physical exercise , especially in competitive sports , is characterized by considerable strain for an athlete , and causes faster ageing of the erythrocytes .
elevated body temperature , metabolic acidosis , hypoglycemia , and hemoconcentration , observed during physical exercise , reduce the osmotic resistance of erythrocytes ( reeder and wilson 2001 ; robinson et al .
( 2007 ) suggested that intravascular hemolysis observed during long - term exercise results from the injury of older erythrocytes , which are less elastic and thus more susceptible to damage .
the same authors documented a very strong inverse correlation ( r  =  0.911 , p  <  0.05 ) between hemolysis and the levels of spectrin , a peripheral protein of the erythrocyte membrane that is expressed on its internal surface and forms the cytoskeleton .
these findings support the hypothesis that structural alterations of erythrocyte membranes increase the susceptibility of these cells to hemolysis , which results in elevated plasma levels of free iron .
iron released from the cells can be stored in ferritin molecules , or bound to low - molecular - weight cytoplasmic ligands , forming the so - called labile iron pool ( lip ) .
the iron contained in the lip is further utilized to reconstruct iron - dependent enzymes or is involved in the generation of reactive oxygen species ( reeder and wilson 2005 ) .
an increase in iron levels is associated with enhanced synthesis of hepcidin , a peptide that inhibits the secretion of iron from enterocytes and its release to plasma from monocytes and macrophages . moreover ,
intensified binding of iron to its storage protein , ferritin , is observed in enterocytes ( deicher and hrl 2006 ) .
recent studies have revealed that hepcidin plays a pivotal role in the regulation of iron homeostasis , reducing its transport and absorption .
this process is likely driven by the presence of enhanced hemolysis and acute phase response .
the latter is associated with increased levels of pro - inflammatory cytokines , which in turn stimulate the synthesis of hepcidin ( van deuren et al .
anemia of inflammation is characterized by normocytosis and normochromia , shorter survival of erythrocytes , low serum concentrations of iron and transferrin , and elevated levels of ferritin ( nikolaidis et al . 2003 ) .
notably , low levels of iron are a cause of reduced exercise capacity in athletes .
it is estimated that a 12  g  % decrease in blood hemoglobin concentration can lead to a 20  % reduction of exercise capacity ( gardner et al .
1977 ) . consequently , detailed analysis of exercise - induced changes in iron metabolism becomes vitally important .
available data about the effect of short - term high - intensity exercise on hepcidin levels are sparse , although this type of load is a frequent component of sports training .
therefore , the aim of this study was to analyze the relationship between high - intensity physical exercise and changes in hepcidin levels and selected parameters of iron metabolism in rowing athletes .
the study included 20 male athletes , members of the national polish rowing team ( 16 heavyweight and 4 lightweight rowers ) , who took part in a training camp .
all of the participants were free of any health problems and were not taking any anti - inflammatory drugs , vitamins , or medications known to affect iron metabolism for 2  weeks prior to the tests .
the participants were under no dietary restrictions.table  1basic characteristics of the study participants ( means    standard deviations)parameters 
 n  =  20age , years21.3    0.82body mass , kg87.7    9.63body height , m1.91    0.06duration of training , years7.2    1.3 basic characteristics of the study participants ( means    standard deviations ) the athletes performed a controlled 2,000  m time test .
each participant had to cover the distance on a rowing ergometer ( concept ii , usa ) in the shortest time possible .
as the results of this test were taken into consideration during selection to the championship team , the athletes were well motivated to perform it to the best of their abilities .
the test on a rowing ergometer was conducted in the morning hours , between 11:30 am and 12:00 pm .
the air temperature during testing was 17  c and relative humidity amounted to 55  % .
before the main test , each participant had 5  min for individual warm - up .
blood samples from the athletes were obtained three times : ( 1 ) at rest ( in the morning , after an overnight fast ) , ( 2 ) immediately after the exercise ( 1  min after completing the test ) , and ( 3 ) after a 24-h recovery period .
blood samples were obtained from the antecubital vein , with dipotassium ethylene diamine tetra - acetic acid ( k2edta ) used as an anticoagulant .
blood samples were taken for the analysis of red blood cells ( rbcs ) , mean corpuscular volume ( mcv ) , hemoglobin , and hematocrit in a mythic 18 hematology analyzer ( orphee medical , geneva , switzerland ) .
samples were immediately centrifuged to separate red blood cells from plasma and determine the activity of creatine kinase ( ck ) .
blood samples were collected without additives for the analysis of serum iron ( fe ) , soluble transferrin receptor ( stfr ) , ferritin , total iron - binding capacity ( tibc ) , interleukin ( il)-6 , tumor necrosis factor ( tnf)- , hepcidin , and myoglobin .
serum and plasma samples were frozen immediately after centrifugation and stored at 80  c until use .
additionally , capillary blood samples were obtained from an earlobe prior to warm - up preceding the ergometric test and after ( within 1  min ) each exercise test to assess the athletes lactic acid levels .
serum il-6 was measured using a commercially available enzyme - linked immunosorbent assay ( elisa ; quantikine hs , r&d systems , minneapolis , minnesota , usa ) with an assay range of 0.3810  pg  ml .
serum concentrations of tnf- ( in pg / ml ) were quantified using a commercially available enzyme immunoassay ( quantikine human immunoassay , cat .
serum hepcidin was measured using a commercially available elisa ( wuhan eiaab science co. , china ) with an assay range of 0.18712  ng  ml .
si257 and ti1010 , randox , antrim , uk ) ; the results were expressed in g / dl . the unsaturated iron - binding capacity ( uibc ) was calculated from the formula : uibc  =  tibc  fe .
the myoglobin concentration was determined immunochemically , with the help of the myoglobin elisa kit ( cat .
11170 , oxisresearch , percipio bio , foster city , california , usa ) ; the results were expressed in ng / ml . serum ferritin levels were determined immunochemically , with an aid of a commercially available diagnostic kit ( cat .
de7750 , hlzel diagnostika , kln , germany ) ; the results were expressed in ng / ml . the average interassay cv precision was 7.5  % .
concentrations of soluble transferrin receptor ( stfr ) were determined immunochemically with a commercially available diagnostic kit ( cat .
creatine kinase ( ck ) activity was determined in plasma samples with a commercially available kit ( cat .
lcn 282 , hach lange , dsseldorf , germany ) ; the results were expressed in u / l . the lactic acid in capillary blood was determined immediately after sample collection using a commercially available kit ( cat .
lkm 140 , hach lange , dsseldorf , germany ) ; lactic acid concentrations were expressed in mmol / l . if necessary , the results were adjusted for hemoconcentration , using the exercise - induced changes in hematocrit as a covariate .
statistical analyses were performed using the statistica v. 10.0 software package ( stat  soft , cracow , poland ) .
homogeneity of variance was verified with levene s test . if significant changes were documented in one - way analysis of variance ( anova ) , fisher s post hoc test was applied to locate the source of significant differences .
the non - parametric friedman anova was used in the case of variables with distributions other than normal .
the statistical significance of all the tests was set at p    0.05 .
the study included 20 male athletes , members of the national polish rowing team ( 16 heavyweight and 4 lightweight rowers ) , who took part in a training camp .
all of the participants were free of any health problems and were not taking any anti - inflammatory drugs , vitamins , or medications known to affect iron metabolism for 2  weeks prior to the tests .
the participants were under no dietary restrictions.table  1basic characteristics of the study participants ( means    standard deviations)parameters 
 n  =  20age , years21.3    0.82body mass , kg87.7    9.63body height , m1.91    0.06duration of training , years7.2    1.3 basic characteristics of the study participants ( means    standard deviations )
each participant had to cover the distance on a rowing ergometer ( concept ii , usa ) in the shortest time possible .
as the results of this test were taken into consideration during selection to the championship team , the athletes were well motivated to perform it to the best of their abilities .
the test on a rowing ergometer was conducted in the morning hours , between 11:30 am and 12:00 pm .
the air temperature during testing was 17  c and relative humidity amounted to 55  % .
before the main test , each participant had 5  min for individual warm - up .
blood samples from the athletes were obtained three times : ( 1 ) at rest ( in the morning , after an overnight fast ) , ( 2 ) immediately after the exercise ( 1  min after completing the test ) , and ( 3 ) after a 24-h recovery period .
blood samples were obtained from the antecubital vein , with dipotassium ethylene diamine tetra - acetic acid ( k2edta ) used as an anticoagulant .
blood samples were taken for the analysis of red blood cells ( rbcs ) , mean corpuscular volume ( mcv ) , hemoglobin , and hematocrit in a mythic 18 hematology analyzer ( orphee medical , geneva , switzerland ) .
samples were immediately centrifuged to separate red blood cells from plasma and determine the activity of creatine kinase ( ck ) .
blood samples were collected without additives for the analysis of serum iron ( fe ) , soluble transferrin receptor ( stfr ) , ferritin , total iron - binding capacity ( tibc ) , interleukin ( il)-6 , tumor necrosis factor ( tnf)- , hepcidin , and myoglobin .
serum and plasma samples were frozen immediately after centrifugation and stored at 80  c until use .
additionally , capillary blood samples were obtained from an earlobe prior to warm - up preceding the ergometric test and after ( within 1  min ) each exercise test to assess the athletes lactic acid levels .
serum il-6 was measured using a commercially available enzyme - linked immunosorbent assay ( elisa ; quantikine hs , r&d systems , minneapolis , minnesota , usa ) with an assay range of 0.3810  pg  ml .
serum concentrations of tnf- ( in pg / ml ) were quantified using a commercially available enzyme immunoassay ( quantikine human immunoassay , cat .
serum hepcidin was measured using a commercially available elisa ( wuhan eiaab science co. , china ) with an assay range of 0.18712  ng  ml . the average interassay cv precision was 2.7  % .
si257 and ti1010 , randox , antrim , uk ) ; the results were expressed in g / dl . the unsaturated iron - binding capacity ( uibc )
the myoglobin concentration was determined immunochemically , with the help of the myoglobin elisa kit ( cat .
11170 , oxisresearch , percipio bio , foster city , california , usa ) ; the results were expressed in ng / ml . serum ferritin levels were determined immunochemically , with an aid of a commercially available diagnostic kit ( cat .
de7750 , hlzel diagnostika , kln , germany ) ; the results were expressed in ng / ml . the average interassay cv precision was 7.5  % .
concentrations of soluble transferrin receptor ( stfr ) were determined immunochemically with a commercially available diagnostic kit ( cat .
creatine kinase ( ck ) activity was determined in plasma samples with a commercially available kit ( cat .
lcn 282 , hach lange , dsseldorf , germany ) ; the results were expressed in u / l . the lactic acid in capillary blood was determined immediately after sample collection using a commercially available kit ( cat .
lkm 140 , hach lange , dsseldorf , germany ) ; lactic acid concentrations were expressed in mmol / l . if necessary , the results were adjusted for hemoconcentration , using the exercise - induced changes in hematocrit as a covariate .
statistical analyses were performed using the statistica v. 10.0 software package ( stat  soft , cracow , poland ) .
if significant changes were documented in one - way analysis of variance ( anova ) , fisher s post hoc test was applied to locate the source of significant differences .
the non - parametric friedman anova was used in the case of variables with distributions other than normal .
the statistical significance of all the tests was set at p    0.05 .
power output , blood lactate levels , and total run time are shown in table  2 .
data on a training load of the examined athletes during 30  days preceding the ergometric test are presented in table  3.table  2power output , blood lactic acid levels , and total run time ( means    standard deviations)parameters 
 n  =  20power , watts430    34.1w / kg4.88    0.28lamin , mmol / l1.6    0.14lamax , mmol / l14.9    2.62time ,
s373.4    10.38table  3training schedule during the 3  days preceding blood sample collectiondays before ergometer test321total training time , min / day210150120ergometer test2,000  mtime rowed , min / day7090100distance rowed , km / day161820training for force development , min / day70extensive endurance rowing training time , min / day4090100high - intensity endurance rowing training time , min / day30unspecific training ( running , etc . ) , min / day706020 power output , blood lactic acid levels , and total run time ( means    standard deviations ) training schedule during the 3  days preceding blood sample collection the values of blood parameters that indicate iron metabolism in the studied athletes are presented in table  4 and fig .  1 ( a : iron , b : ferritin , and c : hepcidin ) .
participation in the high - intensity exercise test resulted in a significant increase in most of the studied parameters , and their subsequent return to pre - exercise levels after 1  day of recovery .
however , an exercise - induced increase in serum iron level proved insignificant during statistical analysis , and the values for this parameter ( documented at the end of the recovery period ) were significantly lower than its baseline and post - exercise measurements.table  4changes in iron metabolism status during exhaustive exercisevariablespre - exercise m (   sd)post - exercise m (   sd)recovery m (   sd ) 
 p ( anova)rbc , 10/l5.29 ( 0.29)5.65 ( 0.30 ) 
 5.26 ( 0.30 ) 
 0.00000hgb , g / dl15.47 ( 0.67)16.54 ( 0.81 ) 
 15.34 ( 0.67 ) 
 0.00000hct ,  % 44.33 ( 1.95)48.23 ( 2.09 ) 
 43.99 ( 2.26 ) 
 0.00000mcv , fl85.78 ( 2.69)87.30 ( 2.87 ) 
 85.51 ( 2.79 ) 
 0.00000tibc , g / dl320.8 ( 32.83)352.2 ( 34.57 ) 
 325.5 ( 26.82 ) 
 0.00155uibc , g / dl175.0 ( 17.92)192.1 ( 18.87 ) 
 177.6 ( 14.66 ) 
 0.00155stfr , g / ml0.93 ( 0.14)1.26 ( 0.24 ) 
 0.93 ( 0.17 ) 
 0.00002myoglobin , ng / ml245.2 ( 71.38)407.7 ( 131.21 ) 
 373.7 ( 104.86 ) 
 0.00026 
 rbc red blood cells , hgb hemoglobin , hct hematocrit , mcv mean corpuscular volume , uibc unsaturated iron - binding capacity , tibc total iron - binding capacity , stfr soluble transferrin receptor 
 pre - exercise  post - exercise 
 pre - exercise  recovery 
 post - exercise  recoveryfig . 
1serum iron a , ferritin b , and hepcidin c levels in rowers at baseline , immediately after exercise , and after a 1-day recovery period .
* statistically significant difference between trials ( p  <  0.05 ) changes in iron metabolism status during exhaustive exercise 
 rbc red blood cells , hgb hemoglobin , hct hematocrit , mcv mean corpuscular volume , uibc unsaturated iron - binding capacity , tibc total iron - binding capacity , stfr soluble transferrin receptor 
 pre - exercise  post - exercise 
 pre - exercise  recovery 
 post - exercise  recovery serum iron a , ferritin b , and hepcidin c levels in rowers at baseline , immediately after exercise , and after a 1-day recovery period .
* statistically significant difference between trials ( p  <  0.05 ) furthermore , the exercise test resulted in an increase in serum il-6 . upon recovery
in contrast , we did not document a significant effect of physical exercise on tnf- levels ( fig .  2b).fig .  2interleukin 6 a and tumor necrosis factor  b levels in rowers at baseline , immediately after exercise , and after a 1-day recovery period .
* statistically significant difference between trials ( p  <  0.05 ) interleukin 6 a and tumor necrosis factor  b levels in rowers at baseline , immediately after exercise , and after a 1-day recovery period .
* statistically significant difference between trials ( p  <  0.05 ) finally , creatine kinase activity ( fig .  3 ) and serum myoglobin levels ( table  4 ) increased significantly in response to the exercise test and remained elevated after 1  day of recovery.fig . 
3creatine kinase levels at baseline , immediately after exercise , and after a 1-day recovery period .
* statistically significant difference between trials ( p  <  0.05 ) creatine kinase levels at baseline , immediately after exercise , and after a 1-day recovery period .
enhanced inflammatory response , observed during intense exercise load , causes the secretion of many mediators from circulating macrophages , including il-6 , which stimulates the expression of hepcidin in hepatocytes and its release into the circulation ( banzet et al .
the ergometric exercise test caused a significant increase in il-6 and hepcidin levels , along with a 22  % increase in ferritin , a marker of systemic iron deposits that is classified as an acute phase protein .
we also observed a positive correlation between il-6 and hepcidin levels ( r  =  0.481 , p  <  0.05 ) .
( 2013 ) studied trained and untrained individuals exposed to the high - intensity interval exercise test , and observed post - exercise increases in il-6 and hepcidin in both groups .
however , the hepcidin levels in trained participants returned to baseline , pre - exercise values as soon as after 1  day of recovery , versus after as many as 5  days of recovery for untrained individuals .
these results are consistent with the findings documented for our rowers , in whom the concentrations of il-6 ( fig . 
in contrast to our findings regarding il-6 , we did not document any significant changes in tnf- concentration , either in response to exercise stimulation or during the recovery period ( fig . 
( 2008 ) showed that post - exercise tnf- concentrations in rowers are also significantly modulated by the training load before the trial .
the post - exercise increase in this cytokine was observed solely during the period of high - volume training .
it is noteworthy that we examined our rowers during the general preparation phase , which is predominated by aerobic exercise ( table  3 ) and probably insufficient to induce changes in tnf- concentration . according to available data , the mechanism that controls the post - exercise expression of hepcidin
is associated not only with enhancement of the inflammatory response , but also with a hemolysis - induced increase in iron levels ( reeder and wilson 2005 ) . while the ergometric test resulted in only an insignificant increase in serum iron concentrations among our rowers ( fig . 
1a ) , the remaining parameters that characterize systemic iron metabolism ( tibc , uibc , stfr ) increased significantly .
( 2012 ) , ferritin ( and probably also transferrin ) , but not hemolysis or rhabdomyolysis , can constitute the main sources of free iron during physical exercise .
higher post - trial levels of ck and myoglobin observed in our rowers confirmed the presence of exercise - induced injury to the sarcolemma .
however , a further increase in these parameters during the recovery period ( fig .  3 ; table  4 ) did not result in corresponding changes of the remaining parameters of iron metabolism ; this finding is consistent with the above - mentioned hypothesis regarding the regulatory roles of hepcidin and ferritin . according to suedekum and dimeff ( 2005 ) ,
an increase in ferritin concentration can result from the enhanced permeability of cellular membranes associated with exercise - induced injury of the reticuloendothelial system cells and hepatocytes .
the latter cells exhibit enhanced ferritin synthesis in response to erythrocyte hypercatabolism or mild hemolysis . despite being stable in acute conditions (
das gupta and abbi 2003 ) , the second marker of iron metabolism , a soluble transferrin receptor , is less useful as a measure of exercise - induced changes in iron status owing to its lack of standardization .
we observed a 26  % post - exercise increase in stfr concentration among our rowers , as well as its subsequent return to baseline during the recovery period ( table  4 ) .
a 24-h recovery period resulted in a marked decrease in iron level in our participants , compared with both pre- and post - exercise levels ( fig . 
( 2012 ) observed that a considerable proportion of elite rowing athletes and professional soccer players have iron deficiency , independent of the training mode .
however , only rowers exhibit lower erythrocyte counts and hemoglobin levels during pre - season training than during the recuperation period . according to nemeth et al .
( 2004 ) , impaired hemoglobin synthesis can result from a transient increase in hepcidin activity , which causes rapidly decreasing plasma iron levels and resultant anemia .
( 2006 ) showed that high - intensity exercise causes a decrease in the serum level of iron in rats ; in contrast , serum iron was significantly increased in animals exposed to moderate exercise . decreased iron ion concentration in biological fluids , including blood serum ,
can constitute one of basic protective mechanisms that is activated in order to prevent their involvement in reactions associated with the generation of toxic oxygen species .
a study of iron - injected mice exposed to a treadmill test demonstrated a number of unfavorable changes that were not observed in control animals .
the iron - overloaded mice exhibited significantly higher iron content in muscles and plasma , which ( according the authors ) was caused by enhanced oxidative stress .
furthermore , the excessive , rapid accumulation of iron exerted negative effects on skeletal muscle function in mice , as confirmed by decreased muscle strength and the presence of muscle atrophy ( reardon and allen 2009 ) . similarly , 1  week of excessive iron supplementation ( 120  mg / day ) resulted in increased oxidative stress and inflammatory response in young , healthy humans ( schmann et al .
intense physical exercise on a rowing ergometer resulted in increases in all analyzed erythrocyte parameters of our rowers , with their subsequent return to baseline after a 1-day recovery period ( table  4 ) .
boyadjiev and taralov ( 2000 ) showed that highly trained athletes from different sports have lower red blood cell counts , packed cell volumes , and hemoglobin concentrations than untrained controls .
moreover , they observed that the values of these parameters were the lowest among male swimmers and rowers ( i.e. , representatives of disciplines characterized by high - intensity training ) , making them predisposed to so - called sports anemia .
it is noteworthy that depending on the training phase , youth rowers perform between 15 and 25  h of exercise per week .
maintaining proper balance between intensive exercise and recovery appears to be vitally important in view of such a considerable training load .
too heavy an exercise load and too short a recovery period can lead to the accumulation of unfavorable training outcomes , which manifests as , among other things , a decrease in erythrocyte parameters ( rietjens et al . 2005 ) . analyzing the results of previous studies
, one can hardly identify the hematological parameters that might be helpful in diagnosing iron deficiency in athletes .
chronic exercise - induced inflammation is associated with iron deficiency , which should be distinguished from true anemia .
typically , determined hematological parameters ( erythrocyte count , packed cell volume , serum iron concentration ) do not fully reflect the degree and severity of sports anemia . in turn , the levels of ferritin and transferrin are not sufficiently accurate , as they are both elevated in any anemia of inflammation .
finally , stfr concentration , which is elevated in acute states , constitutes a marker of iron deficiency in tissues , rather than a measure of anemia ( skikne 2008 ; zoller and vogel 2004 ) .
we observed a 26  % post - exercise increase in stfr concentration in our rowers , with its subsequent return to baseline during the recovery period ( table  4 ) . according to schumacher et al .
( 2002 ) , the post - test increase in stfr is determined by exercise intensity . in their study , both trained and untrained individuals exhibited increased stfr solely in response to the more intense exercise test . 

a high - intensity exercise test caused significant changes in the hepcidin levels and parameters of iron metabolism of rowing athletes , which subsequently returned to baseline values during the recovery period .
serum iron levels decreased significantly during the recovery period . while a single bout of intense exercise did not cause clinically significant loss of iron in our athletes , the consequences of two to three daily overexertion loads ( e.g. competition or period of intense load ) can accumulate and constitute an important risk factor for anemia .
future studies should be aimed at determining the time that is required to return iron levels to their pre - exercise values and identifying factors that promote the replenishment of iron resources in athletes .
 purposephysical exercise , especially intense physical exercise , causes a number of unfavorable changes , including an increase in the level of pro - inflammatory cytokines with the resultant sequestration of iron in macrophages and decreased iron absorption . 
 this can lead to a reduced supply of iron for erythroid progenitor cells and promote the development of anemia.methodthis study included a group of 20 rowing athletes , members of the national polish rowing team . 
 the participants performed a 2,000-m maximum test on a rowing ergometer . 
 blood samples were taken from the antecubital vein prior to the exercise test , 1  min after completing the test , and after a 24-h recovery period . 
 we determined the levels of hepcidin , interleukin 6 ( il-6 ) , tumor necrosis factor  , soluble transferrin receptor , ferritin , total iron - binding capacity , unbound iron - binding capacity , iron , red blood cells , hemoglobin , hematocrit , mean corpuscular volume , creatine kinase , and myoglobin.resultthe high - intensity exercise test caused significant changes in hepcidin levels , il-6 , and iron metabolism parameters , with their subsequent return to baseline values during the recovery period . 
 the serum iron levels decreased significantly during the recovery compared with pre- and post - exercise levels.conclusionthese results suggest that the high - intensity ergometric test was reflected by a marked decrease in serum level of iron during the recovery period , but did not induce concomitant changes in the remaining erythrocyte parameters . 
Introduction
Materials and methods
Study population
Experimental procedure
Sample treatment
Measurements
Statistical analysis
Results
Discussion
Conclusions
Conflict of interest