Physical Exercise Combined With Whole-Body Cryotherapy In

8102019 Physical Exercise Combined With Whole-Body Cryotherapy In

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Research ArticlePhysical Exercise Combined with Whole-Body Cryotherapy inEvaluating the Level of Lipid Peroxidation Products and OtherOxidant Stress Indicators in Kayakers

PaweB Sutkowy1 Beata Augusty Nska2 Alina Wof niak1 and Andrzej Rakowski3

983089 Te Chair of Medical Biology of Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in orunKarłowicza 983090983092 983096983093 983088983097983090 Bydgoszcz Poland

983090 Te Chair and Department of Biochemistry of Ludwik Rydygier Collegium Medicum in BydgoszczNicolaus Copernicus University in orun Karłowicza 983090983092 983096983093 983088983097983090 Bydgoszcz Poland

983091 ldquoZawiszardquo Civilian and Military Sports Union Gdanska 983089983094983091 983096983093 983097983089983093 Bydgoszcz Poland

Correspondence should be addressed to Paweł Sutkowy pawel983090983091983091983095wppl

Received 983089983092 February 983090983088983089983092 Revised 983092 April 983090983088983089983092 Accepted 983092 April 983090983088983089983092 Published 983089983095 April 983090983088983089983092

Academic Editor Kota V Ramana

Copyright copy 983090983088983089983092 Paweł Sutkowy et al Tis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Te in1047298uence o exercise combined with whole-body cryotherapy (WBC) on the oxidantantioxidant balance in healthy men wasassessed Te study included 983089983094 kayakers o the Polish National eam aged 983090983090983095 plusmn 983090983094 subjected to WBC (minus983089983090983088∘Cndashminus983089983092983093∘C 983091 min)twice a day or the 1047297rst 983089983088 days o a 983089983097-day physical training cycle pre exercise morning stimulation and post exercise afernoonrecovery Blood samples were taken on Day 983088 (baseline) and on Days 983093 983089983089 and 983089983097 Te serum concentration o malondialdehyde(MDA) conjugated dienes (CD) thiobarbituric acid reactive substances (BARS) protein carbonyls vitamin E urea cortisoland testosterone were determined along with the glutathione peroxidase (GPx) activity the total antioxidant capacity (AC) andmorphological blood parameters On 983093th day o exerciseWBC the baseline GPx activity decreased by 983089983093983089 ( lt 005) while on983089983097th day it increased by 983089983097983095 ( lt 005) versus Day 983093 On Day 983089983097 BARS concentration decreased versus baseline and Day 983093 (by 983089983093983097 and 983089983095983092 resp lt 001) On 983089983097 Day urea concentration also decreased versus 983089983089 Day however on 983093th and 983089983089th days thelevel was higher versus baseline Combining exercise during longer training cycles with WBC may be advantageous

1 Introduction

Cryotherapy involves applying extremely low temperatures(between minus983089983088983088∘C and minus983089983094983088∘C) to the body surace or 983089 to983091 minutes Such low temperatures are applied uniormly tothe entire body surace (whole-body cryotherapy WBC) orlocally and are generated using the vapour o liquid nitrogenor liquid synthetic air [983089] Cryotherapy has been used ormany years in sports to treat injuries andpreventovertraining[983090 983091] as well as in the treatment o many diseases due toits analgetic antioedematous and anti-in1047298ammatory effect[983092] A rapid increase in body temperature afer leavingthe cryogenic chamber and intensive cutaneous blood 1047298ow (tissue overperusion lasting or several hours) induce theremoval o metabolites and in1047298ammation mediators rom

damaged tissues WBC also positively affects the centralnervous system (CNS) by decreasing anxiety and stress while

increasing the CNS resistance to exhaustion which may berelated to theincreased level o beta-endorphins [983089] HoweverWBC may also induce oxidative stress [983089 983093] One o thesources o ROS during WBC is a reaction catalyzed by xanthine oxidase resulting in the initial ischaemia occurringduring body exposure to extremely low temperatures aswell as hyperaemia occurring afer leaving the cryogenicchamber [983094] During WBC ROS may also be generatedthrough the oxidation o catecholamines even a singlecryogenic chamber session increases the levels o adrenalinand noradrenalin in the blood serum o young men andwomen [983089] Te higher ROS generation during WBC sessionsmayalso be a resulto stimulationo themetabolism o brown

Hindawi Publishing CorporationOxidative Medicine and Cellular Longevity Volume 2014 Article ID 402631 7 pageshttpdxdoiorg1011552014402631



983090 Oxidative Medicine and Cellular Longevity

adipose tissue which has a high content o mitochondria andcytochromes [983089] Exercise is another actor that stimulatesoxygen metabolism andleads to an increased level o oxygen-derived ree radicals [983093] Te results o postexercise oxidativestress are or example microdamage o skeletal musclesand connective tissues (eg joint cartilages ligaments) [983095]

as well as increased peroxidation o lipids orming muscle1047297bres and also as a result o post-WBC ROS activity bloodplasma lipids (LDL raction) and erythrocyte membranes[983093 983096] Despite the disadvantageous effects o oxidativestress acontrolled increase in ROS concentration may cause adaptivechanges involving improvements in the antioxidant capacity o the organism [983097] Many authors postulate positive adaptivechanges as a result o using WBC during physical exerciseand indicate WBC as a recovery or stimulation method thatis being increasingly employed by athletes [983089 983093 983097] Tus theaim o this study was to determine the effect o a total o 983090983088 WBC sessions combined with physical exercise on theoxidantantioxidant balance in high-level kayakers rom thePolish National eam In blood serum o sportsmen theconcentration o lipid peroxidation products (MDA CD andBARS) protein carbonyls vitamin E urea cortisol andtestosterone as well as the activity o GPx and the ACweredetermined Additionally selected morphological bloodparameters were measured the count o red blood cells(RBC) and white blood cells (WBC) which were divided intoneutrophils and lymphocytes as well as the concentration o hemoglobin (HGB)

2 Material and Methods

983090983089 Study Subject Te study involved 983089983094 kayakers o the

Polish National eam (age 227 plusmn 26 years body height1843 plusmn 52 cm and body weight 860 plusmn 49 kg) Te athletesprepared or the World Championships in Duisburg (heldon 983090983090ndash983091983089 August 983090983088983089983091) between 983090983090 July and 983097 August983090983088983089983091 (983089983097 days) in the Olympic Preparation Centre in WałczPoland During that period the kayakers were subjected tostrict control regarding their diet exercise and recovery by the team o trainers that is coaches a physician and twosport dieticians Te athletes perormed specialized physicaltraining two times a daymdashin the morning and afernoon Teintensity range o the exercise bouts was characterized by measuring lactate acid concentration using portable analyzerTe schedule o the training is presented in able 983089 Te study

was approved by the Bioethics Committee o Ludwik Rydy-gier Collegium Medicum in Bydgoszcz Nicolaus CopernicusUniversity in orun Poland (number KB 983091983095983088983090983088983089983091) Teathletes provided their written consent or the participationin the study

983090983090 Experimental Design During the 1047297rst 983089983088 days o thetraining cycle the kayakers combined their strict exerciseschedule with two WBC sessions per day Te 1047297rst sessionwas conducted in the morning afer breakast immedi-ately beore the 1047297rst exercise bout (preexercise morningstimulation) while the second session was conducted inthe afernoon ollowing a short rest and supper afer the

second (last) exercise bout (postexercise afernoon recovery)Te temperature in the cryogenic chamber was gradually decreased every day rom minus983089983090983088 to minus983089983092983093∘C Beore every entry into the chamber the participants remained in an adaptive

vestibule or 983091983088 seconds at minus983094983088∘C Every WBC session was 983091minutes long (excluding the stay in the adaptive vestibule)

Blood samples or analysis were taken rom the basilic veinat 983092 time points on Day 983088 (baselinemdashthe day beore the starto the training camp) Day 983093 (twice a day WBC or 983093 days)Day 983089983089 (1047297rst day without WBC) and Day 983089983097 (no WBC or983097 successive days o the training camp) Blood samples werecollectedevery timeat midday between the 1047297rst exercise boutand the dinner

983090983091 Determination of the Concentrations of MDA CDBARS and Vitamin E Te levels o MDA and vitamin Ewere determined using high-perormance liquid chromatog-raphy (HPLC) while the BARS level was determined using

the spectrophotometric method by Buege and Aust [983089983088] asmodi1047297ed by Esterbauer and Cheeseman [983089983089] Te methodwas also used or the preparation o serum samples orMDA quanti1047297cation Te CD level was determined using thespectrophotometric method described by Sergent et al [983089983090]Te analytical perormance o the methods used or MDAand vitamin E assessment was satisactory with the intra-assay coefficient o variation (CV) between 983093983094 and 983089983088983092and the interassay CV between 983092983094 and 983089983091983090 As regradsthe CD and BARS determination methods the ranges o intra-and interassay CV were 983095983093to 983089983089983090 and983091983094 to 983089983090983090respectively

Vitamin E quanti1047297cation was conducted by mixing 983090983088 1038389Lworking solution o internal standard (tocopheryl acetate983089983096983094 1038389gmL) with 983090983088983088 1038389L serum Protein denaturation wasinduced by shaking the investigated solution with 983096983088983088 1038389Lacetonitrile Once centriuged the supernatant was 1047297lteredusing an SPE system (Captiva 983090 1038389m) into glass tubes and 983092 mLhexane was added to perorm extraction Subsequently thesamples were shaken centriuged and rozen at minus983096983088∘C orapproximately 983092983093 min Te rozen hexane raction containing

vitamins was decanted into clean tubes and evaporatedto dryness under nitrogen at 983092983088∘C Ten the sample wasdissolved by adding 983089983088983088 1038389L phase mixed ultrasonically and1047297nally injected into an HPLC system using a syringe Tedetection was conducted using a UV-Vis detector at thewavelength 1103925 = 983090983097983090 nm Te concentration o vitamin E was

expressed as 1038389gL o serumDetermination o the BARS concentration was con-

ducted by mixing 983088983093 mL serum with 983092983093 mL reaction mixconsisting o 983088983091983095983093 thiobarbituric acid (BA) and 983089983093trichloroacetic acid (CA) in 983088983090983093 N HCl Te samples wereincubated on a water bath or 983090983088 min at 983089983088983088∘C to optimizethe conditions or the MDA-BA reaction Subsequently thesamples were cooled down and centriuged at 983092 ∘C or 983089983093 minat 983090983088983088983088 timesg Afer centriugation supernatant was collectedTe detection was conducted at the wavelength 1103925 = 983093983091983090 nmBARS consist mainly o MDA thereore or the sake o simplicity the BARS level in serum was expressed as theMDA level (nmolmL)



Oxidative Medicine and Cellular Longevity 983091

983137983138983148983141 983089 Te weekly course o the training cycle combining exercise and cryotherapy in the kayakers rom the Polish National eam between983090983090 July and 983097 August 983090983088983089983091

Day o week ime o exercise ype o exercise Duration (min) Intensity rangelowast

Monday 983089983088983088983088 AM Strength training + specialized on-water training 983089983092983088 IIIV

983089983094983088983088 PM Specialized on-water training 983097983088 II

uesday 983089983088983088983088 AM Specialized on-water training + stretching 983089983088983088 IIII983089983094983088983088 PM Specialized on-water training + 983094 km running 983089983089983088 IIIII

Wednesday 983089983088983088983088 AM Specialized on-water training + stretching 983089983089983088 IIII

983089983094983088983088 PM Specialized on-water training + strength training 983089983088983088 VII

Tursday 983089983088983088983088 A M Ergometer + specialized on-water training + 983094 km running 983089983091983088 IIIII

983089983094983088983088 PM Recovery mdash mdash

Friday 983089983088983088983088 AM Strength training + specialized on-water training 983089983090983088 VIII

983089983094983088983088 PM Specialized on-water training + stretching 983096983088 IIII

Saturday 983089983088983088983088 AM Ergometer + specialized on-water training 983089983089983088 IIIII

983089983094983088983088 PM Specialized on-water training + 983094 km running 983097983088 IIIII

Sunday 983089983088983088983088 AM Specialized on-water training 983089983089983088 III

983089983094983088983088 PM Recovery

lowastLactate acid concentration in blood I lt 983090 mmolL II lt 983092 mmolL III = 983092 mmolL (lactate threshold) IV gt 983092 lt 983094 mmolL and V gt 983094 lt 983096 mmolL

Properly prepared serum samples or MDA quanti1047297-cation were separated on an HPLC system using a C983089983096(983090983093983088mm) column Te detection was conducted using a UV-Vis detector Te concentration o the investigated compoundwas determined using the WorkStation Polaris sofware TeMDA level in blood serum was expressed as nmolmLCD aregenerated in the process o lipid peroxidation as a result o double bond regrouping afer a hydrogen atom is removedrom a residue o a polyunsaturated atty acid Tey orm a

characteristic absorbance peak at the wavelength 1103925 = 983090983091983091 nmTe CD level was expressed as absorbance units per mL o serum (983089983088minus1 AbsmL)

983090983092 Determination of AC Protein Carbonyls Concentrationand GPx Activity Te intra-assay and interassay controlimprecision as CV obtained or the methods used inthe determination o AC and protein carbonyls was 983094983093ndash983089983088983090 and 983096983094ndash983089983089983097 respectively For the GPx determinationmethod the interassay CV was between 983094983095 and 983097983089and the intra-assay CV was between 983091983091 and 983089983088983091 ACand protein carbonyls were determined using commercial

ELISA kits by Cell Biolabs Inc Te AC test involved thereduction o Cu2+ ionstoCu+ ionsby the antioxidants presentin the sample Te quantity o antioxidants in the samplewas directly proportional to the concentration o the newly ormed Cu+ ions that reacted with a chromogen ormingcoloured products Te absorbance o the solution was thenmeasured at the wavelength 1103925 = 983092983097983088 nm and compared withtheabsorbance valueson thecalibrationcurvewhich enabledthe estimation o the antioxidant levels in the investigatedsample Te calibration curve was generated based on thesame procedure but using known concentrations o uric acidas an antioxidant AC in the serum sample was expressed asthe concentration o copper-reducing equivalents (1038389M CRE)

Te quanti1047297cation o protein carbonyls in the sample wasbased on their binding on a 983097983094-well plate in 983090-hour incuba-tion at 983091983095∘C ollowed by their detection using appropriateantibodies and estimation o their quantity rom a standardcurve based on the oxidized and reduced bovine serum albu-min (BSA) standards Te concentration o protein carbonylsin blood serum was expressed as 1038389molmg

Te activity o GPx was determined using the methoddescribed by Paglia and Valentine [983089983091] Te method is based

on the decomposition o hydrogen peroxide by GPx at983090983088∘C with the concurrent oxidation o reduced glutathioneOxidized glutathione is then reduced in a reaction catalyzedby glutathione reductase Reduced nicotinamide adeninedinucleotide phosphate (NADPH) is a coenzyme in thisreaction and turns into an oxidized orm which causes achange in light absorbance at the wavelength 1103925 =983091983092983088nmTeGPx activity was expressed as UL o serum

983090983093 Morphological Blood Parameters Urea and HormonesAn additional analysis o blood cell count (RBC WBC) andHGB concentration as well as the levels o urea cortisol and

testosterone was conducted Te morphological parameterswere determined using the Sysmex XS 983096983088983088i hematology analyzer Te concentrations o testosterone and cortisol weremeasured via the competition method using commercial kitsby Roche In turn the urea concentration was assayed viathe kinetic method with urease using commercial reagents by Roche as well Te assays had the intrarun CV ranging rom983093983095 to 983095983091 and the interrun CV ranging rom 983092983094 to 983096983091

983090983094 Statistical Analysis Te obtained results were tested orthe normality o their distribution (the Kolmogorov-Smirnov test) and the homogeneity o variance (Levenersquos test) Temain statistical analysis was represented by the ANOVA




983137983138983148983141 983090 Oxidative stress indicators and the concentration o urea cortisol and testosterone in the blood serum o kayakers rom the PolishNational eam during a 983089983097-day physical activity including WBC sessions twice a day or the 1047297rst 983089983088 days

Baseline (Day 983088) Day 983093 Day 983089983089 Day 983089983097

MDA [983089983088minus2 nmolmL] 983090983094983091 plusmn 983090983093 983090983095983095 plusmn 983090983092 983090983094983088 plusmn 983089983096 983090983093983093 plusmn 983089983096

CD [983089983088minus1 AbsmL] 983092983094 plusmn 983090983093 983092983094 plusmn 983089983090 983092983092 plusmn 983089983088 983091983094 plusmn 983088983096

BARS [983089983088minus2 nmol MDAmL] 983093983088983091 plusmn

983093983088 983093983089983090 plusmn

983095983088 983092983093983097 plusmn

983093983091 983092983090983091 plusmn

983092983092aabb

Protein carbonyls [1038389molmg] 983093983091983096983092 plusmn 983090983091983091 983093983091983092983096 plusmn 983089983091983094983089 983093983091983093983092 plusmn 983091983094983090 983093983091983093983096 plusmn 983090983093983093

Vit E [1038389gL] 983089983095983095 plusmn 983096983090 983089983092983094 plusmn 983091983095 983089983091983094 plusmn 983091983091 983089983091983089 plusmn 983090983097

GPx [UL] 983090983090983093983097 plusmn 983090983094983092 983089983097983089983094 plusmn 983090983095983096a 983090983089983090983097 plusmn 983092983090983094 983090983090983097983092 plusmn 983091983088983091b

AC [1038389M CRE] 983096983089983091983094 plusmn 983094983095983095 983095983094983097983094 plusmn 983095983096983092 983095983095983092983089 plusmn 983089983088983090983088 983096983092983093983091 plusmn 983089983089983092983088

Urea [mgdL] 983091983091983089 plusmn 983094983091 983091983097983097 plusmn 983094983091a 983092983093983090 plusmn 983093983093aa 983091983095983090 plusmn 983096983092c

Cortisol [nmolL] 983094983090983096983090 plusmn 983094983090983097 983094983094983089983091 plusmn 983096983091983089 983094983096983096983095 plusmn 983095983091983092 983094983091983096983092 plusmn 983089983088983097983090

estosterone [nmolL] 983090983091983096 plusmn 983093983097 983090983090983096 plusmn 983092983097 983090983092983092 plusmn 983092983095 983090983092983097 plusmn 983093983094

Te results are expressed as the mean plusmn standard deviation a statistically signi1047297cant difference versus baseline (a lt 005 aa

lt 001) b statistically signi1047297cant

versus Day 983093th (b lt 005 bb

lt 001) and c statistically signi1047297cant versus Day 983089983089 ( lt 005)

983137983138983148983141 983091 Selected morphological parameters o the blood o kayakers during a 983089983097-day training cycle with two WBC sessions per day or the

1047297rst 983089983088 days one beore an exercise bout (morning stimulation) and one afer an exercise bout (afernoon recovery) Te results are expressedas the mean plusmn standard deviation No statistically signi1047297cant differences were observed

Morphological blood variable erm o the study


RBC (mlnmm983091) 983093983088 plusmn 983088983091 983093983090 plusmn 983088983090 983093983089 plusmn 983088983090 983093983089 plusmn 983088983091

WBC (thousmm983091) 983093983093 plusmn 983089983089 983093983097 plusmn 983089983088 983093983094 plusmn 983088983096 983093983093 plusmn 983089983089

NEU () 983092983089983094 plusmn 983093983088 983092983092983094 plusmn 983096983089 983092983091983089 plusmn 983092983097 983092983090983092 plusmn 983094983097

LYMPH () 983092983092983090 plusmn 983093983088 983092983090983092 plusmn 983095983094 983092983090983095 plusmn 983093983089 983092983092983088 plusmn 983093983097

HGB (gdL) 983089983093983091 plusmn 983088983095 983089983093983097 plusmn 983088983094 983089983093983092 plusmn 983088983093 983089983093983093 plusmn 983088983096

WBC white blood cells RBC red blood cells NEU neutrophils LYMF lymphocytes and HGB haemoglobin

test with post hoc analysis (ukeyrsquos range test) MoreoverPearsonrsquos product-moment correlation coefficients betweenthe investigated parameters were evaluated Te results wereexpressed as the mean plusmn standard deviation and differences ata signi1047297cance level lt 005 were considered as statistically signi1047297cant

3 Results

Te study demonstrated a statistically signi1047297cant decreasein the baseline GPx activity by 983089983093983089 as compared with the

activity measured on 983093th day o the training cycle involvingphysical exercise and WBC ( lt 005) while afer Day 983089983097it increased by 983089983097983095 ( lt 005) versus Day 983093 Moreover on983089983097th day 983097 days afer the discontinuation o cryotherapy astatistically signi1047297cant decrease in the BARS level was oundas compared with the baseline value and the level detected on983093th day o exercise combined with WBC (by 983089983093983097 and 983089983095983092resp lt 001) On 983089983097th day signi1047297cantly lower urea level

versus 983089983089th day was also ound ( lt 005) whereas on 983093thand 983089983089th days the values were higher than baseline ( lt 005and lt 001 resp able 983090)

Troughout the experiment no statistically signi1047297cantchanges in the levels o MDA CD protein carbonyls vitamin

E cortisol and testosterone were ound in the blood serumo the study subjects No AC changes in the blood serumo the athletes (able 983090) and no changes in the hematologicalparameters were observed either (able 983091) ( gt 005)

Moreover the study indicated many statistically signi-icant linear correlations through the whole experiment atthe baseline time point o the study between AC and GPx(907317 = minus0697 lt 005) on Day 983093 between vitamin E andMDA (907317 = minus0645 lt 005) BARS and vitamin E (907317 =minus0608 lt 005) andbetween BARSand AC(907317 = minus0710 lt 001) on Day 983089983097 between MDA and AC (907317 = minus0634

lt 005) as well as between BARS and AC (907317 = minus0683 lt 001)

4 Discussion

Te obtained results show a possible pro1047297table effect o exercise combined with WBC Combining exercise and WBCmay potentially improve sports perormance because o theprolongation o exercise duration or intensity Probably itmay result rom maintenance o oxidantantioxidant balance

In the study on the 983093th day o physical exercise combinedwith WBC sessions a decrease in the GPx activity by 983089983093983089 versus baseline was observed ( lt 005) Te lower




activity o GPx is a maniestation o the decreased activity o antioxidant mechanisms in the studied sportsmen Inturn on 983089983097th day o the training cycle (the 983097th day o theexercise bouts without WBC) the GPx activity increasedby 983089983097983095 versus 983093th day ( lt 005) but concurrently itdid not change in a statistically signi1047297cant manner relative

to baseline (able 983090) It demonstrates a certain degree o disturbance in oxidantantioxidant balance during the 1047297rstten days o the training cycle associated with WBC and itsrecovery at the end o the cycle that is during nine suc-cessive days o exercise bouts ollowing the discontinuationo WBC sessions Both the physical exercise undertakenby the athletes and the whole-body effect o extremely low temperatures may be thesource o the increased generation o ROS During aerobic exercise the main source o ROS is therespiratory chain whose natural by-products are ree radicals[983095] Physical exercise intensi1047297es the metabolism o oxygenEndurance exercise increases the demand o oxygen in theorganism between 983089983088 and 983090983088 times At the same time oxygenconsumption in skeletal muscles increases 983089983088983088ndash983090983088983088-old [983095]Duringanaerobic exercise that is above the lactate thresholdthe main source o ROS is xanthine oxidase producedrom xanthine dehydrogenase in vascular endothelium underischemic conditions [983089983092] A similar occurrence may also beobserved during WBC sessions along with the subsequenthyperemia [983094] Te physical exercise undertaken by thekayakers during the training cycle was at a variable level o intensitymdashrom aerobic to anaerobic (able 983089) Te BARSlevel on the 983097th day o the post-WBC training cycle decreasedin a statistically signi1047297cant manner compared to the valuesmeasured either beore the training cycle or on Day 983093 o the exerciseWBC combination Tereore the lower level o lipid peroxidation products demonstrates the decreased levelo oxidative tissue damage in kayakers through the actiono these two stressors However cryotherapy may improvethe efficiency o the BARS elimination mechanism whosemain component is MDA which in turn is metabolized in theliver and probably also in the skeletal muscles o physically well-trained people [983093] In turn the results o changes inserum urea concentration in kayakers indicate that WBC alsointensi1047297es metabolism o proteins Te obtained results o GPx activity and BARS concentration suggest that addingthe effect o extremely low temperatures to physical exercisehelps to maintain the balance in oxidoreduction processesIt may be explained by adaptive changes in the organismwhich are described by hormesis theory A stressor can have

a tempering effect i it is used regularly or longer period atoptimal intensity [983089983093] Such effect o WBC is also indicated by other authors who all in all highlight the antioxidant effecto whole-body cryotherapy In a study involving multipleWBC sessions but no physical exercise (983089983088 WBC sessionsminus983089983091983088∘C983091 min once a day) in both men ( = 24) andwomen ( = 2 2) a statistically signi1047297cant increase in thetotal antioxidant status (AS) and the plasma level o uricacid as compared with the control group not subjected toWBC (men = 22 women = 26) was observed [983089983094]Te results demonstrating the antioxidant properties o WBChave also been presented by other authors Wozniak et al[983089] or example conducted a study involving a group o

proessional kayakers ( = 20) whowere subjected toa 983089983088-day physical activity with WBC sessions conducted three times aday (983089 WBC session beore the 1047297rst exercise bout and 983090 WBCsessions beore the second exercise bout with temperaturesdecreasing rom minus983089983090983088 to minus983089983092983088∘C 983091 min) and a similar 983089983088-day control physicalactivity without WBCTe studyshowed that

the GPx activity afer the 983089983088th day o the physical activity including WBC was lower than that observed afer the 983089983088thday o the physical activity without WBC [983089] Te activity o the enzyme afer Day 983089983088 o the latter physical training cyclewas higher in a statistically signi1047297cant manner than beorethis physical activity whereas afer the 983089983088th day o the exercisebouts includingthree WBC sessions a day theactivity showedno statistically signi1047297cant difference rom that measuredbeore thestudy Te article by Wozniaketal[983089] alsoindicateslower levels o plasma BARSCD and erythrocyte CD witha lower activity o the erythrocytic superoxide dismutase(SOD) afer the 983094th day o exercise bouts including WBC ascompared with the values obtained on the same day o thephysical activity without the cryogenic chamber stimulationsessions Te authors claim that the oxidative stress inducedby extremely low temperatures causes changes in the cells o the organism which may protect them against the disruptiono the oxidantantioxidant balance during physical exer-cise [983089] Other data indicate an anti-in1047298ammatory effect o cryotherapy (983093 sessionsweek once a day minus983089983089983088∘C983090 min) [983089983095]Te authors demonstrated a statistically signi1047297cant decreasein the levels o proin1047298ammatory cytokines and an increasein the levels o anti-in1047298ammatory cytokineschemokines inthe blood o the Italian national rugby team members where = 10 [983089983095] Te same authors also claim not to haveobserved any changes in the values o selected immunesystem parameters antibodies (IgA IgM IgG) C-reactiveprotein prostaglandin E983090 (PGE983090) and muscle enzymescreatine kinase (CK) and lactate dehydrogenase (LAD)[983089983095]

Te effect o a single WBC session on the oxidationand reduction processes in the human organism has alsobeen described Mila-Kierzenkowska et al [983089983096] designed anexperiment in which proessional volleyball players ( =18) were subjected to a single WBC session (minus983089983091983088∘C 983090 min)immediately ollowed by a 983092983088-min submaximal physicalexercise on a cycloergometer and then a control exercisebout excluding WBC conducted 983090 weeks later Te authorsdemonstrated the antioxidant and anti-in1047298ammatory effecto WBC higher catalase (CA) and SOD activity was observed

afer the control exercise bout than afer the exercise boutpreceded by a WBC session Te levels o proin1047298ammatory cytokines interleukin 983094 and 983089 were also higher afer thecontrol exercise bout [983089983096] A single WBC session (minus983089983091983088∘C983091 min) with no exercise involved was also administered tohealthy nonathletes ( = 10 210 plusmn 09) in whom anincrease in the activity o GPx and erythrocytic glutathionereductase (R-GSSG) was observed along with an increase inthe levels o nonenzymatic plasma antioxidants (glutathioneuric acid albumins and extra-erythrocyte hemoglobin) [983089983097]Te authors indicated WBC as a source o ROS but alsoconsidered cryotherapy as a actor stimulating the antiox-idant deense mechanisms o the organism [983089983097] Te only




nonenzymatic antioxidant that level was determined in thisstudy was vitamin E No statistically signi1047297cant changes inits level were observed However it was also demonstratedthat on Day 983093 o the training cycle combined with WBCthe levels o vitamin E and BARS correlated in a statistically signi1047297cant manner (907317 = minus0608 lt 005) Tis demonstrates

the role o vitamin E in the removal o ROS generated by physical exercise and whole-body cryotherapy Moreoversta-tistically signi1047297cant correlations that were ound are evidenceor correct physiological unctions in kayakers during wholeexperiment baselinemdashAC versus GPx (907317 = minus0697) Day 983093mdashvitamin E versus MDA (907317 = minus0645) and BARS versusAC (907317 = minus0710) and Day 983089983097mdashMDA versus AC (907317 =minus0634) and BARS versus AC (907317 = minus0683)

Despite the ambiguous effect o WBC on the oxi-dantantioxidant balance which depends on the condi-tions o the study and the characteristics o the inves-tigated group the authors o most papers conclude thatthe exposure to extremely low temperatures increases theantioxidant capacity o the organism although it is asource o ROS at the same time Te authors unanimously emphasize that WBC does not generate any signi1047297cant oxi-dantantioxidant imbalance and in the long term accordingto hormesis theory may induce adaptive changes Tereorethe WBC sessions are bene1047297cial or health and improvethe speed o postexercise recovery [983089 983089983093 983089983094 983089983097] It canbe supposed that the results o this study con1047297rm thishypothesis because on 983093th day a clear normalization o the observed changes was noticedmdashthe oxidantantioxidantbalance in kayakers was recovering despite the continuanceo both intensive exercise bouts and until 983089983088th day WBCsessions

5 Conclusions

Combining exercise with whole-body cryotherapy sessionsmay have a positive effect on the oxidantantioxidant balanceduring physical effort

Possible pro1047297table effect o combining exercise withcryotherapy could extend exercise duration or intensity thusimproving sports perormance

Conflict of Interests

Te authors declare that there is no con1047298ict o interestsregarding the publication o this paper

Acknowledgments

Te authors thank all the sportsmen who participated in thestudy and declare no 1047297nancial support or the project Tepurchase o the ELISA kit rom Cell Biolabs Inc or thedetermination o AC was 1047297nanced with a scholarship orthe development o PhD students under the ProjectldquoProgramrozwoju Collegium Medicum UMKrdquo (no POKL983088983092983088983089983088983089-983088983088-983089983097983089983088983096-983088983088) Te project was co1047297nanced by EU unds underthe European Social Fund

References

[983089] A Wozniak B Wozniak G Drewa and C Mila-Kierzenkowska ldquoTe effect o whole-body cryostimulation onthe prooxidantmdashantioxidant balance in blood o elite kayakersafer trainingrdquo European Journal of Applied Physiology vol 983089983088983089no 983093 pp 983093983091983091ndash983093983091983095 983090983088983088983095

[983090] C Swenson L Sw ard and J Karlsson ldquoCryotherapy in sportsmedicinerdquo Scandinavian Journal of Medicine and Science inSports vol 983094 no 983092 pp 983089983097983091ndash983090983088983088 983089983097983097983094

[983091] R Meeusen and P Lievens ldquoTe use o cryotherapy in sportsinjuriesrdquo Sports Medicine vol 983091 no 983094 pp 983091983097983096ndash983092983089983092 983089983097983096983094

[983092] X Guillot N ordi L Mourot et al ldquoCryotherapy in in1047298am-matory rheumatic diseases a systematic reviewrdquo Expert Reviewof Clinical Immunology vol 983089983088 no 983090 pp 983090983096983089ndash983090983097983092 983090983088983089983092

[983093] A Wozniak G Drewa B Wozniak et al ldquoEffect o cryogenictemperatures and exercise on lipid peroxidation in kayakersrdquoBiology of Sport vol 983090983090 no 983091 pp 983090983092983095ndash983090983094983088 983090983088983088983093

[983094] G Bhaumik K K Srivastava W Selvamurthy and S SPurkayastha ldquoTe role o ree radicals in cold injuriesrdquo Inter-

national Journal of Biometeorology vol 983091983096 no 983092 pp 983089983095983089ndash983089983095983093983089983097983097983093

[983095] A Mastaloudis S W Leonard and M G raber ldquoOxidativestress in athletes during extreme endurance exerciserdquo FreeRadical Biology and Medicine vol 983091983089 no 983095 pp 983097983089983089ndash983097983090983090 983090983088983088983089

[983096] A Wozniak ldquoSigns o oxidative stress afer exerciserdquo Biology of Sport vol 983090983088 no 983090 pp 983097983091ndash983089983089983090 983090983088983088983091

[983097] C M Bleakley F Bieuzen G W Davison and J CostelloldquoWhole-body cryotherapy empirical evidence and theoreticalperspectivesrdquo Journal of Sports Medicine vol 983093 pp 983090983093ndash983091983094 983090983088983089983092

[983089983088] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo Methods in Enzymology vol 983093983090 pp 983091983088983090ndash983091983089983088 983089983097983095983096

[983089983089] H Esterbauer and K H Cheeseman ldquoDetermination o

aldehydic lipid peroxidation products malonaldehyde and 983092-hydroxynonenalrdquo Methodsin Enzymology vol 983089983096983094 pp 983092983088983095ndash983092983090983089983089983097983097983088

[983089983090] O Sergent I Morel P Cogrel et al ldquoSimultaneous measure-ments o conjugated dienes and ree malondialdehyde used asa micromethod or the evaluation o lipid peroxidation in rathepatocyte culturesrdquo Chemistry and Physics of Lipids vol 983094983093no 983090 pp 983089983091983091ndash983089983091983097 983089983097983097983091

[983089983091] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization o erythrocyte glutathione per-oxidaserdquo Te Journal of Laboratory and Clinical Medicine vol983095983088 no 983089 pp 983089983093983096ndash983089983094983097 983089983097983094983095

[983089983092] B Halliwell andJ M C Gutteridge Free Radicals in Biologyand Medicine Clarendon Press Oxord UK 983089983097983097983091

[983089983093] C Mila-Kierzenkowska A Wozniak M Szpinda et al ldquoEffectso thermal stress on the activity o selected lysosomal enzymesin blood o experienced and novice winter swimmersrdquo Scandi-navian Journal of Clinical and Laboratory Investigation vol 983095983090pp 983094983091983093ndash983094983092983089 983090983088983089983090

[983089983094] E Miller Ł Markiewicz J Saluk and I Majsterek ldquoEffecto short-term cryostimulation on antioxidative status and itsclinical applications in humansrdquo European Journal of Applied Physiology vol 983089983089983090 no 983093 pp 983089983094983092983093ndash983089983094983093983090 983090983088983089983090

[983089983095] G Ban1047297 G Melegati A Barassi et al ldquoEffects o whole-body cryotherapy on serum mediators o in1047298ammation and serummuscle enzymes in athletesrdquo Journal of Termal Biology vol 983091983092no 983090 pp 983093983093ndash983093983097 983090983088983088983097




[983089983096] C Mila-Kierzenkowska A Jurecka A Wozniak M SzpindaB Augusty nska and B Wozniak ldquoTe effect o submaximalexercise preceded by single whole-body cryotherapy on themarkers o oxidative stress and in1047298ammation in blood o volleyball playersrdquo Oxidative Medicine and Cellular Longevity vol 983090983088983089983091 Article ID 983092983088983097983093983094983095 983089983088 pages 983090983088983089983091

[983089983097] A Lubkowska B Dolegowska Z Szygula and A KlimekldquoActivity o selected enzymes in erythrocytesand levelo plasmaantioxidants in response to single whole-body cryostimulationin humansrdquo Scandinavian Journal of Clinical and Laboratory Investigation vol 983094983097 no 983091 pp 983091983096983095ndash983091983097983092 983090983088983088983097



Submit your manuscripts at

httpwwwhindawicom




adipose tissue which has a high content o mitochondria andcytochromes [983089] Exercise is another actor that stimulatesoxygen metabolism andleads to an increased level o oxygen-derived ree radicals [983093] Te results o postexercise oxidativestress are or example microdamage o skeletal musclesand connective tissues (eg joint cartilages ligaments) [983095]

as well as increased peroxidation o lipids orming muscle1047297bres and also as a result o post-WBC ROS activity bloodplasma lipids (LDL raction) and erythrocyte membranes[983093 983096] Despite the disadvantageous effects o oxidativestress acontrolled increase in ROS concentration may cause adaptivechanges involving improvements in the antioxidant capacity o the organism [983097] Many authors postulate positive adaptivechanges as a result o using WBC during physical exerciseand indicate WBC as a recovery or stimulation method thatis being increasingly employed by athletes [983089 983093 983097] Tus theaim o this study was to determine the effect o a total o 983090983088 WBC sessions combined with physical exercise on theoxidantantioxidant balance in high-level kayakers rom thePolish National eam In blood serum o sportsmen theconcentration o lipid peroxidation products (MDA CD andBARS) protein carbonyls vitamin E urea cortisol andtestosterone as well as the activity o GPx and the ACweredetermined Additionally selected morphological bloodparameters were measured the count o red blood cells(RBC) and white blood cells (WBC) which were divided intoneutrophils and lymphocytes as well as the concentration o hemoglobin (HGB)

2 Material and Methods

983090983089 Study Subject Te study involved 983089983094 kayakers o the

Polish National eam (age 227 plusmn 26 years body height1843 plusmn 52 cm and body weight 860 plusmn 49 kg) Te athletesprepared or the World Championships in Duisburg (heldon 983090983090ndash983091983089 August 983090983088983089983091) between 983090983090 July and 983097 August983090983088983089983091 (983089983097 days) in the Olympic Preparation Centre in WałczPoland During that period the kayakers were subjected tostrict control regarding their diet exercise and recovery by the team o trainers that is coaches a physician and twosport dieticians Te athletes perormed specialized physicaltraining two times a daymdashin the morning and afernoon Teintensity range o the exercise bouts was characterized by measuring lactate acid concentration using portable analyzerTe schedule o the training is presented in able 983089 Te study

was approved by the Bioethics Committee o Ludwik Rydy-gier Collegium Medicum in Bydgoszcz Nicolaus CopernicusUniversity in orun Poland (number KB 983091983095983088983090983088983089983091) Teathletes provided their written consent or the participationin the study

983090983090 Experimental Design During the 1047297rst 983089983088 days o thetraining cycle the kayakers combined their strict exerciseschedule with two WBC sessions per day Te 1047297rst sessionwas conducted in the morning afer breakast immedi-ately beore the 1047297rst exercise bout (preexercise morningstimulation) while the second session was conducted inthe afernoon ollowing a short rest and supper afer the

second (last) exercise bout (postexercise afernoon recovery)Te temperature in the cryogenic chamber was gradually decreased every day rom minus983089983090983088 to minus983089983092983093∘C Beore every entry into the chamber the participants remained in an adaptive

vestibule or 983091983088 seconds at minus983094983088∘C Every WBC session was 983091minutes long (excluding the stay in the adaptive vestibule)

Blood samples or analysis were taken rom the basilic veinat 983092 time points on Day 983088 (baselinemdashthe day beore the starto the training camp) Day 983093 (twice a day WBC or 983093 days)Day 983089983089 (1047297rst day without WBC) and Day 983089983097 (no WBC or983097 successive days o the training camp) Blood samples werecollectedevery timeat midday between the 1047297rst exercise boutand the dinner

983090983091 Determination of the Concentrations of MDA CDBARS and Vitamin E Te levels o MDA and vitamin Ewere determined using high-perormance liquid chromatog-raphy (HPLC) while the BARS level was determined using

the spectrophotometric method by Buege and Aust [983089983088] asmodi1047297ed by Esterbauer and Cheeseman [983089983089] Te methodwas also used or the preparation o serum samples orMDA quanti1047297cation Te CD level was determined using thespectrophotometric method described by Sergent et al [983089983090]Te analytical perormance o the methods used or MDAand vitamin E assessment was satisactory with the intra-assay coefficient o variation (CV) between 983093983094 and 983089983088983092and the interassay CV between 983092983094 and 983089983091983090 As regradsthe CD and BARS determination methods the ranges o intra-and interassay CV were 983095983093to 983089983089983090 and983091983094 to 983089983090983090respectively

Vitamin E quanti1047297cation was conducted by mixing 983090983088 1038389Lworking solution o internal standard (tocopheryl acetate983089983096983094 1038389gmL) with 983090983088983088 1038389L serum Protein denaturation wasinduced by shaking the investigated solution with 983096983088983088 1038389Lacetonitrile Once centriuged the supernatant was 1047297lteredusing an SPE system (Captiva 983090 1038389m) into glass tubes and 983092 mLhexane was added to perorm extraction Subsequently thesamples were shaken centriuged and rozen at minus983096983088∘C orapproximately 983092983093 min Te rozen hexane raction containing

vitamins was decanted into clean tubes and evaporatedto dryness under nitrogen at 983092983088∘C Ten the sample wasdissolved by adding 983089983088983088 1038389L phase mixed ultrasonically and1047297nally injected into an HPLC system using a syringe Tedetection was conducted using a UV-Vis detector at thewavelength 1103925 = 983090983097983090 nm Te concentration o vitamin E was

expressed as 1038389gL o serumDetermination o the BARS concentration was con-

ducted by mixing 983088983093 mL serum with 983092983093 mL reaction mixconsisting o 983088983091983095983093 thiobarbituric acid (BA) and 983089983093trichloroacetic acid (CA) in 983088983090983093 N HCl Te samples wereincubated on a water bath or 983090983088 min at 983089983088983088∘C to optimizethe conditions or the MDA-BA reaction Subsequently thesamples were cooled down and centriuged at 983092 ∘C or 983089983093 minat 983090983088983088983088 timesg Afer centriugation supernatant was collectedTe detection was conducted at the wavelength 1103925 = 983093983091983090 nmBARS consist mainly o MDA thereore or the sake o simplicity the BARS level in serum was expressed as theMDA level (nmolmL)


















983089983094983088983088 PM Recovery




















983093983088 983093983089983090 plusmn

983095983088 983092983093983097 plusmn

983093983091 983092983090983091 plusmn

983092983092aabb























3 Results








4 Discussion



















5 Conclusions





Acknowledgments


References




























httpwwwhindawicom


















983089983094983088983088 PM Recovery




















983093983088 983093983089983090 plusmn

983095983088 983092983093983097 plusmn

983093983091 983092983090983091 plusmn

983092983092aabb























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4 Discussion



















5 Conclusions





Acknowledgments


References




























httpwwwhindawicom









983093983088 983093983089983090 plusmn

983095983088 983092983093983097 plusmn

983093983091 983092983090983091 plusmn

983092983092aabb























3 Results








4 Discussion



















5 Conclusions





Acknowledgments


References




























httpwwwhindawicom

















5 Conclusions





Acknowledgments


References




























httpwwwhindawicom







5 Conclusions





Acknowledgments


References




























httpwwwhindawicom









httpwwwhindawicom




httpwwwhindawicom

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Physical Exercise Combined With Whole-Body Cryotherapy In