Research Article Alterations in Red Blood Cells and Plasma ...downloads.hindawi.com/journals/tswj/2013/168376.pdf · haemoglobin (Hb) concentration, and haematocrit (HTC) were determined

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 168376 10 pageshttpdxdoiorg1011552013168376

Research ArticleAlterations in Red Blood Cells and Plasma Properties afterAcute Single Bout of Exercise

Krzysztof Gwozdzinski1 Anna Pieniazek2 Joanna Brzeszczynska1

Sabina Tabaczar1 and Anna Jegier3

1 Department of Molecular Biophysics University of Lodz Pomorska 141143 90-236 Lodz Poland2Department of Thermobiology University of Lodz 90-236 Lodz Poland3Department of Sport Medicine Medical University of Lodz 90-647 Lodz Poland

Correspondence should be addressed to Krzysztof Gwozdzinski kgwozdzbiolunilodzpl

Received 17 September 2013 Accepted 12 November 2013

Academic Editors H Grant K Kiselyov and L A Videla

Copyright copy 2013 Krzysztof Gwozdzinski et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this study was to investigate alterations in haemoglobin conformation and parameters related to oxidative stress inwhole erythrocytes membranes and plasma after a single bout of exercise in a group of young untrained men Venous bloodsamples from eleven healthy young untrained males (age = 22 plusmn 2 years BMI = 23 plusmn 25 kgm2) were taken from the antecubitalvein before an incremental cycling exercise test immediately after exercise and 1 hour after exercise Individual heart rate responseto this exercise was 195 plusmn 12 beatsmin and the maximum wattage was 292 plusmn 27W Immediately after exercise significant increasein standard parameters (haemoglobin haematocrit lactate levels and plasma volume) of blood was observed as well as plasmaantioxidant capacity one hour after exercise Reversible conformational changes in haemoglobin measured using a maleimide spinlabel were found immediately following exercise The concentration of ascorbic acid inside erythrocytes significantly decreasedafter exercise A significant decline in membrane thiols was observed one hour after exercise but simultaneously an increase inplasma thiols immediately after and 1 h after exercise was also observed This study shows that a single bout of exercise can lead tomobilization of defensive antioxidant systems in blood against oxidative stress in young untrained men

1 Introduction

A substantial amount of evidence in the existing literatureindicates that exercise is associated with increased generationof reactive oxygen species (ROS) including nitrogen andchlorine species [1 2] This increase appears to be due to dis-ruption of the oxidant-antioxidant homeostasis and results ininduction of oxidative stress in the human body In humansexercise has been reported to increase skeletal muscle super-oxide dismutase (SOD) activity and the activity of variousprotective enzymes in the blood Reactive oxygen speciesgenerated during exercise can cause oxidative stress anddamage to structural and functional integrity at the cellularlevel Moreover there is no doubt that moderate exercise hasnumerous health benefits and that regular physical activity isan important factor in the prevention and treatment of car-diovascular diseases It improves physical and psychologicalwell-being as well as delays the ageing process [3 4]

Oxygen consumption during exercise increases ten- tofifteenfold but oxygen supply to active tissue may rise bya factor of one hundred [5] ROS production in responseto strenuous exercise can proceed through different waysthe mitochondrial respiratory chain or NAD(P)H oxidasexanthine oxidase and autooxidation of catecholamines [6]ROS can also be released by macrophages and neutrophils ina respiratory burst or during destruction of iron-containingproteins [7 8] Moreover increased production of ROS hasbeen observed in both strenuous prolonged exercise or briefmaximal exercise [9 10] In the case of intensive exercise ahigh level of oxygen consumption mechanical (shear) stressdisruption of iron-containing proteins and an increase inprostanoid and catecholamine levels have been detected [11]

In our previous work we examined the effect of exercise-induced oxidative modifications on the physicobiochemicalproperties of erythrocyte membrane in young untrainedmales [12] However there is little evidence concerning

2 The Scientific World Journal

the influence of physical activity on the disintegration ofinternal erythrocyte components In addition it is notknown whether alteration in haemoglobin conformationoccurs during exercise However it has been reported that3 of total haemoglobin (5mmolL) is converted daily tomethaemoglobin releasing superoxide anion radicals [13] Asa precursor of other reactive oxygen species superoxide iscontinuously produced in erythrocytes due to a high oxygentension in arterial blood and haeme iron content whichacts as a free radical catalyst [14] Under these conditionsROS can damage erythrocyte components and after leavingthe cell these have the potential also to damage otherplasma components in circulation [15] This paper is focusedprimarily on the modulation of the conformational proper-ties of internal cellular components (mainly haemoglobin)following a single bout of exercise in young untrainedmalesIt seems that a higher consumption of oxygen and free radicalgeneration can lead to changes in haemoglobin conformationduring exercise As an indicator of the internal antioxidantsystem ascorbic acid and glutathione concentrations insideerythrocytes were measured as well as parameters relatedto oxidative stress in the cellular membrane Changes in theerythrocyte membrane and haemoglobin conformation wereexamined against the background of plasma parameters

2 Materials and Methods

21 Chemicals 4-Maleimido-2266-tetramethylpiperidine-1-oxyl (MSL) 4-iodoacetamide-2266-tetramethylpiperi-dine-1-oxyl (ISL) and 4-amino-2266-tetramethylpiperi-dine-1-oxyl (Tempamine) were obtained from Sigma Chem-ical Co (St Louis MO) All other chemicals were analyticalgrade products from POCh (Gliwice Poland)

22 Subjects and Protocol Eleven healthy males (mean age22 plusmn 2 years mean height 181 plusmn 7 cm mean mass 83 plusmn85 kg mean body mass index 23 plusmn 25 kgm2) volunteeredto participate in this study Subjects recruited were clinicallyhealthy according to a medical doctorrsquos examination that hadthe following exclusion criteria resting blood pressure higherthan 14090 resting heart rate higher than 90 beatsminsmoking or using antioxidant supplements and medica-ments All subjects were untrained (ie not performingany regular physical activity) Ethical approval was obtainedfrom the Medical University of Lodz All subjects signed aninformed consent form prior to participation

23 Experimental Procedures Subjects refrained from per-forming strenuous exercise and drinking alcohol for 24 hoursprior to testing and came to the lab in the morning after anovernight fast (no breakfast tea or coffee)

Exercise was performed using a friction-braked cycleergometer (Monark Sweden) Subjects started pedaling at 60Watts and 60 rpm (revolutions per minute) for 1min afterwhich the workload was increased by 30 Watts every minuteuntil volitional exhaustion Subjects maintained a pedal fre-quency of 60 rpm throughout the test and they were verballyencouraged to cycle until exhaustionThe heart rate at peak ofexercise was higher than 90 of the predicted maximal heart

rate according to the subjectsrsquo age Individual response tothis exercise was HRmax 195 plusmn 12 beatsmin and maximumwattage (Wmax) was 292 plusmn 27W maximum wattage perkilogram was 343 plusmn 057 (Wkg) During the exercise testheart rate was measured using ECG (system Case 16DRGcomp) Total work performed during exercise amountedto 92782 plusmn 16465 J Average time of exercise duration was873 plusmn 09min The protocol used was the Bruce treadmilltest protocol as this is the method for estimating VO

2max in

athletes The VO2max range 2589minus3262 (mLkgmin) was

calculated according to the following formula (1)

VO2max = 148 minus (1379 sdot 119879) + (0451 sdot 1198792) minus (0012 sdot 1198793)

(1)

119879 is total time on cycle ergometer measured as a fraction of aminute

Following exercise subjects remained seated at rest for1 hour and were allowed to drink only water This specificexercise protocol was used as a model of an acute stressorinducing oxidative stress

Venous blood samples were taken from an antecubitalvein before the exercise test directly after exercise andone hour after exercise The plasma lactate concentrationhaemoglobin (Hb) concentration and haematocrit (HTC)were determined in the collected blood using autoanalyserHb and HTC were used to calculate the ΔPV value accordingto the Dill and Costill formula [16]

ΔPV = [(Hb1

Hb2

) sdot (100 minusHTC

2

100 minusHTC1

) minus 1] sdot 100 (2)

where Hb1 HTC

1are preexercise values and Hb

2 HTC

2are

postexercise or 1 h recovery valuesFor other experiments the blood was centrifuged and the

plasma was separated Erythrocytes were washed three timeswith phosphate buffered saline (PBS pH 74)

24 Haemolysate Preparation Erythrocytes were haemol-ysed with water in the following relation (1 15) then vor-texed for 10min and centrifuged at 4000 rpm Haemolysatewas centrifuged at 16000timesg for separation of erythro-cyte membranes Haemoglobin concentration was measuredusing Drabkinrsquos method [17] The crude haemoglobin with-out purification was used for estimation of conformationalchanges of this protein

25 Spin Labelling of Crude Haemoglobin In order to inves-tigate conformational changes of haemoglobin MSL and ISLwere used which bind covalently to the -SH groups TheEPR spectra of spin labelled crude haemoglobin are shownin Figures 1 and 2

Haemolysate was labelled using 01molL ethanol solu-tions of MSL or ISL (50 1) and incubated for one hour atroom temperature The unbound spin labels were removedthrough dialysis using 20mmolL phosphate buffer pH = 74for 24 h at 4∘C until the EPR signal in dialysis buffer disap-peared The effect of oxidative stress on the conformationalchanges of haemoglobin was studied by an estimation of therelative rotational correlation time 120591

119888for both spin labels

The Scientific World Journal 3

h0 hminus1

w0

(a)

h0

w0

hminus1

(b)

Figure 1 Electron paramagnetic resonance spectrum of (a) 4-maleimido-2266-tetramethylpiperidine-1-oxyl (MSL) and (b) 4-iodoacet-amide-2266-tetramethylpiperidine-1-oxyl (ISL) attached to haemoglobin

0005

10

15

20

25

30

Preexercise Postexercise

120591c

(ns)

lowast

Recovery (1h)

Figure 2 Influence of physical exercise on the mobility of internalerythrocyte peptides and proteins measured with MSL spin labelbefore after and 1 hour after exercise lowast indicates significant dif-ference in 120591

119888between preexercise and postexercise (119875 lt 0001)

indicates significant difference in 120591119888between postexercise and 1 hour

recovery (119875 lt 001)

The spectra of MSL and ISL attached to the crude hae-moglobin indicated that these spin labels were immobilizedEstimation of the level of themobility for these spin labels waspossible by calculating relative rotational correlation times(120591119888) according to the Kivelson formula (3) [18]

120591119888= 1198961199080radicℎ0

ℎminus1

minus 1 (3)

where 120591119888is time when the spin label undergoes full rotation

119896 is constant equal to 65 times 10minus10 s 1199080is width of the midline

of spectrum ℎ0is height of the midline of spectrum and ℎ

minus1

is height of the high-field line of spectrum

26 Spin Labelling of Erythrocyte Internal Components inWhole Erythrocytes Changes of internal cytoplasmic pep-tides and proteins were investigated using a maleimidespin label in order to estimate the erythrocyte componentsmobility More than 90 of the bound spin label remained inthe cytosol and it can be inferred thatMSL binds inside of theerythrocyte mainly with glutathione less with haemoglobinand other proteins [19] Erythrocytes were labelled with MSLduring a one-hour period at room temperature Next anyexcess of spin label was removed by several washings withhuge amounts of cold PBS pH = 74 up to a diminishing ofthe EPR signal in the supernatant From the EPR spectra therelative rotational correlation time was calculated accordingto formula (3)

27 Erythrocyte Internal Viscosity Erythrocyte internal vis-cosity was monitored as described by Morse using Tem-pamine which easily permeates the membrane and remainsunbound inside the erythrocyte [20] The relative rotationalcorrelation times (120591

119888) was calculated using the Kivelson

equation (3)The erythrocyte internal viscosity were calculated accord-

ing to the following formula

120578 =

120591119888(RBC)

120591119888(H2O)120578H2O (4)

where 120591119888(RBC) is rotational correlation time for Tempamine

inside the erythrocyte 120591119888(H2O) is rotational correlation time

for Tempamine in water and 120578H2O is water viscosity equal to

1 cP

28 EPR Measurements EPR spectra were performed on theBruker ESP 300 E spectrometer at room temperature (22 plusmn2∘C) operating at a microwave frequency of 973GHz Theinstrumental settings were as follows the microwave powerwas 10mW and the centre field was set at 3480G with a


range of 80G a 100 kHzmodulation frequency amodulationamplitude of 101 G and a time scan of 256 s

29 Ascorbic Acid Measurements The method is based onthe ability of ascorbic acid reduction of the dye 26-dichlo-rophenolindophenol (indophenol) to a colourless compound[21] The 10 m-phosphoric acid was added to 1mL ofhaemolysate on ice bath to remove haemoglobin and otherproteins The mixture was centrifuged at 4000timesg for 5minThe citrate-acetate buffer (pH 415) with p-chloromercuri-benzoic acid (p-CMB) sodium salt and indophenol solutionat a concentration of 01 gL were added to the supernatantThe absorbance of solution was measured after 30 s at 520 nm(1198601) against water Next the ascorbic acid (in substantia) was

added to the colourless solution (1198602) The blank sample was

prepared from a buffer containing p-CMB and indophenolwhich was measured at 520 nm (119861

1) and repeated measure-

ments were performed after the addition of ascorbic acid (insubstantia) (119861

2)

Δ119860 = (1198611minus 1198612) minus (119860

1minus 1198602) (5)

The concentration of ascorbate in the sample was estimatedfrom the standard curve prepared for different concentrationsof the ascorbic acid (in the range of 0ndash100120583molL) andcalculated as nmolmg Hb

210 Erythrocyte Membrane Preparation Erythrocyte ghostswere prepared using the method described in [22] Erythro-cyte ghosts were used for the measurements of thiol groupshydroperoxides and acetylcholinesterase (AChE) activityMembrane protein concentration was evaluated by the spec-trophotometric method according to Lowry et al using theFolin reagent [23]

211 Thiols Measurements Erythrocyte membrane and plas-ma thiol groups were measured using the method of Ell-man [24] Samples were diluted with a 10mmolL phos-phate buffer pH 80 containing SDS Following this DTNB(551015840-dithiobis(2-nitrobenzoic acid)) from a 10mmolL stocksolution was added and samples were incubated for one hourat 37∘CThe thiols reacted with DTNB to form anions with astrong yellow colour which were optically active at 412 nmThe basal optical activity of the samples was measuredbefore the addition of DTNB A calibration curve was pre-pared using different concentrations of reduced glutathioneThe concentration of the thiol groups was calculated andexpressed as 120583molmg proteins of plasma or as nmolmgproteins of erythrocyte membranes

212 GSHMeasurements Reduced glutathione was analyzedin haemolysate using the method of Ellman [24] The sam-ples were treated with trichloroacetic acid (TCA) and theprotein precipitate was removed by centrifugation Thismethod is analogous to determination of the protein thiolconcentration The concentration of GSH was estimated inmmolpacked cells (PC)

213 Peroxide Measurements For the determination of theerythrocyte membrane and plasma hydroperoxides themethod with xylenol orange was employedThe reaction wasbased on the rapid oxidation of Fe(II) to Fe(III) in the pres-ence of peroxides [25 26] 25mmolL ammonium iron(II)sulfate in 25molL H

2SO4and 125 120583molL xylenol orange

with 100mmolL sorbitol were mixed (1 100) to preparea working solution The samples were then mixed with aworking reagent and incubated for 30min in the dark Reac-tion of Fe(III) with xylenol orange yielded a violet-colouredcomplex which was quantified spectrophotometrically at560 nm The concentration of peroxides was expressed as120583molmg protein for erythrocyte membranes and as 120583molLin the case of plasma

214 Acetylcholinesterase Activity The membrane acetyl-cholinesterase (AChE) activity was measured using Ellmanspectrophotometric method [27]

215 The Reducing Potential of Plasma (RPP) The reducingpotential of plasma (RPP) was measured using a spec-trophotometric method with 11-diphenyl-2-picrylhydrazyl(DPPH) a stable colour radical that reacts with antioxi-dants Plasma was added to 150120583molL DPPH in methanol(1 20) and incubated at room temperature for 30min Theabsorbance of samples was measured at 517 nm Suppressionof colour intensity was proportional to antioxidant content inplasma A calibration curve was prepared from differentTrolox concentrations in the range of 0ndash1000120583molL [28]The reducing potential of plasma was expressed as Troloxequivalents (in 120583molL of plasma)

216 Plasma Viscosity Plasma viscosity was monitored asdescribed by Morse using Tempamine [20] Plasma viscositywas calculated using data obtained from EPR spectra accord-ing to formula (4)

217 Plasma Lipid Peroxidation Lipid peroxidation wasmea-sured using the method with thiobarbituric acid (TBA)described in [29]

218 Plasma Carbonyl Compounds Plasma protein carbonylcontent was estimated using 24-dinitrophenylhydrazine(DNPH) [30]

219 Statistical Analysis All measurements are presented asmeans [plusmn] and standard deviation (SD) For statistical analy-sis we used the SPSS package A repeated-measures analysisof variance (ANOVA) with a Tukey test was used to evaluatethe differences between themeans Statistical significancewasaccepted at 119875 lt 005

3 Results

The investigated parameters associated with oxidative stresswere monitored in whole red blood cells isolated haemo-globin membranes and plasma This was done for blood


Table 1 Effect of physical exercise on lactate haemoglobin concentration as well as haematocrit and delta plasma volume (ΔPV) beforeafter and 1 hour after exercise

Parameter Preexercise119909plusmn SD

Postexercise119909plusmn SD

Recovery (1 h)119909plusmn SD

Lactate (mmolL) 247 plusmn 078 1119 plusmn 178lowast

333 plusmn 080

Haemoglobin (gdL) 1544 plusmn 087 1675plusmn078lowastlowast

1524 plusmn 087

Haematocrit () 4556 plusmn 222 4972plusmn240lowastlowast

4490 plusmn 246

ΔPV () minus1447 plusmn 395 302 plusmn 419

lowastSignificant difference between preexercise and postexercise (119875 lt 00002) lowastlowastpreexercise and postexercise (119875 lt 00005) significant difference between post-exercise and 1 h recovery (119875 lt 00002)

collected at three time points before exercise immediatelyafter exercise and one hour after exercise

The amount of red blood cells and haemoglobin con-centration were determined as parameters that indirectlyindicated oxygen transport by blood Lactate concentrationwas measured as a muscle metabolite Another importantparameter was that the level of plasma volume changes afterexercise and this was determined by the original methodproposed by Dill and Costill [16] Table 1 shows the physic-ochemical parameters of blood at different stages beforeimmediately after and one hour after exercise Significantalterations in all the studied parameters were observed imme-diately after exercise One hour later all returned to the orig-inal control values except in the case of the plasma volume

EPR spectroscopy was used to investigate conformationalchanges of haemoglobin internal viscosity of erythrocytesand plasma viscosity Tempamine was used to investigatechanges in internal viscosity of erythrocytes Measurementsof intercellular viscosity were based on calculation of relativerotational correlation time for this spin label [20] Our resultsdid not show statistically significant changes at different timepoints after exercise Moreover the results obtained for Tem-pamine were confirmed by detection of mobility of erythro-cyte internal components for example proteins and peptidesusingmaleimide spin label Analysis of themaleimide spectraand calculation of the relative rotational correlation timesshowed nonsignificant changes in this parameter immedi-ately after exercise and one hour later in comparison tocontrol

Conformational changes of haemoglobin were estimatedin cell lysates using iodoacetamide andmaleimide spin labelsboth of which covalently bind to -SH groups on cysteine-93of 120573-globin chains [31 32] Figure 1 shows the EPR spectra ofhaemoglobin labelled withMSL and ISL [33] BothMSL- andISL-labelled haemoglobin spectra had two different spatialorientations relative to the haemoglobin molecule [31 32]However both spin labels attached to the haemoglobinexhibited considerable differences in local anisotropicmotionwith respect to the site to which they were attached Thebroad signals marked by the arrows are due to maleimidebeing strongly immobilized at -SH groups on cysteine 93(Figure 1) A relative rotational correlation time was calcu-lated from the spectra for both labels This parameter wassignificantly decreased for MSL immediately after exercise incomparison to controls (119875 lt 0 001) but one hour later anincrease in 120591

119888was found (Figure 2) In the case of ISL there

00

01

02

03

04

05

06

07

Preexercise

Asc

orbi

c aci

d (n

mol

mg H

b) lowast

Recovery (1h)Postexercise

Figure 3 Changes in ascorbic acid concentration inside the ery-throcytes before after and 1 hour after exercise lowast indicates signifi-cant difference between preexercise and postexercise (119875 lt 0005)

were no significant changes at any of the investigated timepoints These results suggest that the acute exercise causedreversible changes in the conformational state of globin chainof haemoglobin

Investigation of low molecular weight of antioxidantsinside the erythrocytes for example glutathione and ascorbicacid showed a significant decline of ascorbic acid immedi-ately after exercise (119875 lt 0 005) and slight increase one hourlater (Figure 3) The level of glutathione increased just afterexercise and decreased one hour after exercise but these datawere statistically insignificant

The levels of thiol groups peroxides and acetylcholin-esterase activity in the erythrocyte membrane were alsoestimated The concentration of -SH groups was significantlylower (119875 lt 005) one hour after exercise in comparison tothe controls before and immediately after exercise (Figure 4)The level of erythrocyte membrane peroxides was notchanged significantly at any of the investigated time pointsafter exercise The erythrocyte acetylcholinesterase activity isa useful marker of oxidative stress This enzyme is locatedon the outer surface of the plasma membrane and its activityis generally decreased by oxidizing agents However AChEactivity in membranes was only insignificantly lower imme-diately upon exercise and one hour after exercise

The effect of a single bout of exercise on the plasmaparameters was also examined An increase in the reduc-ing potential of plasma measured by DPPH radical using


40

50

60

70

80

90


-SH

(nm

olm

g pro

tein

)

Recovery (1h)

Figure 4 Influence of physical exercise on thiol group concentra-tion of erythrocyte membranes measured before after and 1 hourafter exercise indicates significant difference between postexerciseand 1 h recovery (119875 lt 005)

the spectrophotometric method was observed 1 hour afterexercise (119875 lt 005) and additionally significant differ-ences in RPP levels were detected between the time pointsboth immediately after and one hour after exercise (119875 lt005) (Figure 5) Nevertheless analysis of the parameters ofplasma related to oxidative stress revealed a non-significantincrease in TBARS level This change was accompanied bya significant decrease (119875 lt 002) in plasma peroxidesobserved both immediately after and 1 hour after exercise(Figure 6) Furthermore neither the levels of carbonyl com-pounds nor the viscosity of plasma changed following asingle bout of exercise (data not shown) The total level ofplasma thiol groups was significantly elevated immediatelyafter exercise (119875 lt 0002) and one hour after exercise (119875 lt001) compared with preexercise values (Figure 7)

4 Discussion

The question of the role of ROS and their interactions withcell components and physiopathological consequences fol-lowing physical exhaustion is an important issue in free rad-ical biochemistry Generally the intensity of ROS generationduring exercise depends on the level of antioxidant enzymeactivities diet time and intensity of exercise and oxygenconsumption

Commonly investigations of alterations in plasma mus-cles and other tissues in body organs are of relevance toathletes It seems that a study on a young untrained manmaybe useful in explaining changes in the blood of nonathletesundergoing rehabilitation

In our study young untrained healthy males were sub-jected to physical exhaustion by a single bout of exercise Wedescribed early mechanisms of oxidative stress developmentin erythrocyte membranes reflected by changes in theirproperties immediately after exercise and during recoverytime [12]

Lactate is a metabolic product of glucose conversion inmuscles during glycolysis and its levels have been studied insport medicine since the 1930s It is a useful parameter for

0

100

200

300

400

500

600

700

800


Trol

ox (120583

lowast

Recovery (1h)

mol

L)

Figure 5 Effect of physical exercise on reducing potential of plasmaestimated before after and 1 hour after physical exercise lowast indicatessignificant difference between preexercise and 1 hour recovery (119875 lt00002) indicates significant difference between postexercise and1 h recovery (119875 lt 0001)

0

10

20

30

40

50

60

70


Pero

xide

s (120583

mol

L)

Recovery (1h)

Figure 6 Effect of physical exercise on peroxide concentration inplasma measured before after and 1 hour after physical exercise indicates significant difference between postexercise and 1 h recov-ery (119875 lt 002)

00

05

10

15

20

25

30

35


-SH

(120583m

olm

g pro

tein

)

lowastlowastlowast

Recovery (1h)

Figure 7 Effect of physical exercise on thiol group concentration inplasma measured before after and 1 hour after physical exercise lowastindicates significant difference between preexercise and postexercise(119875 lt 002) lowastlowast preexercise and 1 hour recovery (119875 lt 001)


short exercise experiments An approximate fourfold increasein this parameter was observed immediately after exerciseand a decrease in the control value was detected after onehour of recovery time In addition a significant increase innumber of erythrocytes haematocrit and concentration ofhaemoglobin was observed immediately after exercise Onehour after exercise the levels of these parameters werecomparable to the control values These observations are inagreement with other studies [34]

Another important parameter was the change in plasmavolume This parameter reflected changes in the total serumprotein concentration and indicated water leaching from thevascular system during exercise More pronounced changesin ΔPV were observed immediately after exercise while onehour later the changes were moderate

The changes in internal components of erythrocytes forexample in cytoplasmic peptides and proteins includinghaemoglobin can occur as a consequence of acute exerciseBecause haemoglobin remained in continuous interactionwith increased oxygen supply the changes in the conforma-tional state of this molecule were studied

Erythrocytes can be damaged by both internal and exter-nal ROS sources Internally haemoglobin may be autoox-idized to methaemoglobin resulting in the production ofsuperoxide anions which are precursors of other reactiveoxygen species (hydrogen peroxide hydroxyl radical andsinglet oxygen) [35] Externally ROS (including hypochlor-ous acid) can be released by activated neutrophils Strongoxidative agents can easily oxidize cellular proteins andunsat-urated lipids However erythrocytes can be protected againstROS by enhanced activity of antioxidant enzymes and highconcentration of glutathione and other lowmolecular weightantioxidants It seems that a higher consumption of oxygen bythe body during exercise can increase the level of the super-oxide anion released fromhaemoglobin inside red blood cellsas well as increase the release of ROS by activated neutrophilsin plasma Under these conditions RBC may be damaged byinternal and external sources of ROSThus haemoglobin canalso be considered as a potent free radical catalyst This factmotivated us to investigate the internal components of RBC

Potential changes in the dynamics of red blood cellcomponents after exercise were studied by electron paramag-netic resonance spin labelling spectroscopy Spin labels wereused in the investigation of internal viscosity the mobilityof internal components and the conformational state ofhaemoglobinThey bound tomacromolecules and played therole of reporting groups providing information about thechanges in the microenvironment of the nearest region

Two spin labels (Tempamine MSL) were applied for theanalysis of internal viscosity and the physical state of RBCinternal components With the usage of Tempamine a ten-dency to increase the internal viscosity of erythrocytes bothimmediately after exercise and one hour later was observedbut these results were not statistically significant It wasshown that at physiological pH maleimide spin label reactswith thiol groups of peptides and proteins [36] MSL easilypenetrates cellular membrane of erythrocytes and attachesmainly to glutathione [19] A slight increase in the motionof this label was found immediately after exercise but again

in this case the results were not statistically significantGenerally any modulation following a single bout of exercisein young untrained males did not influence the properties ofthe internal fluid of erythrocytes

Possible conformational changes of haemoglobin can beestimated using maleimide and iodoacetamide spin labelsA decrease in relative rotational correlation time value wasdetected for MSL-labelled haemoglobin which indicatesconformational changes in the structure of globin chainsimmediately after exercise However one hour after exer-cise the observed tendency returned to normal levels Thisresult may suggest reversible conformational changes in thehaemoglobin chain However the modulation of conforma-tional properties of Hb following a single bout of exercisein young untrained males did not influence internal fluidproperties

Statistically significant changes in the concentration ofascorbic acid were detected inside erythrocytes shown bya decrease immediately after exercise and an increase onehour after exercise It explains the fact that following a singlebout of exercise the internal defence system of the cellswas activated as a result of elevated oxidative stress in theplasma Tissue ascorbate concentration exceeds its plasmalevels by three to ten times It is possible that during oxidativestress (strenuous exercise) ascorbate might be released fromtissues to plasma and subsequently to erythrocytes becauseequilibrium between its concentration outside and insideRBCs exists

Decreased level of GSH inside erythrocytes was associ-ated with a significant decrease of -SH groups in the mem-brane proteins one hour after exercise as a consequence ofinduced oxidative stress

Erythrocyte acetylcholinesterase is a useful marker ofoxidative stress The inhibition of this enzyme by oxidizingagents such as hydrogen peroxide and lipid peroxides wasdescribed in the 1960s [37] A decline in AChE activity wasalso observed in ischemic patients and in the process of age-ing [38 39] Moreover we found that the acetylcholinesteraseactivity in the cellular membrane exhibited a decreasingtendency which also indicates the presence of oxidative stressin the cellular membrane On the other hand peroxide levelsin the membrane did not change at any of the investigatedtime points

In our study we also observed an insignificant increase ofTBARS in the plasmaThis result is in linewith numerous ear-lier studies on both maximal and submaximal exercise [2 4041] Furthermore similar to previous studies hydroperoxidelevels were found to be significantly decreased [2 40 41] Itis possible that part of the hydroperoxides through a processof fragmentation is converted to aldehydes Nevertheless anapproximate twofold increase in MDA plasma levels afterexercise was observed by Sureda et al [42] In addition someother studies found elevated or unchanged hydroperoxidelevels [43 44]

Moreover the level of carbonyl compounds or plasmafluidity did not significantly change at any of the investigatedtime points after exercise However while an increase inprotein carbonyls was observed by some authors others didnot report such results in trained subjects [45 46]


Generally a decrease in antioxidant capacity wasobserved in response to strenuous exercise [47 48] In ourstudy there was no change in the antioxidant capacity ofplasma immediately after exercise Similar results werereported by Waring et al [49] However a statistically sig-nificant increase in plasma antioxidant capacity was detectedone hour after exercise This observation confirms resultsfrom earlier findings There are a number of studies wherean increase in plasma antioxidant capacity was found duringrecovery time by applying different biochemical methods[12 40 49]

We also observed an increase in total thiols both imme-diately after and one hour after exercise Elevation of bloodplasma thiols was also reported by Rajguru et al who sug-gested that an increase in thiols can be related to triggeringthe repair system in the damaged RBC membrane [50]Therefore an increase of both antioxidant capacity and totalthiols suggests the existence of strong defencemechanisms inplasma

We hypothesize that the oxidative stress generated duringa single bout of exercise is mainly caused by shear stresswhich leads to neutrophil activation and ROS release Thelevel of myeloperoxidase in blood was reported to doubleafter exercise [42 51] This result shows that hypochlorousacid is one of the oxidizing agents in plasma Shear stress maylead to ROS generation and the activation of cyclooxygenase2 and endothelial cell nitric oxide synthase [52 53]

Exercise increases heart rate and blood flow whichtogether lead to vascular shear stress and ROS generation byan endothelium-dependent mechanism [52] The expressionof endothelial nitric oxide synthase (eNOS) and nitric oxideproduction as a consequence of shear stress have also beenextensively discussed [53 54] Thus peroxynitrite can beanother molecule responsible for oxidative stress in plasma

The increase in reducing plasma potential and in thi-ols concentration in response to oxidative stress providesevidence for mobilization of a defence system and can beexplained as an adaptation reaction of the human body andthe vascular system to exerciseWe conclude that a single boutof exercise mobilizes antioxidant defence systems in plasmaand erythrocytes

In summary RBCs provide an effective protection systemagainst externally triggered oxidative stress However itseems that during maximal or prolonged submaximal exer-cise RBCsmay be exposed to greater damage from externallyinduced oxidative stress

Such studies are arguably useful in explaining changes inthe blood of nonathletes undergoing rehabilitation

Acknowledgment

This work was supported in part by Grant nos N 404 11733 and N 404 178 440 of Ministry of Science and HigherEducation (Poland)

References

[1] R J Bloomer P G Davis L A Consitt and LWideman ldquoPlas-ma protein carbonyl response to increasing exercise duration in

aerobically trained men and womenrdquo International Journal ofSports Medicine vol 28 no 1 pp 21ndash25 2007

[2] G W Davison T Ashton B Davies and D M Bailey ldquoInvitro electron paramagnetic resonance characterization of freeradicals relevance to exercise-induced lipid peroxidation andimplications of ascorbate prophylaxisrdquo Free Radical Researchvol 42 no 4 pp 379ndash386 2008

[3] D Zieker E Fehrenbach J Dietzsch et al ldquocDNA microarrayanalysis reveals novel candidate genes expressed in humanperipheral blood following exhaustive exerciserdquo PhysiologicalGenomics vol 23 no 3 pp 287ndash294 2005

[4] K Masuda K Tanabe and S Kuno ldquoExercise and reactiveoxygen species in eldery-exercise as prevention of oxidativestressrdquo International Journal of Sport andHealth vol 4 pp 348ndash359 2006

[5] C K Sen T Rankinen S Vaisanen and R Rauramaa ldquoOxida-tive stress after human exercise effect of N-acetylcysteine sup-plementationrdquo Journal of Applied Physiology vol 76 no 6 pp2570ndash2577 1994

[6] M J Jackson ldquoExercise and oxygen radical production bymusclerdquo in Handbook Oxidants and Antioxidant in Exercise CK Sen L Packer and O Hannien Eds pp 57ndash68 Elsevier Sci-ence Amsterdam The Netherlands 2000

[7] A M Niess H-H Dickhuth H Northoff and E FehrenbachldquoFree radicals and oxidative stress in exercise-immunologicalaspectsrdquo Exercise Immunology Review no 5 pp 22ndash56 1999

[8] JM Peake ldquoExercise-induced alterations in neutrophil degran-ulation and respiratory burst activity possible mechanisms ofactionrdquo Exercise Immunology Review vol 8 pp 49ndash100 2002

[9] K Suzuki S Naganuma M Totsuka et al ldquoEffects of exhaus-tive endurance exercise and its one-week daily repetition onneutrophil count and functional status in untrained menrdquoInternational Journal of Sports Medicine vol 17 no 3 pp 205ndash212 1996

[10] K SuzukiH Sato TKikuchi et al ldquoCapacity of circulating neu-trophils to produce reactive oxygen species after exhaustiveexerciserdquo Journal of Applied Physiology vol 81 no 3 pp 1213ndash1222 1996

[11] J Peake and K Suzuki ldquoNeutrophil activation antioxidantsupplements and exercise-induced oxidative stressrdquo ExerciseImmunology Review vol 10 pp 129ndash141 2004

[12] J Brzeszczynska A Pieniazek L Gwozdzinski K Gwozdzin-ski and A Jegier ldquoStructural alterations of erythrocyte mem-brane components induced by exhaustive exerciserdquo AppliedPhysiology Nutrition and Metabolism vol 33 no 6 pp 1223ndash1231 2008

[13] H P Misra and I Fridovich ldquoThe generation of superoxideradical during the autoxidation of hemoglobinrdquo The Journal ofBiological Chemistry vol 247 no 21 pp 6960ndash6962 1972

[14] J W Bayens ldquoOxygen and liferdquo in Medical Biochemistry J WBayens and M H Domoniczak Eds pp 497ndash506 ElsevierPhiladelphia Pa USA 2005

[15] R M Johnson G Goyette Jr Y Ravindranath and Y-S HoldquoHemoglobin autoxidation and regulation of endogenousH

2O2

levels in erythrocytesrdquo Free Radical Biology and Medicine vol39 no 11 pp 1407ndash1417 2005

[16] D B Dill and D L Costill ldquoCalculation of percentage changesin volumes of blood plasma and red cells in dehydrationrdquoJournal of Applied Physiology vol 37 no 2 pp 247ndash248 1974

[17] D L Drabkin ldquolsquoSpectrophotometric studiesrsquo XIVThe crystallo-graphic and optical properties of the haemoglobin of man those


of other speciesrdquo The Journal of Biological Chemistry vol 164no 2 pp 703ndash723 1946

[18] D Kivelson ldquoTheory of ESR linewidths of free radicalsrdquo TheJournal of Chemical Physics vol 33 no 4 pp 1094ndash1106 1960

[19] K Gwozdzinski ldquoA spin label study of the action of cupric andmercuric ions on human red blood cellsrdquoToxicology vol 65 no3 pp 315ndash323 1991

[20] P D Morse II ldquoDetermining intracellular viscosity from therotational motion of spin labelsrdquo Methods in Enzymology vol127 no C pp 239ndash249 1986

[21] S T Omaye J David Turnbull and H E Sauberlich ldquoSelectedmethods for the determination of ascorbic acid in animal cellstissues and fluidsrdquo Methods in Enzymology vol 62 pp 3ndash111979

[22] J T Dodge C Mitchell and D J Hanahan ldquoThe preparationand chemical characteristics of hemoglobin-free ghosts ofhuman erythrocytesrdquo Archives of Biochemistry and Biophysicsvol 100 no 1 pp 119ndash130 1963

[23] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[24] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[25] C Gay J Collins and J M Gebicki ldquoHydroperoxide assay withthe ferric-xylenol orange complexrdquoAnalytical Biochemistry vol273 no 2 pp 149ndash155 1999

[26] C Gay and J M Gebicki ldquoA critical evaluation of the effectof sorbitol on the ferric-xylenol orange hydroperoxide assayrdquoAnalytical Biochemistry vol 284 no 2 pp 217ndash220 2000

[27] G L Ellman K D Courtney V Andres Jr and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no 2pp 88ndash95 1961

[28] T Yamaguchi H Takamura T Matoba and J Terao ldquoHPLCmethod for evaluation of the free radical-scavenging activity offoods by using 11-diphenyl-2-picrylhydrazylrdquo Bioscience Bio-technology and Biochemistry vol 62 no 6 pp 1201ndash1204 1998

[29] C A Rice-Evans A T Diplock and M C R Symons ldquoTech-niques in free radical researchrdquo in Laboratory Techniques inBiochemistry andMolecular Biology R H Burdon and P H vanKnippenberg Eds vol 22 Elsevier Amsterdam The Nether-lands 1991

[30] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[31] S Ohnishi J C Boeyens and H M McConnell ldquoSpin-labeledhemoglobin crystalsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 56 no 3 pp 809ndash813 1966

[32] S Ogawa and H M McConnell ldquoSpin-label study of hemo-globin conformations in solutionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 58 no1 pp 19ndash26 1967

[33] M J Telen and R E Kaufman ldquoThe mature erythrocyterdquo inWintrobersquoS Clinical Hematology J P Greer and J Foerster Edspp 217ndash247 Lippincot Williams amp Wilkins Philadelphia PaUSA 1999

[34] S Choudhary R Rajnee and B K Binawara ldquoEffect of exerciseon serum iron blood haemoglobin and cardiac efficiencyrdquo Jour-nal of Postgraduate Medical Institute vol 26 no 1 pp 13ndash162012

[35] P Hochstein and S K Jain ldquoAssociation of lipid peroxidationand polymerization of membrane proteins with erythrocyteagingrdquo Federation Proceedings vol 40 no 2 pp 183ndash188 1981

[36] L J Berliner ldquoThe spin-label approach to labeling membraneprotein sulfhydryl groupsrdquo Annals of the New York Academy ofSciences vol 414 pp 153ndash161 1983

[37] B W OrsquoMalley C E Mengel W D Meriwether and L GZirkle Jr ldquoInhibition of erythrocyte acetylcholinesterase byperoxidesrdquo Biochemistry vol 5 pp 40ndash45 1966

[38] M de Carvalho Correa P Maldonado C Saydelles da Rosaet al ldquoOxidative stress and erythrocyte acetylcholinesterase(AChE) in hypertensive and ischemic patients of both acute andchronic stagesrdquo Biomedicine and Pharmacotherapy vol 62 no5 pp 317ndash324 2008

[39] R Jha and S I Rizvi ldquoAge-dependent decline in erythrocyteacetylcholinesterase activity correlation with oxidative stressrdquoBiomedical Papers vol 153 no 3 pp 195ndash198 2009

[40] J G Steinberg S Delliaux and Y Jammes ldquoReliability of dif-ferent blood indices to explore the oxidative stress in responseto maximal cycling and static exercisesrdquo Clinical Physiology andFunctional Imaging vol 26 no 2 pp 106ndash112 2006

[41] M G Nikolaidis A Kyparos M Hadziioannou et al ldquoAcuteexercise markedly increases blood oxidative stress in boys andgirlsrdquo Applied Physiology Nutrition and Metabolism vol 32 no2 pp 197ndash205 2007

[42] A Sureda P Tauler AAguilo et al ldquoRelation between oxidativestress markers and antioxidant endogenous defences duringexhaustive exerciserdquo Free Radical Research vol 39 no 12 pp1317ndash1324 2005

[43] H M Alessio A E Hagerman B K Fulkerson J Ambrose RE Rice and R L Wiley ldquoGeneration of reactive oxygen speciesafter exhaustive aerobic and isometric exerciserdquo Medicine andScience in Sports and Exercise vol 32 no 9 pp 1576ndash1581 2000

[44] R L Wilber P L Holm D M Morris et al ldquoEffect of F1O2 onoxidative stress during interval training at moderate altituderdquoMedicine and Science in Sports and Exercise vol 36 no 11 pp1888ndash1894 2004

[45] H Miyazaki S Oh-ishi T Ookawara et al ldquoStrenuous endur-ance training in humans reduces oxidative stress followingexhausting exerciserdquo European Journal of Applied Physiologyvol 84 no 1-2 pp 1ndash6 2001

[46] N Rahnama A A Gaeini and M R Hamedinia ldquoOxidativestress responses in physical education students during 8 weeksaerobic trainingrdquo Journal of Sports Medicine and PhysicalFitness vol 47 no 1 pp 119ndash123 2007

[47] M G Tozzi-Ciancarelli M Penco and C Di Massimo ldquoInflu-ence of acute exercise on human platelet responsiveness possi-ble involvement of exercise-induced oxidative stressrdquo EuropeanJournal of Applied Physiology vol 86 no 3 pp 266ndash272 2002

[48] T A Watson R Callister R D Taylor D W Sibbritt L KMacdonald-Wicks andM LGarg ldquoAntioxidant restriction andoxidative stress in short-duration exhaustive exerciserdquoMedicineand Science in Sports and Exercise vol 37 no 1 pp 63ndash71 2005

[49] W S Waring A Convery V Mishra A Shenkin D J Webband S R J Maxwell ldquoUric acid reduces exercise-inducedoxidative stress in healthy adultsrdquo Clinical Science vol 105 no4 pp 425ndash430 2003

[50] S U Rajguru G S Yeargans andNW Seidler ldquoExercise causesoxidative damage to rat skeletal muscle microsomes whileincreasing cellular sulfhydrylsrdquo Life Sciences vol 54 no 3 pp149ndash157 1994


[51] J Pincemail GCamusA Roesgen et al ldquoExercise induces pen-tane production and neutrophil activation in humans Effect ofpropranololrdquo European Journal of Applied Physiology and Occu-pational Physiology vol 61 no 3-4 pp 319ndash322 1990

[52] F R M Laurindo M D A Pedro H V Barbeiro et al ldquoVascu-lar free radical release ex vivo and in vivo evidence for a flowmdashdependent endothelial mechanismrdquo Circulation Research vol74 no 4 pp 700ndash709 1994

[53] I Fleming and R Busse ldquoMolecular mechanisms involved inthe regulation of the endothelial nitric oxide synthaserdquo Ameri-can Journal of Physiology Regulatory Integrative and Compara-tive Physiology vol 284 no 1 pp R1ndashR12 2003

[54] G Kojda and R Hambrecht ldquoMolecular mechanisms of vas-cular adaptations to exercise Physical activity as an effectiveantioxidant therapyrdquoCardiovascular Research vol 67 no 2 pp187ndash197 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the influence of physical activity on the disintegration ofinternal erythrocyte components In addition it is notknown whether alteration in haemoglobin conformationoccurs during exercise However it has been reported that3 of total haemoglobin (5mmolL) is converted daily tomethaemoglobin releasing superoxide anion radicals [13] Asa precursor of other reactive oxygen species superoxide iscontinuously produced in erythrocytes due to a high oxygentension in arterial blood and haeme iron content whichacts as a free radical catalyst [14] Under these conditionsROS can damage erythrocyte components and after leavingthe cell these have the potential also to damage otherplasma components in circulation [15] This paper is focusedprimarily on the modulation of the conformational proper-ties of internal cellular components (mainly haemoglobin)following a single bout of exercise in young untrainedmalesIt seems that a higher consumption of oxygen and free radicalgeneration can lead to changes in haemoglobin conformationduring exercise As an indicator of the internal antioxidantsystem ascorbic acid and glutathione concentrations insideerythrocytes were measured as well as parameters relatedto oxidative stress in the cellular membrane Changes in theerythrocyte membrane and haemoglobin conformation wereexamined against the background of plasma parameters

2 Materials and Methods

21 Chemicals 4-Maleimido-2266-tetramethylpiperidine-1-oxyl (MSL) 4-iodoacetamide-2266-tetramethylpiperi-dine-1-oxyl (ISL) and 4-amino-2266-tetramethylpiperi-dine-1-oxyl (Tempamine) were obtained from Sigma Chem-ical Co (St Louis MO) All other chemicals were analyticalgrade products from POCh (Gliwice Poland)

22 Subjects and Protocol Eleven healthy males (mean age22 plusmn 2 years mean height 181 plusmn 7 cm mean mass 83 plusmn85 kg mean body mass index 23 plusmn 25 kgm2) volunteeredto participate in this study Subjects recruited were clinicallyhealthy according to a medical doctorrsquos examination that hadthe following exclusion criteria resting blood pressure higherthan 14090 resting heart rate higher than 90 beatsminsmoking or using antioxidant supplements and medica-ments All subjects were untrained (ie not performingany regular physical activity) Ethical approval was obtainedfrom the Medical University of Lodz All subjects signed aninformed consent form prior to participation

23 Experimental Procedures Subjects refrained from per-forming strenuous exercise and drinking alcohol for 24 hoursprior to testing and came to the lab in the morning after anovernight fast (no breakfast tea or coffee)

Exercise was performed using a friction-braked cycleergometer (Monark Sweden) Subjects started pedaling at 60Watts and 60 rpm (revolutions per minute) for 1min afterwhich the workload was increased by 30 Watts every minuteuntil volitional exhaustion Subjects maintained a pedal fre-quency of 60 rpm throughout the test and they were verballyencouraged to cycle until exhaustionThe heart rate at peak ofexercise was higher than 90 of the predicted maximal heart

rate according to the subjectsrsquo age Individual response tothis exercise was HRmax 195 plusmn 12 beatsmin and maximumwattage (Wmax) was 292 plusmn 27W maximum wattage perkilogram was 343 plusmn 057 (Wkg) During the exercise testheart rate was measured using ECG (system Case 16DRGcomp) Total work performed during exercise amountedto 92782 plusmn 16465 J Average time of exercise duration was873 plusmn 09min The protocol used was the Bruce treadmilltest protocol as this is the method for estimating VO

2max in

athletes The VO2max range 2589minus3262 (mLkgmin) was

calculated according to the following formula (1)

VO2max = 148 minus (1379 sdot 119879) + (0451 sdot 1198792) minus (0012 sdot 1198793)

(1)

119879 is total time on cycle ergometer measured as a fraction of aminute

Following exercise subjects remained seated at rest for1 hour and were allowed to drink only water This specificexercise protocol was used as a model of an acute stressorinducing oxidative stress

Venous blood samples were taken from an antecubitalvein before the exercise test directly after exercise andone hour after exercise The plasma lactate concentrationhaemoglobin (Hb) concentration and haematocrit (HTC)were determined in the collected blood using autoanalyserHb and HTC were used to calculate the ΔPV value accordingto the Dill and Costill formula [16]

ΔPV = [(Hb1

Hb2

) sdot (100 minusHTC

2

100 minusHTC1

) minus 1] sdot 100 (2)

where Hb1 HTC

1are preexercise values and Hb

2 HTC

2are

postexercise or 1 h recovery valuesFor other experiments the blood was centrifuged and the

plasma was separated Erythrocytes were washed three timeswith phosphate buffered saline (PBS pH 74)

24 Haemolysate Preparation Erythrocytes were haemol-ysed with water in the following relation (1 15) then vor-texed for 10min and centrifuged at 4000 rpm Haemolysatewas centrifuged at 16000timesg for separation of erythro-cyte membranes Haemoglobin concentration was measuredusing Drabkinrsquos method [17] The crude haemoglobin with-out purification was used for estimation of conformationalchanges of this protein

25 Spin Labelling of Crude Haemoglobin In order to inves-tigate conformational changes of haemoglobin MSL and ISLwere used which bind covalently to the -SH groups TheEPR spectra of spin labelled crude haemoglobin are shownin Figures 1 and 2

Haemolysate was labelled using 01molL ethanol solu-tions of MSL or ISL (50 1) and incubated for one hour atroom temperature The unbound spin labels were removedthrough dialysis using 20mmolL phosphate buffer pH = 74for 24 h at 4∘C until the EPR signal in dialysis buffer disap-peared The effect of oxidative stress on the conformationalchanges of haemoglobin was studied by an estimation of therelative rotational correlation time 120591

119888for both spin labels


h0 hminus1

w0

(a)

h0

w0

hminus1

(b)


0005

10

15

20

25

30


120591c

(ns)

lowast

Recovery (1h)






120591119888= 1198961199080radicℎ0

ℎminus1

minus 1 (3)




minus1







120578 =

120591119888(RBC)

120591119888(H2O)120578H2O (4)




1 cP









2)

Δ119860 = (1198611minus 1198612) minus (119860

1minus 1198602) (5)














3 Results








333 plusmn 080


1524 plusmn 087


4490 plusmn 246








00

01

02

03

04

05

06

07

Preexercise

Asc

orbi

c aci

d (n

mol

mg H

b) lowast








40

50

60

70

80

90


-SH

(nm

olm

g pro

tein

)

Recovery (1h)



4 Discussion





0

100

200

300

400

500

600

700

800


Trol

ox (120583

lowast

Recovery (1h)

mol

L)


0

10

20

30

40

50

60

70


Pero

xide

s (120583

mol

L)

Recovery (1h)


00

05

10

15

20

25

30

35


-SH

(120583m

olm

g pro

tein

)

lowastlowastlowast

Recovery (1h)
























Acknowledgment


References

















2O2



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


h0 hminus1

w0

(a)

h0

w0

hminus1

(b)


0005

10

15

20

25

30


120591c

(ns)

lowast

Recovery (1h)






120591119888= 1198961199080radicℎ0

ℎminus1

minus 1 (3)




minus1







120578 =

120591119888(RBC)

120591119888(H2O)120578H2O (4)




1 cP









2)

Δ119860 = (1198611minus 1198612) minus (119860

1minus 1198602) (5)














3 Results








333 plusmn 080


1524 plusmn 087


4490 plusmn 246








00

01

02

03

04

05

06

07

Preexercise

Asc

orbi

c aci

d (n

mol

mg H

b) lowast








40

50

60

70

80

90


-SH

(nm

olm

g pro

tein

)

Recovery (1h)



4 Discussion





0

100

200

300

400

500

600

700

800


Trol

ox (120583

lowast

Recovery (1h)

mol

L)


0

10

20

30

40

50

60

70


Pero

xide

s (120583

mol

L)

Recovery (1h)


00

05

10

15

20

25

30

35


-SH

(120583m

olm

g pro

tein

)

lowastlowastlowast

Recovery (1h)
























Acknowledgment


References

















2O2



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2)

Δ119860 = (1198611minus 1198612) minus (119860

1minus 1198602) (5)














3 Results








333 plusmn 080


1524 plusmn 087


4490 plusmn 246








00

01

02

03

04

05

06

07

Preexercise

Asc

orbi

c aci

d (n

mol

mg H

b) lowast








40

50

60

70

80

90


-SH

(nm

olm

g pro

tein

)

Recovery (1h)



4 Discussion





0

100

200

300

400

500

600

700

800


Trol

ox (120583

lowast

Recovery (1h)

mol

L)


0

10

20

30

40

50

60

70


Pero

xide

s (120583

mol

L)

Recovery (1h)


00

05

10

15

20

25

30

35


-SH

(120583m

olm

g pro

tein

)

lowastlowastlowast

Recovery (1h)
























Acknowledgment


References

















2O2



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







333 plusmn 080


1524 plusmn 087


4490 plusmn 246








00

01

02

03

04

05

06

07

Preexercise

Asc

orbi

c aci

d (n

mol

mg H

b) lowast








40

50

60

70

80

90


-SH

(nm

olm

g pro

tein

)

Recovery (1h)



4 Discussion





0

100

200

300

400

500

600

700

800


Trol

ox (120583

lowast

Recovery (1h)

mol

L)


0

10

20

30

40

50

60

70


Pero

xide

s (120583

mol

L)

Recovery (1h)


00

05

10

15

20

25

30

35


-SH

(120583m

olm

g pro

tein

)

lowastlowastlowast

Recovery (1h)
























Acknowledgment


References

















2O2



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


40

50

60

70

80

90


-SH

(nm

olm

g pro

tein

)

Recovery (1h)



4 Discussion





0

100

200

300

400

500

600

700

800


Trol

ox (120583

lowast

Recovery (1h)

mol

L)


0

10

20

30

40

50

60

70


Pero

xide

s (120583

mol

L)

Recovery (1h)


00

05

10

15

20

25

30

35


-SH

(120583m

olm

g pro

tein

)

lowastlowastlowast

Recovery (1h)
























Acknowledgment


References

















2O2



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Acknowledgment


References

















2O2



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Acknowledgment


References

















2O2



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Alterations in Red Blood Cells and Plasma ...downloads.hindawi.com/journals/tswj/2013/168376.pdf · haemoglobin (Hb) concentration, and haematocrit (HTC) were determined