min of recovery concentrations of atp , adp , amp , tan and imp are expressed as mol / l rbc values are given as mean    sd ; n  =  22 tan ( total adenine nucleotides )  =  [ atp ]  +  [ adp ] )  +  [ amp ] aec ( adenylate energy charge ) was evaluated according to the formula by atkinson aec  =  ( [ atp ]  +  0.5[adp])/([atp ]  +  [ adp ]  +  [ amp ] ) *  p  <  0.01 ; * * p  <  0.006 ; * * * p 
<  0.0001 , significantly different from before exercise p  <  0.01 ; p  <  0.001 ; p  <  0.0001 , significantly different from after exercise table  4 shows concentrations of ino , hyp , ado and guo in the blood .
ino concentration measured in blood immediately after exercise reached 0.5    0.26  mol / l and did not differ significantly from the value measured before exercise ( 0.1    0.04 
ino concentration was significantly higher 30  min after exercise , both in comparison to the resting value ( p  <  0.001 ) and after the effort ( p  <  0.001).table  4concentrations of guanosine , inosine , adenosine and hypoxanthine in blood before exercise , after exercise and 30  min of recoverybefore exerciseafter exercise30  min of recoveryguanosine ( guo ) mol / l whole blood0.11 
  0.030.14    0.030.13    0.04inosine ( ino ) mol / l whole blood0.11 
  0.030.50    0.261.12    0.59***adenosine ( ado ) mol / l whole blood0.07 
  1.6116.25    7.86***18.54    8.78***concentrations of guo , ino , ado and hyp are expressed in mol / l whole bloodvalues are given as mean    sd ; n  =  22 * * *  p  < 
0.0001 , significantly different from before exercisep  <  0.01 , significantly different from after exercise concentrations of guanosine , inosine , adenosine and hypoxanthine in blood before exercise , after exercise and 30  min of recovery concentrations of guo , ino , ado and hyp are expressed in mol / l whole blood values are given as mean    sd ; n  =  22 * * *  p  <  0.0001 , significantly different from before exercise p  <  0.01 , significantly different from after exercise physical exercise contributed to a nearly eightfold increase in hyp concentration in the blood in relation to resting levels ( from 2.2    1.61 to 16.3    7.86  mol / l ) ( p  <  0.0001 ) .
high concentrations of hyp were observed even 30  min after the exercise ( 18.5    8.78  mol / l ) , albeit this increase was still significant in relation to resting values ( p  <  0.0001 ) but not to post - exercise levels .
there was no significant relationship between the concentration of hyp in the blood and the concentration of imp in red blood cells .
there were no significant changes in the concentrations of ado and guo in the blood after exercise and at the 30th min of rest .
the concentration of inorganic phosphorous measured before the exercise was 1.14    0.13  mm .
immediately after exercise it was 1.39    0.22  mm and on the 30th min of restitution it was 1.46    0.28  mm , so in both times the concentration of the inorganic phosphorous significantly increased in comparison with resting values ( p  <  0.01 ) .
the post - exercise increase in the concentration of ino and hyp in the blood has already been observed by many researchers ( sutton et al .
1980 ; hellsten - westing et al . 1989 ; bangsbo et al . 1992 ; hellsten et al . 1999 ; zhao et al . 2000 ) .
many studies also demonstrated that ino and hyp are produced in muscles during moderate to intense exercise , while there is great individual variation of changes in plasma concentrations of hyp induced by intense exercise ( sahlin et al .
( 1989 ) , the serum levels of hyp increased up to five times the resting levels after arm cranking and more than ten times after short distance post - maximum running .
hellsten - westing et al . ( 1993a ) and sahlin et al . ( 1991 ) showed that the significant peak in the hyp concentration appears 20  min after conclusion of the exercise .
( 1999 ) showed that 100  s after intense exercise , femoral venous ino and hyp concentrations had increased and significant net release of hyp and ino was observed after 70  s. in recovery , venous ino and hyp increased further , and the release of ino and hyp peaked after 1220  min and were still significant after 90  min of recovery .
a significant increase in hyp concentration in the blood observed in this study immediately after the exercise and accompanied by no significant changes in the concentrations of ino , exhibits differences in the rate of entry of these metabolites into the plasma .
( 2000 ) in their studies of muscle adenine nucleotide metabolism during maximal exercise and in recovery from maximal exercise in humans showed a significant increase in the concentration of hyp and ino in plasma , respectively , at the 5th- and 10th min of recovery , while hellsten et al .
( 1999 ) showed a multiple significant increase in plasma concentrations of hyp immediately after high - intensity intermittent exercise .
the differences in the rate of entry of these metabolites into the blood may be due , inter alia , to different scheme of the exercise or its duration .
however , in this study a significant increase in the concentration of extracellular markers of muscle adenine nucleotide catabolism ( ino and hyp ) indicates the occurrence of severe energy stress that occurred during exercise .
the observed increase in the concentration of hyp immediately after the exercise and at the 30th min of recovery , points to a significant increase in the degradation of atp .
the results show that hyp was already released from working muscles during exercise , and also 30  min after its completion .
our study shows a significant increase in imp concentration in red blood cells immediately after exercise .
these results confirm the increase in the post - exercise concentration of imp observed by yamamoto et al .
( 1983 ) , who after 2  min of intense physical exertion showed no increase in the concentration of erythrocyte imp . one of the reasons for the differences may be the duration of the effort . as our research shows , 25  min of exercise with increasing intensity results in an increase in imp concentration of red blood cells .
imp concentration in red blood cells is the result of the synthesis and degradation of this nucleotide .
one of the reasons leading to an increase in imp concentration in red blood cells may be a decrease in ph , high external phosphate concentrations , and low \documentclass[12pt]{minimal } 
				 \usepackage{amsmath } 
				 \usepackage{wasysym } 
				 \usepackage{amsfonts } 
				 \usepackage{amssymb } 
				 \usepackage{amsbsy } 
				 \usepackage{mathrsfs } 
				 \usepackage{upgreek } 
				 \setlength{\oddsidemargin}{-69pt } 
				 \begin{document}$$ { \text{p}}_{{{\text{o}}_{2 } } } $ $ \end{document } , it has been demonstrated that the imp synthesis is regulated by ph , inorganic phosphate and oxygen tension ( berman et al .
( 1994 ) , a 25-min exercise on a cycloergometer with increasing intensity until refusal leads to a significant increase in pi in red blood cells and plasma , and a decline in blood ph .
in our study we also showed a significant increase in pi in plasma , not only immediately after effort but also on the 30th min of restitution .
moreover , in vitro studies on the effects of inorganic phosphate on the concentration of nucleotides showed that in red blood cells incubated in a solution containing 2  mm pi , an increase in imp concentration occurs ( yamamoto et al .
( 1988 ) showed that the uptake of hyp and accumulation of imp in red blood cells are significantly increased at acidic ph levels , high external phosphate concentrations , and low \documentclass[12pt]{minimal } 
				 \usepackage{amsmath } 
				 \usepackage{wasysym } 
				 \usepackage{amsfonts } 
				 \usepackage{amssymb } 
				 \usepackage{amsbsy } 
				 \usepackage{mathrsfs } 
				 \usepackage{upgreek } 
				 \setlength{\oddsidemargin}{-69pt } 
				 \begin{document}$$ { \text{p}}_{{{\text{o}}_{2 } } } $ $ \end{document } , the conditions that accompany intense physical effort .
given the fact that one way of removing hyp from blood is its uptake by red blood cells , the significant increase in hyp concentration in the blood observed immediately after exercise , and the significant increase in imp concentration in red blood cells suggests that hyp was included in the imp pool in a reaction catalysed by hgprt .
it does not seem that a significant increase in imp concentration in red blood cells immediately after exercise was caused by the increased activity of amp deaminase ( ec 3.5.4.6 ) .
first , we did not observe a significant decrease in the concentration of amp , which accompanies an increase in amp deaminase activity ( tavazzi et al .
secondly , it has been shown that intense physical activity leads to the inhibition in amp deaminase in skeletal muscle , caused directly by an increase in the concentration of orthophosphate and a decline in ph ( hellsten et al .
it has also been demonstrated that pi , imp and 2.3 bpg are the inhibitors of the enzyme ( bontemps et al .
thus , the increase in pi concentration in red blood cells , and a drop in blood ph , as well as an increase in the concentration of imp observed immediately after the exercise , suggest the inhibition of amp deaminase .
it seems that an irreversible reaction leading to the hydrolytic deamination of amp to imp in erythrocytes is inhibited because of the necessity to protect the cellular atp .
it is known that under physiological conditions , the pool of adenine and guanine nucleotides is kept constant .
the concentration ratio of individual nucleotides within this pool is maintained primarily by adenylate kinase , guanylate kinases , and enzymes involved in the catabolism of these compounds .
our study showed no erythrocyte changes in the concentration of adenine and guanine nucleotides immediately after exercise .
significant decreases in amp and adp in the absence of significant changes in atp and tan were observed only at the 30th min of rest , which confirms results from other authors ( makarewicz et al .
( 1980 ) showed a significant decrease in amp and adp in red blood cells in the absence of significant changes in the concentration of atp immediately after intense exercise and 30  min of rest .
yamamoto et al . ( 1994 ) , incubating the red blood cells in a solution containing 2  mm pi , showed a decrease in amp and adp in the absence of significant changes in the concentrations of atp .
in addition to the pi - induced decrease in amp and adp in vitro , they showed a decline in amp and adp in red blood cells in the group of 6 healthy subjects after a 25-min bicycle exercise on a cycloergometer . in our study , a drop in the concentration of amp and adp , with the simultaneous increase in atp / adp and adp / amp ratios after 30  min of rest indicates an increased phosphorylation amp and adp to atp ( table  3 ) .
although we observed no significant increase in atp , an 8% increase in the concentration of this nucleotide was observed in red blood cells during recovery in comparison to resting values . because the only source of erythrocyte atp is glycolysis , it can be supposed that physical activity results in an increased rate of this conversion in erythrocytes . in summary
, it can be concluded that a standardized physical exercise with increasing intensity leads to an increase in imp concentration in red blood cells immediately after exercise , which with a significant increase in hyp concentration in the blood suggests that hyp was included in the imp pool . during the rest ,
an increase in atp / adp and adp / amp ratios occur , which indicates an increased phosphorylation of amp and adp to atp .
the exercise used in this study did not lead to erythrocyte changes in the concentration of guanosine and pyridine nucleotides .
 maximal physical exertion is accompanied by increased degradation of purine nucleotides in muscles with the products of purine catabolism accumulating in the plasma . 
 thanks to membrane transporters , these products remain in an equilibrium between the plasma and red blood cells where they may serve as substrates in salvage reactions , contributing to an increase in the concentrations of purine nucleotides . in this study , we measured the concentrations of adenine nucleotides ( atp , adp , amp ) , inosine nucleotides ( imp ) , guanine nucleotides ( gtp , gdp , gmp ) , and also pyridine nucleotides ( nad , nadp ) in red blood cells immediately after standardized physical effort with increasing intensity , and at the 30th min of rest . 
 we also examined the effect of muscular exercise on adenylate ( guanylate ) energy charge  
 aec ( gec ) , and on the concentration of nucleosides ( guanosine , inosine , adenosine ) and hypoxanthine . 
 we have shown in this study that a standardized physical exercise with increasing intensity leads to an increase in imp concentration in red blood cells immediately after the exercise , which with a significant increase in hyp concentration in the blood suggests that hyp was included in the imp pool . 
 restitution is accompanied by an increase in the atp / adp and adp / amp ratios , which indicates an increase in the phosphorylation of amp and adp to atp . physical effort applied in this study 
 did not lead to changes in the concentrations of guanine and pyridine nucleotides in red blood cells . 
Introduction
Methods
Subjects
Exercise protocol
Blood analysis
Statistical analysis
Results
Discussion