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Fitness status and post-exercise inflammatory markers in 18-35-year-old males Rose, Peter Geoffrey Donald 2008

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FITNESS STATUS AND POST-EXERCISE INFLAMMATORY MARKERS IN 18-35YEAR-OLD MALESbyPETER GEOFFREY DONALD ROSEA THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIRMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Human Kinetics)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)June 2008© Peter Geoffrey Donald RoseAbstractAcute physical exercise results in transient systemic elevations of cytokines.Themost significant elevation is seen in interleukin-6 (il-6). Elevatedvalues of il-6 have beenreported to enhance fatigue and diminish performance during enduranceexercise. Adelayed increase in C-reactive protein (CRP) has also been shown in response toil-6.Persistent elevations in systemic interleukin-6 and C-reactive protein values have beenassociated with increased risk of cardiovascular disease.The purpose of this investigation was to determine whether differences exist inresting and post-exercise measures of il-6 and CRP between trained male enduranceathletes and age matched untrained males.Twenty-five eligible males were recruited; thirteen trained (T) mean (SD): age =26.6(4.9) yrs, mass = 73.0(7.8) kg, height 179.0(5.7) cm, BMI = 22.6(1.4) V02 =68.6(5.6) mFkg’min’and twelve untrained (U): age = 23.4(3.8) yrs, mass 77.9(15.0)kg, height = 179.0(8.7) cm, BMI = 23.9(3.0) V02 = 42.4(4.6) mlkg’min’. The twogroups were matched for age and body mass index (BMI) and differed significantly inaerobic fitness and hours of exercise per week.Days after an initial aerobic fitness assessment subjects were challengedwith a 45 minute cycle ergometer exercise bout at an intensity corresponding toindividual ventilatory threshold (VT). Serum il-6 was measured pre-exercise, 30 minutespost-, and 24 hours post-exercise. Serum CRP was measured pre-exercise and 24 hourspost-exercise. 11-6 values were analyzed using a 2x3 mixed design ANOVA and CRPusing a 2x2 mixed design ANOVA.11-6 values increased significantly in both groups 30 minutes post-exercise[T Q<0.05) and U (p<O.OS)] and returned to baseline at 24 hours. 11-6 was not differentbetween groups at any time point. CRP values did not increase significantly in eithergroup between pre- and 24 hours post-exercise. CRP values were significantly higher inthe untrained group pre- (p<O.OS) and 24 hours post-exercise (p<0.05) compared to thetrained group.These results demonstrate no significant difference in il-6 between T and U at restand or after exercise. This study also demonstrates a reduction in resting and postexercise CRP in endurance trained males compared to untrained males matched for ageand BMI.11Table ofContentsAbstractiiTable ofContentsiiiList of TablesvList of FiguresviAcknowledgementsviiChapter1: Introduction11.1 Statementof Problem21.2 Purposeof the Investigation31.3 ResearchQuestionlllypotheses3Chapter2: Reviewof Literature,5Chapter 3:Methodology193.1 StudyDesign193.2 Participants,203.3 EligibilityCriteri213.4 Procedures223.5 Outcomemeasures243.6 DataAnalysis26Chapter4: Results284.1 SubjectDemographicCharacteristics284.2 SubjectPhysiologicaland PerformanceCharacteristics284.3 SubjectCompliance,304.3.1 Protocol304.3.2 Performance304.4 OutcomeMeasures314.4.1 Interleukin-6314.4.2 C-ReactiveProtein314.4.3 Correlations324.4.4 BloodGlucose324.4.5 Ratingsof PerceivedExertion33111Chapter 5: Discussion445.1 General Findings445.2 11-6445.3 CRP475.4 Training Adaptations535.5 Overtraining Implications575.6 Health Implications605.7 Limitations625.8 Future Directions64Chapter 6: Conclusion70Bibliography,71Appendices80Appendix A - 11-6 concentrationsFollowing Cycle ExerciseinDifferent Fitness Groups,81Appendix B - ParticipantQuestionnaire82Appendix C - EthicalApproval Certificate,84ivList of TablesTable 4.1 Subject Demographic Characteristics35Table 4.2 Subject Physiologicaland Performance Characteristics36Table 4.3 PerformanceOutcome Measures Within Groups37Table 4.4 Outcome Measures Between Groups38Table 4.5 Outcome Measures 11-6and CRP Within Groups39Table 4.6 Outcome Measure Blood GlucoseWithin Groups40VList of FiguresFigure 4.1 Subjectsscreened, consented,assigned, andcompleted34Figure 4.2 Interleukin-6Concentrations41Figure 4.3 C-ReactiveProtein Concentrations42Figure 4.4 BloodGlucose Concentrations43viAcknowledgementsI would like to acknowledge and thank my supervisor, Dr. Jack Taunton, whogave me unconditional support throughout my graduate work. After each of our manymeetings I left with a heightened interest in my project and medicine as a whole. Youhave inspired me to continue in this field. Thank you to my committee members, Dr. TedRhodes and Dr. Donna Maclntyre, who helped guide me through this project with theirinvaluable input. I would also like to recognize the generous financial support of the B.CSports Medicine Research Foundation and the Faculty of Education for making thisproject possible. Last but not least, I would like to thank my family, Mom, Dad, Jamie,and Elizabeth, for your constant support of every kind.viiChapter 1: IntroductionAcute physical exercise of various modes has consistentlybeen shown to result intransient systemic elevations in both pro- and anti-inflammatorycytokines. The mostsignificant elevation is seen in interleukin-6(il-6). Elevated values of circulatingil-6have recently been reported to enhance fatigue anddiminish performance duringendurance exercise (73). Inaddition, the post-exercise increasein circulating il-6 hasbeen hypothesized by some researchersas an underlying cause of the unexplainedovertraining syndrome (43, 73).A delayed increase in circulatingacute phase proteins,most notably C-reactive protein(CRP), has also been demonstratedin response toelevations in circulatingil-6 and other pro-inflammatorycytokines after a strenuousboutof exercise (48). Persistent elevationsin systemic CRP valueshave been associated withan increased risk of cardiovascular diseasewhen compared to adults maintaininglowerlevels of C-reactive protein(64).Much research in past yearshas explored various methodsof manipulating thepost-exercise increase of inflammatorymediators. Of this research,only one studyinvolving healthy human subjectshas utilized prospectiveendurance exercisetraining toattenuate this response.The main finding ofthis prospective training studyby Fischer etal, (23) was that a 10-weekendurance exercise trainingprogram can reducepost-exerciseil-6 mRNA expression inhuman skeletal muscle inresponse to a concentricexercisestimulus of the same relativeworkload. Although post-exerciseplasma il-6 valuesinthese subjects were similarbefore and after training,this study indicatesthat trainingstatus plays a role in skeletalmuscle il-6 protein production.Cross-sectional analysis of1post-exercise plasma il-6 values between trainedand untrained subjects in differentstudies indicate that trained subjects havean attenuated post-exercise il-6 elevation incomparison to lesser trained subjects. However,cross-sectional comparisons are difficultdue to differing subject criteria, exercise modes andintensities, and differing samplingschedules. To date no research has directlycompared the systemic post-exerciseil-6 andCRP response to an identical relativeexercise stimulus in trainedand untrained humansmatched for age, sex and body composition.A comparison of post-exercise immunologicalmarkers between trainedanduntrained subjects will provideopportunity to analyze the relationshipbetween these twovariables. The main objectiveof this research is to determinewhether post-exerciseincreases of systemic il-6 andCRP differ significantlybetween trained anduntrainedyoung males. Consequently,this research will contributeto the current understandingofthe immunological and endocrineadaptations associatedwith exercise training.1.1 Statement of Problem1) Prior to this study, nohuman study has directlycompared the systemicpost-exercise il-6 and CRPin trained and untrainedindividuals in responseto anexercise challenge of similarrelative workload.2) Although a cross-sectionalanalysis of current researchstudies indicates thatarelationship exists betweentraining status and post-exerciseelevations of plasmail-6 and CRP, such a relationshiphas not yet been scientificallyevaluated with acontrolled study in menmatched for age andbody mass index.23) 11-6 is a known stimulatorof CRP; however, previous research has failedtoevaluate a significant correlational relationshipbetween the two inflammatorymarkers in response to physical exercise.4) No available research has examinedthe differences in the ratio of peak post-exercise il-6 and CRP between trained anduntrained individuals.1.2 Purpose of the InvestigationThe primary purpose of this study was to investigate the differences betweenaerobically trained and untrained young males in resting and post-exercise systemicconcentrations of both il-6 and CRP in response to a predetermined concentric exercisestimulus. The secondary purpose of this study was to quantitatively evaluate thecorrelational relationship between circulating concentrations of il-6 30 minutes post-exercise and CRP 24 hours post-exercise.1.3 Research Question/HypothesesThe following research hypotheses were formulated prior to the investigation basedupon available research literature:1) Subjects in the trained study group will demonstrate a significantly lower (CL .05)concentration of systemic il-6 post-exercise challenge (30-minutes post-exercise)compared the untrained subject group.2) Males in the trained study group will demonstrate a significantly lower (CL .05)concentration of systemic CRP post-exercise challenge (24-hours post-exercise)compared to the untrained subject group.33) Following each exercise stimulus circulatingconcentrations of plasma il-6 in bothgroups will increase significantly (cL .05) compared to pre-exercise values.4) Systemic CRP concentrations will increase significantly (cL .05) inresponse to theexercise challenge in the untrained group but will not increase significantly in thetrained group.5) Resting values of plasma iI-6 and serum CRP will not be significantly differentbetween training groups.6) Differences in pre- and post-exercise concentrations of plasma il-6 will show asignificant correlation(.05) with differences in pre- and post-exerciseconcentrations of CRP in both study groups.4Chapter 2: Review ofLiterature11-6 is a —27 kDa glycoproteinconsisting of 212amino acids, includinga 28amino acid signal peptide.11-6 is in a sub-classof cytokines that sharea similar helicalprotein structure and a similarsignal transducer (glycoprotein130). Cytokines area classof polypeptide messenger proteinsresponsible forsignal transmission amongstvariousimmune system and otherorgan cells. Cytokinesmay act in autocrine, paracrine,orendocrine fashion. In responseto trauma or infectionpro-inflammatory cytokinestumornecrosis factor-cL (TNF-ct) and interleukin-1f3(il-i j3) are producedlocally (65). Theinitial increase of11-113and TNF-cL is followedby increases of il-6,and then anti-inflammatory cytokinesil-i receptor antagonist(Il-ira), tumor necrosisfactor receptors(TNF-R) and il-lO. Assuch, il-6 has been termedinflammation-sensitiverather than pro-inflammatory (61).11-113, TNF-cL, and bacterial endotoxin arethe classic stimuli foril-6release (2). Althoughsynthesized bya variety of cells, il-6is mainly secretedby activatedmonocytes, fibroblasts,and vascular endothelialcells in response toinjury or infection(2). 11-6 individually,or in combination withinflammatory cytokinesil-i f3 and TNF-a,stimulates a delayedhepatocyte productionof acute-phase proteinstermed an acute-phaseresponse. CRP isan acute-phase proteinthat increases upto 1000-fold with traumaorinfection (24). 11-6has been identifiedas the chief stimulatorof acute-phase proteinproduction (26).Systemic il-6 also hasthe ability to activatethe hypothalamic-pituitary-adrenalaxis (43). Prior researchhas indicated that il-6can permeate the blood-brainbarrier (4)and, when administeredin doses equivalentto those during exercise,results in increasedsensations of fatigue,depressed moodstate, and decreased abilityto concentrate (73). Ina study by Robson-Ansley(73) subjects were injectedwith recombinanthuman il-6 in a5dosage equivalent to that followingstrenuous exercise significantlyimpairedperformance in a 10km runningtime trial. The precisemechanism resultingin decreasedperformance followingil-6 administrationis unclear but Robson-Ansleyindicates thatserotonergic pathwaysare likely activated.Marked post-exerciseelevations of plasmail-6 concentrationare a consistentfinding throughout previousresearch (55, 60, 62,63, 85, 52). Thecytokine responsetoexercise differs fromthat of trauma or sepsis.Most notably, il-6 isthe first cytokine toappear in circulationin response to exerciserather than il-i13or TNF-ct (62). Thispatternsuggests that il-6synthesis during exerciseis stimulated by factorsother than il-if3 andTNF-a. Furthermore,il-6 increases greatlywith exercise, whileil-i13and TNF-cLgenerally increase minimallyor not at all (66).Early researchindicated that il-6 productionduring exercise wasassociated with,and likely a resultof muscle damage (10).However, later researchfailed to reportasimilar association,suggesting thatil-6 may be producedin response to musclecontractions withoutmuscle damage (61,17). Post-exerciseplasma il-6 increasesinresponse to exercisewithout muscledamage have beena common finding(12, 55, 58, 61,62, 63). In orderto elucidate the sourceof il-6 production Steensberg(83) designed astudy that monitoredthe arterial-venous(a-v) differenceof il-6 in both a workingandresting leg. The exercisebout consisted of one-leggedknee extensionsfor 5 hours at 40%of peak poweroutput. The a-v differencein il-6 was equal betweenlegs pre-exercise.Throughout the exercisebout the a-v differencein the active legincreased significantlyand peaked at9.77 ng/l, whereasthe a-v difference inthe resting legremained similartopre-exercise. Althougha specific source couldnot be identifiedin this study, therelationship betweena-v differences in il-6from the active limband systemic il-66concentrations indicated that systemic post-exercise il-6 elevations could be accountedfor by production in the active limb.In both a human model and an animal model biopsies of the active muscle havedemonstrated marked elevations in il-6 mRNA following strenuous concentricandeccentric exercise (62, 40). Furthermore, Penkowa (65) detected il-6 withinmuscle cellsand displayed an increase following contraction. With the use of microdialysis cathetersimplanted in skeletal muscle and peritendinous tissue, Langberg (44)have suggested thatperitendinous tissue also contributes to the post-exerciseincrease in systemic il-6. In all,the available research has established that il-6 productionand release occurs in skeletalmuscle cells in response to exercise and is likely thechief source of il-6 productionduring non-damaging exercise.Other sources of il-6 release during exercisehave been suggested; however, theirrelative contribution to post-exercise il-6 elevationappears to be minimal. Lyngso (49)examined the il-6 release from adipose tissue duringexercise by comparing the a-vdifference between a radial artery and a subcutaneousabdominal vein in nine healthysubjects. Systemic il-6 peaked 30 minutesafter a 1 hour cycle ergometer exercise at 60%ofVO2max, increasing significantly above resting values. Elevations in adipose tissue il-6production were unchanged until 1 hourpost-exercise, at which point systemic il-6concentrations were far below peak values.The demonstrated time-course of il-6 releasefrom adipose tissue indicates that it doesnot contribute significantly to systemic peakpost-exercise il-6 elevations.Nybo (59) also used a-v difference between arterialand internal jugular venousconcentrations of plasma il-6 to show that the brainproduces il-6 during enduranceexercise. 11-6 release and uptake by the brain at rest wasnil but increased significantly7(p<.O5)after 1 hour on a cycle ergometer at 50% Of VO2m. Measurements of a-vdifferences in combination with absolute measures of cerebral blood flow indicated a netrelease of il-6 from the brain of .3 ng/min. Considering that peak il-6 output from anexercising leg reached approximately 30 ng/min (83), cerebral il-6 production likelyaccounts for a minute portion of elevated systemic il-6 concentration during exercise.The time-course and degree of il-6 elevation after exercise is dependent uponexercise type, intensity, and duration. In response to damaging eccentric exercisecirculating il-6 concentrations are elevated immediately after exercise, increasesteadilyand peak approximately 6 hours post-exercise (51, 17). Following concentric-basedexercise, circulating il-6 concentrations demonstrate an exponential increase,setting on10-30 minutes into exercise and reaching maximum concentrationsupon cessation orwithin 30 minutes post-exercise (65, 62, 89). The intensity of exercise has shownasignificant correlation with peak post-exercise circulating il-6 concentrationsduringrunning exercise (63). Concentrations of circulating il-6 have beenreported to increase asmuch as 128-fold following a marathon run in healthy young males (61). Soonaftercessation of activity in both cycling and running-based exercise modelscirculating il-6levels decline quickly, reaching resting levels between 6 and 24 hourspost-exercise (61,62, 89).No research to date has directly compared systemic il-6 concentrations during orfollowing exercise in subjects with different fitness levels. A cross-sectional analysisbetween modes of exercise and fitness levels is difficult as systemic il-6 levels aredependent upon a variety of factors not controlled between studies including musclemassinvolved and contraction type. However; in order to review the available literaturea8cross-sectional analysis of comparable research incorporatingsimilar exercise modes andintensities will follow.In a study by Vassilakopoulos (89) healthy untrained malesaged 28-44 yearswere subjected to a 45-minute exercise bouton an upright cycle ergometer at 70%ofVO2max in order to examine systemic cytokine responses.The men in this study had amean VO2mof 38.9 mi/mm/kg and were notregularly participating in exercise. Restingpre-exercise plasma concentrationsof il-6 had a mean concentration of--’lpg/mi. Plasmail-6 reached 4 pg/mi upon cessationof exercise and peaked 30 minutespost-exercise at7.3 pg/mi. Contrary to similarstudies, Vassilakopoulos (89) hadsubjects ingest acarbohydrate rich breakfast priorto the exercise bout. Based onresearch by NehisenCannarella (55) and Nieman (58)that indicate an attenuation ofil-6 production withcarbohydrate ingestion priorto exercise, post-exercise plasmail-6 concentrations likelywould have increased to a greaterextent had subjects completed theexercise bout after anovernight fast.In a similar study Febbraio(20) recruited seven males aged22.1+/-3 .8 years witha mean VO2max of 47.9 ml/kg/min.The exercise bout consistedof 120 minutes of cyclingon a semi-recumbent ergometerat 65% of individualVO2m. In this study subjectscompleted the predeterminedexercise bout following an overnightfast. Systemic plasmaiI-6 was recorded at 0 (pre-exercise),30, 60, 90, and 120 minutes into exercisewithrespective concentrationsof 2, 4, 5, 7.5, and 12.5 pg/mi. No bloodsamples were recordedafter cessation of exercise. Althoughexercise intensity was5% less in this studycompared to that ofVassilakopoulos et al. (89), the exercise boutwas completedfollowing an overnight fast andthe exercise duration was more than2-fold. Mean il-6concentrations reached7.5 pg/mi at time points in both studies;at 75 minutes (309minutes post-exercise) in Vassilakopoulos’study and 90 minutes in Febbraio’sstudy.Considering subjects in Vassilakopoulos’study completed the exercise bout 30 minutesprior to this measure and subjects inFebbraio’ s study had been cyclingcontinuously for90 minutes prior to the measure,it would be expected that subject’sin Febbraio’s studywould demonstrate higher concentrationsof plasma il-6. Furthermore, subjects inFebbraio’ s study completed the exercise bout followingan overnight fast, likely leadingto greater post-exercise il-6 increasesthan if fed prior to exercise.In another study incorporating a similar exercise boutMacDonald (50) examinedpost-exercise il-6 concentrations in elite athletes. These subjectswere well trained andparticipated in physical training 5-8 times per week. Subjects includedeight males aged28 +1- 1 year with mean VO2mvalues of 65+1-1 ml/kg/min. The exercise stimulusconsisted of 1 hour of upright cycle ergometer exercise at 70%of individual VO2m;completed in the morning following a light breakfast consisting of 75% carbohydrateswith a total energy intake of 713 KJ. Systemic plasma il-6 was recorded at 0 (preexercise), 10, 20, 30, 45, and 60 minutes of exercise. Thecorresponding il-6concentrations were .8, 1, 1.2, 1.6, 2.7, and 4 pg/ml respectively. Considering thesimilarmode and relative exercise intensity in this study compared toVassilakopoulos’, it wouldbe expected that both groups would demonstrate similar post-exercise il-6concentrations.At similar time points of exercise (45 minutes into exercise) inVassilakopoulos’ andMacDonald’s study, circulating plasma il-6 concentrations were4 and 2.7 pg/mlrespectively. This further suggests that training status has a large influenceon plasma il6 concentrations in response to exercise.Another similar study by Starkie (83) examined circulating plasma il-6during 2hours of upright cycle ergometer exercise at 70% of individual VO2max.The subjects in10this study were endurance trained males aged 25 +1-5 years with a meanVO2max value of60.2 mi/mm/kg. Subjects completed the 2 hour trial in the morningfollowing anovernight fast. Circulating plasma ii-6 was sampled pre-exercise,after 60 minutes ofexercise, and upon completion of exercise at 120 minutes.The corresponding plasma il-6concentrations were .1, .2 and 1.5 pg/ml. Theseresults are similar to another study byStarkie et al., (83) in which males with a meanVO2max value of 61.03 mi/kg/mm showedpeak post-exercise plasma il-6 values of.8 pg/mi in response to 90 minutes of cyclingexercise at 70%VO2m. Unfortunately, in the latter study no pre-exercise nutritionalmeasures were reported. These studies incorporatedthe same exercise mode and relativeintensity as Vassilikapoulos et a!., (89)with a markedly increased duration. Asa result, itwould be expected that mean post-exerciseplasma il-6 concentrations would be muchgreater in these subjects. In contrast, themean peak plasma il-6 concentrationsfor Starkie(82) and Starkie (83) after 120 and90 minutes of cycle exercise correspondedto 20% and11% of peak post-exercise plasma il-6 concentrationin Vassilikapoulos’ research.In combination these studies displayan important relationship between physicalfitness, measured as maximal oxygen consumption,and circulating ii-6 concentrationsinresponse to concentric-based cycling exercise.The mean systemic plasma il-6 valuesthroughout exercise in these studies aredisplayed in a table and plotted on one graph(Appendix A), which clearly demonstratesa relationship between training statusandpost-exercise plasma il-6 values at similarrelative exercise intensities and durations.It isnot possible to quantitatively comparevalues between research by Vassilakopoulos(89)and the others as blood sampling timepoints were not identical; however, theplasma ii-6increases in the more fit subjects appearmarkedly diminished when compared totheuntrained subjects in Vassilakopoulos’work.11Two prospective training studies have been undertaken in order to investigate theeffects of training on post-exercise plasma il-6 levels. Croisier (17) designeda study inwhich 5 moderately active males underwent an injury exercise protocol.The exerciseprotocol incorporated three stages of 30 maximal eccentric contractionsof the kneeflexors and extensors of both legs, before and after a submaximaleccentric trainingprogram. The training protocol consisted of5 training sessions over three weeks; eachtraining session involved 5 stages of 10 submaximaleccentric contractions similar tothose of the injury exercise protocol. After trainingpost-exercise values of serummyoglobin and delayed onset muscle soreness weresignificantly reduced. Croisier (17)attributed this reduction to a training-inducedresistance of skeletal muscle to eccentriccontraction. In contrast, post-exercise valuesof plasma il-6, measured immediately, 30minutes, 48 hours, and 72 hours afterexercise, were not different before and after thetraining regimen. This study demonstratedthat short term eccentric training does notattenuate the eccentric exercise-inducedplasma il-6 response.In another prospective training study, Fischer (22) examinedcirculating skeletalmuscle il-6 mRNA and plasma concentration of il-6in 7 males following a 3 hour boutofisolated knee extensor contractions before and after a10-week endurance trainingprogram. The contractions were performedat a rate of 60 contractions per minute and anintensity of 50% of the subject’s maximum wattage sustainablefor 1 minute. The trainingprotocol consisted of 1 hour of isolated knee extensions5 days per week at 75% of preexercise maximum wattage sustainable for 1 minute.The training workload wasincreased 5-10% every fortnight dependingon subject progress. Upon completion of the10-week training program subjects were retested anda post-training maximal wattagewas determined. The workload corresponding to 50%of the subjects’ maximum wattage12sustainable for 1 minute, and subsequent post-training exercise challenge, was increasedby a mean of 44%, indicating that the training protocol improved functional performance.Resting il-6 mRNA was similar pre- and post-training in all subjects; however,post-exercise skeletal muscle mRNA increased only 8-fold post-training as opposedto 76-foldpre-training. Circulating plasma il-6 at rest, immediately post-exercise,and 2 hours post-exercise were similar before and after training. This isof great interest considering thatthe workload performed in the post-training exercisechallenge was 44% higher than preexercise. Considering research by Ostrowski(63), that indicates post-exercise plasma il-6concentration is highly correlated with exercise intensity(r = .30,p< .05), it would beexpected that a 44% increase in exercise intensitywould result in a markedly higherincrease in post-exercise plasma il-6.The systemic CRP response to exerciseis lesser studied and is less sensitive thanthat of ii-6. Resting CRP concentrationsare generally <2 mg/i in healthy individuals.Inresponse to moderate or prolonged enduranceexercise systemic serum CRPconcentrations may rise in similar fashionto that seen in trauma or infection. In responseto an ultradistance 246 km foot raceCRP values reached a mean of 97.3 +1- 57.6mg/iMargeli (33). The post-exercise elevationin serum CRP tends to occur 16-24 hoursafterthe exercise bout (80, 19, 12). In another study thatexamined inflammatory markers intwo separate marathon groups the meanCRP concentrations 16 hours followingastandard marathon were 15 mg/i and 11.8mg/i respectively (12). Interestingly, 1hour ofcycle ergometer exercise at 60% OfVO2m stimulated a significant (j=.O4)increase inserum CRP 24 hours post-exercise in untrained subjects (<5hrs/week of recreationalactivity) (80).13Although il-6 has been identified as the chief mediator of hepatocyte productionof CRP, a significant correlation between the two inflammatory markers has not beendemonstrated. Czarkowska-Paczek (18) designed a study examining the correlationbetween post-exercise values of il-6 and CRP. In the study 14 trained male cyclistsperformed a graded cycling test to exhaustion. Athletes began riding at 20 kmlh andgrade was increased every 3 minutes until exhaustion. Blood samples were drawn froman antecubital vein before, immediately after and 2 hours post-exercise. Serum CRPconcentrations were 3.25, 2.39, and 2.70 mg/dl at the respective sampling times. Serumil-6 concentrations were 0.48, 1.21, and 10.07 pg/mi at the respective sampling times. Noother sampling times were examined. As no correlation was evident between measures atcessation of exercise (r = -.1588,p.588) or 2 hours post-exercise (r = .358,p= .209),the authors concluded that there is no significant correlation between post-exercise il-6and CRP. Since the investigators only monitored CRP up to 2 hours post exercise it is notsurprising there was no recorded increase in this marker. No data points were recorded16-24 hours post-exercise. It is likely that an increase in CRP would be seen during thistime since hepatocyte production of CRP in response to an inflammatory response hasbeen shown to occur >6 hours following the inflammatory stimulus (92). A moreappropriate measure of correlation between post-exercise il-6 and CRP would be tocompare peak values of each marker; 0-1 hour post-exercise for il-6 and 16-24 hourspost-exercise for CRP.A mechanism for the apparent attenuated post-exercise circulating il-6 responsedisplayed in trained individuals has yet to be elucidated. As a result, researchers havedesigned studies in order to manipulate variables that potentially have an effect on il-6production. In unpublished research, Gleeson & Bishop (2000) reported that cycling14exercise in a glycogen-depleted state enhances the post-exerciseplasma il-6 response. Inthis study cyclists performed 60 mmof exercise at 75%V02mfollowed by a time trial(equivalent to 30 mm work at 80%V02m). Peak post-exercise elevations of plasma il-6were approximately 11-fold higher in the lowCHO group (in which subjects were given<1 g CHO/kg/day for three days priorto testing) in comparison to subjects inthe highCR0 group (receiving approximately8 g CHOIkg/day for three days priorto testing).Although no data indicating a differencein skeletal muscle glycogenstores betweengroups was recorded, it is likely thatthe low CHO group began the exercisebout withlower levels of stored skeletalmuscle glycogen. This researchrepresents a cmderelationship between pre-exerciseglycogen stores and post-exercisesystemic il-6.Steensberg (84) further exploredthe relationship between skeletalmuscleglycogen content and post-exercisemuscular il-6 production byhaving young physicallyactive males complete a two-leggedconcentric knee-extensor exercise,where one legwas depleted of glycogen priorto the exercise bout. The pre-exerciseglycogen content ofthe glycogen depleted leg was -40%lower (P<.05) than the controlleg; 200 and 350mmol glocosyl U (kg dry wt)’ respectively.The exercise stimulus consistedofconcentric knee-extensions overa range of60 degrees (90-30degree angle) at 40% ofmaximum power until exhaustion(4-5 hours). Net il-6 release fromeach leg wasevaluated by measuring the arterial-venousdifference in il-6 concentrationin each legbefore and during the exercisebout. Throughout the exercise boutthe workload did notdiffer between legs. Prior to exerciseno net il-6 release from either leg wasdetected. At 1hour into the exercise protocolan arterial-venous difference wasdemonstrated in theglycogen depleted leg. In contrast,an arterial-venous difference inthe control leg was notobserved until subjects reachedexhaustion. These findings suggestthat pre-exercise15glycogen content in the active muscle influences il-6 releasefrom that muscle regardlessof circulating substrates and hormones.Considering this research in combination withprevious research by Steensberg (85), demonstrating thatexercise-induced elevations incirculating plasma il-6 can be accounted for by the activeskeletal muscle; it appears thatpre-exercise skeletal muscle glycogen storesaffect the systemic plasma il-6 response toconcentric exercise.The multiple adaptations that occur in response to endurance trainingare wellestablished. One of these adaptations is an increase in skeletal muscle glycogen storage.As evidenced by Putman (70), 7 or 8 consecutive days of cycle training for 2 hours persession at an intensity of 60% of individual VO2max was enough to elevate skeletal muscleglycogen content both at rest and during exercise. The subjects in this study were healthyuntrained males. In another training study, Fischer (23) had young healthy untrainedmales complete 1 hour of isolated knee extensions 5 days per week at 75% of preexercise maximum wattage sustainable for 1 minute. Training was performed on amodified ergometer and the duration of the training program was 10 weeks. The trainingworkload was increased 5-10% every fortnight depending on subject progress. Meanvalues of resting skeletal muscle glycogen increased significantly (p<.O5) from 400 to750 mmol/kg dry wt. throughout the training program. Since endurance training elevatesskeletal muscle glycogen, and pre-exercise glycogen stores dramatically affect postexercise il-6 values, training-induced increases in skeletal muscle glycogen stores likelycontribute to the attenuated il-6 elevation in trained subjects.Oxidative stress has also been indicated as a stimulus for skeletal muscle il-6production. Kosmidou (41) investigated the effect of reactive oxygen species on variousskeletal muscle cells in response to work by Thannickal (88) that examined the cellular16signaling ability of free radicals. Results of this study demonstrated that skeletalmyotubes, differentiated from C2C12 murine skeletal myobiasts, produced il-6 inresponse to pyrogallol (PYR), xanthine/xanthine oxidase (X/XO), or hydrogen peroxide(11202).In contrast, skeletal myobiasts and endothelial cells showed no il-6 response tosimilar exposure. It is also of interest that superoxide dismutase (SOD) and catalase(CAT) inhibited the reactive oxygen species-induced increase in ii-6 from myotubes.Thus, it is suggested that oxidative stress plays a role in skeletal muscle il-6 production.A number of researchers have administered exogenous antioxidants beforeexercise in order to attenuate the elevation of circulating il-6 post-exercise. The results ofsuch research have demonstrated varying results. Vassilakopoulos (89) examined thecytokine response to 45 minutes of cycle ergometer exercise at 70% ofVO2max in thesame subjects with and without antioxidant supplementation. Subjects were young,healthy, but untrained males with meanVO2mvalues of 38.9 ml/kg/min. The two trialswere completed in the same order for each subject and separated by one month.Antioxidant supplementation consisted of 200mg vitamin E, 50,000 IU vitamin A, and1,000mg vitamin C daily for 60 days; allopurinol 600mg/day for 1 5days; and Nacetylcystein 2g/day for 3 days and 800mg the day before the second exercise bout.Without supplementation subjects displayed a peak post-exercise il-6 concentration of 7.3pg/mi. With supplementation the peak il-6 concentration post-exercise was 2.5 pg/ml;significantly diminished (p<.05). Fischer et al. (22) also found a blunted post exercise il-6elevation with antioxidant supplementation. Subjects in this study were physically activenon-athletes. Subjects were assigned to either treatment, vitamin C 500mg/day andvitamin E 400 lU/day for 29 days, or placebo. The exercise bout consisted of 3 hours ofconcentric knee extensions at 50% of maximum power. Peak post-exercise plasma il-617values for the placebo and treatment group were 21 pg/ml and 11 pg/mi respectively(p<.05). These studies indicate that antioxidant supplementation attenuates the post-exercise ii-6 response to concentric exercise in untrained males.In contrast, other research indicates that post-exercise il-6 elevations in highlytrained athletes are unaffected by similar antioxidant supplementation (57, 54, 56). Theexercise bouts incorporated in these studies included a 50km ultramarathon, a 60kmultramarathon, and an ironman triathion respectively. Researchers have suggested that thediscrepancy between the varied results may be due to enhanced muscular and systemicantioxidant capacity achieved from strenuous physical training (89). Much evidence hasbeen reported suggesting that skeletal muscle antioxidant enzymes superoxide dismutase(SOD) and glutathione peroxidase (GPX) are enhanced in response to regular endurancetraining (67, 38, 45, 79). As such, enhanced skeletal muscle antioxidant status due totraining may play a role in the attenuated il-6 response to concentric-based muscleactivity.18Chapter 3: Methodology3.1 Study DesignThis study was cross-sectional mixed model design intended to monitor bothresting and post-exercise measures of systemic inflammation at different time points priorto and in response to a sub maximal endurance cycle ergometer challenge in bothaerobically trained and untrained males aged 19-3 5. The independent variables weretraining status and training history while the dependent variables, measuring systemicinflammation, were the systemic serum concentrations of il-6 and CRP pre-exercise andat assigned time points after a submaximal exercise bout. Blood glucose was alsorecorded before and immediately after the exercise bout to ensure similar blood glucoselevels between groups. Subjects were assigned to either a trained or untrained groupbased upon measures of maximal oxygen consumption (VO2)and training history.Assignment to the trained group required a cycle VO2max 65 mllkg/min and a VO2m45 ml/kg/min for the untrained group. Experimental groups were matched for age andbody mass index (BMI). Following assignment to experimental group, blood sampleswere analyzed for blood glucose, il-6 and CRP at rest (pre-exercise) and following apredetermined exercise challenge. For post-exercise measures il-6 samples were drawn30 minutes and 24 hours post-exercise, and CRP samples were drawn 24 hours postexercise. Blood glucose was recorded immediately pre- and immediately post-exercise.The exercise stimulus was a 45 minute bout on a cycle ergometer at an intensitycorresponding to individual ventilatory threshold.193.2 ParticipantsHealthy males aged 18-35 were selected from the University of British Columbiacampus and the greater Vancouver area. Male participants only were selected in order toeliminate potential antioxidant effects of female sex hormone fluctuations. Estrogen hasantioxidant properties potentially reducing oxidative stress while systemic estrogen iselevated (13). In addition, there seems to be a reduced il-6, il-i, and TNF-ct response totypical stimuli in females compared to males (77). After providing informed consentparticipants were selectively enrolled into the study in accordance with the predeterminedeligibility criteria for the respective experimental groups. After eligibility determinationon the first day of testing subjects were assigned to groups based upon fitness status. Atotal sample size (N) of 26, 13 subjects per group, was determined via power analysis fora 2x3 mixed design analysis of variance (ANOVA) design. This sample size wasdesigned to result in a power of .80 in detecting a difference between groups on the mainfactor of post-exercise plasma il-6 concentration, with a main effect size of(f2=.32).Of the 26 subjects that completed the study 13 trained and 12 untrained subjectswere included in the data analysis. One subject from the untrained group was excludedfrom the study at a later date due to a breach of protocol.203.3 Eligibility Criteria(1) Males aged 18-35years old,(2) Normal BMI: 18.5-24.9 kg/rn2 [Weight(kg)/height (rn)2],(3) Maximal Oxygenconsumption 45 mi/kg/mm (cycle ergometer) and notparticipating regularly in physical exercise 2 hoursper week OR MaximalOxygen consumption 65 ml/kg/min (cycle ergometer)and regularly training atan elite level,(4) Current and expected Vancouver Resident for duration of study,(5) No antioxidant/Vitamin supplementation 6 weeks prior to study,(6) No use of steroidal anti-inflammatory medication 6 weeksprior to study,(7) Healthy,i. No history of chronic disease (CVD, endocrine, hepatic,inflammatory, etc...)ii. No traumatic or chronic injuries present 6 months prior to studyiii. No febrile illness/infection 6 weeks prior to beginning of study(8) Non-smoker(9) No current or planned caloric intake restriction,(10) No shift workers (potential for circadian rhythm ii-6abnormalities)213.4 ProceduresAfter initial contact and voluntaryagreement to participate, subjects providedinformed consent and were immediatelyenrolled into the study. All procedures wereapproved by the Clinical Research EthicsBoard of the University of British Columbiaand Providence Healthcare. Prior to anyphysical testing subjects completed a physicalactivity readiness questionnaire (PAR-Q). Upon completionof a PAR-Q and medicalclearance each subject underwent baseline aerobic fitness and bodycompositionmeasures in the John M. Buchanan exercise physiology laboratory.Baseline fitnessmeasures were completed following 24 hours without exercise and a 2 hour fast. Bodycomposition was recorded as both BMI (mass (kg)/height (rn)2)and the sum ofthreeskinfolds (axilla, abdominal, and mid-thigh). Also at this time subjectscompleted aquestionnaire (Appendix B) in order to establish training history. Aerobic fitness wasassessed using a maximal oxygen consumption (VO2max) cycle ergometer protocol.Subjects were allowed a five minute self-selected warm up before the VO2max protocolbegan. The VO2max protocol began at 50 watts with workloadincreases of 25 wattsevery minute until volitional fatigue. A maximal test was confirmed based on predetermined indicators; respiratory exchange ratio (RER) above 1.1, HR within ten beatsof age predicted maximum HR (220-age), and a plateau inVO2max [either a decrease oran increase of <2 ml/kg/min]. VO2max was determined byaveraging the highest V02values over two consecutive 20 s intervals.In addition to measurement ofVO2max, ventilatory threshold was calculatedusing ventilatory equivalence (VeIVO2)in order to establish a subsequent individualexercise challenge workload for each subject.Ventilatory equivalence was assessed bytrained exercise physiologists. The ergometer utilized inthis protocol was an 800 series22Sensormedics electronically braked cycle ergometer. Expired gases were analyzed with aSensormedics Vmax 29 metabolic cart.At least 72 hours after baseline fitness measures were recorded subjectsunderwent the exercise challenge on a cycle ergometer at a predetermined workload. Thework bout was a 45 minute cycle at a wattage corresponding to individual cycleventilatory threshold. In preparation for the execise challenge subjects were asked toavoid strenuous physical activity 72 hours prior to and physical activity entirely for 48hours prior to the exercise challenge. Also, subjects were asked to refrain from alcoholconsumption within 48 hours of testing.On the day of the exercise challenge subjects reported to the St. Paul’s HospitalHealthy Heart fitness gym between 8am and 10am following an overnight fast (wateronly past 11pm the previous night). Subjects were encouraged to travel to the hospitalusing the least physically strenuous means available. At this time a technician drew oneresting venous blood sample from an antecubital vein in the subjects’ preferred arm; thesample was later divided for baseline serum CRP measurement and serum il-6measurement. After this a finger prick blood sample was analyzed for blood glucose.Glucose was recorded to determine whether any subjects began the exercise bout withabnormal blood glucose concentrations or had a significant change throughout the bout.This was of great importance as blood glucose status has been shown to have a largeaffect upon skeletal muscle il-6 production. Upon completion of the blood samplingsubjects completed a 5 minute self-selected warm up on an electronically braked cycleergometer. After warming up subjects completed the predetermined exercise challenge(45 minutes at a wattage corresponding to individual cycle ventilatory threshold). Heartrate was recorded every minute and rating of perceived exertion (RPE), on a Borg 1523point scale, was recorded every fiveminutes during the exercise bout.If the assignedworkload was not manageable individualworkload was adjusted in orderto maintain aheart rate within ten beats per minuteof that corresponding to ventilatorythresholddetermined on day one. Immediatelyafter the exercise boutanother finger prick bloodsample was analyzed forblood glucose. After thisglucose analysis subjects remainedseated in the laboratory for15 minutes still fasting.Approximately 15 minutespost-exercise subjects were weighedin order to monitor fluidloss. Water was consumedaccordingly to replenishfluid loss and achieve pre-exerciseweight. Thirty minutespost-exercise another blood samplewas drawn for measurementof post-exercise serumil-6.After this sample subjectswere asked to returnto the clinic in 24 hoursand were free toleave. Subjects were askedto refrain from vigorousphysical activity andalcoholconsumption within this24 hour period. Twenty-fourhours following the cessationof theexercise bout subjectsreturned to the hospitallab for another bloodsample to measure 24hour post-exercise serum il-6and CRP. A protocol timelineis included as Chart 4.1.3.5 Outcome measuresIntereukin-6:For il-6 analysis bloodwas collected in 5 ml samplesinto serum separator tubescontaining silica and polymergel for serum separation. Sampleswere drawn byvenipuncture from anantecubital vein of the subjectsarm at predetermined pointsintime. Serum separator tubeswere then inverted 5 times andleft to clot for 30 minutes.Atthat time samples were centrifugedat 1100-1300 x gravityfor 10-15 minutes. Serumwasthen aliquoted and stored ina freezer at -80 C until assayed.Serum il-6 concentration24was determined using a commercially availablequantikine high-sensitivity enzymelinked immunosorbent assay (ELISA)kit (R & D Systems, Minneapolis,Minn. USA).The ELISA kit used has a mean intra-assaycoefficient of variation of 7.4and a meaninter-assay coefficient of varianceof 7.3. Samples were tested in duplicate.C-reactive protein:For CRP analysis blood was collectedin 5 ml samples into serum separatortubescontaining silica and polymer gelfor serum separation. Sampleswere drawn byvenipuncture from an antecubitalvein of the subjects armat predetermined points intime. Serum separator tubes werethen inverted 5 times andleft to clot for 30 minutes.Atthat time samples were centrifugedat 1100-1300 x gravity for 10-15 minutes.Serum wasthen aliquoted and stored ina freezer at -20 C until assay within24 hours. Serum CRPwas evaluated using an Immulite2000 High Sensitivity solid-phase,chemiluminescentimmunometric assay (DiagnosticProducts Corporation, Los Angeles,California. USA).The selected assay hasmean inter- and intra-assaycoefficients of variation of < 5%.Blood Glucose:Low blood glucose concentrationsduring exercise have beenshown to enhancethe post-exercise increasein il-6. Blood glucose concentrationswere recorded using aportable hand held glucose meter(Bayer Glucometer Elitewith Bayer Ascensia ELITEtest strips, Bayer Corporation, Elkhart,Indiana. USA). Finger prick capillarysampleswere drawn from a cleanand dry self-selected finger for eachanalysis. In combinationthe Bayer Glucometer Elite and BayerAscensia ELITE test stripshave an intra-assaycoefficient of variation of4.0% and an inter-assay coefficientof variation of 5.3% atnormal blood glucose concentrations.25Rate of Perceived Exertion:During the 45-minute cycle ergometer challengesubjects indicated ratings ofperceived exertion every five minutes usinga 15-point Borg scale. The mean of all RPEscores for each subject was taken as that subject’s RPEscore for the exercise challenge.3.6 Data AnalysisData analysis was completed using SPSSsoftware (version 12.0). All data wasfirst tested for normality of distribution and homogeneityof variance prior to furtherstatistical analysis. All measurementssubjected to analyses were reportedas means.Demographic and physiological descriptivestatistics compared between groupswereanalyzed by independent t-tests. Significancefor each t-test was set at a..05. Systemicil-6 concentrations were analyzed usinga 2 (Groups: trained and untrained)x 3 (Time:pre-, 30 minutes post-exercise, and 24hours post-exercise) mixed designanalysis ofvariance (ANOVA) with repeated measureson the second variable. If needed, violationsfor sphericity were corrected using the Greenhouse-Geissercorrection. A 2 (Groups:trained and untrained) x 2 (Time: pre-and 24 hours post-exercise) mixed design ANOVAwas used to analyze serum CRP concentrations.A 2 (Groups: trained and untrained)x 2(Time: pre- and immediate post-exercise)mixed design ANOVA was alsoused toanalyze blood glucose concentrations.Significant main or interaction effectsfor il-6,CRP, and blood glucose were further analyzed usingpost-hoc t-tests. Significance foreach t-test was set at a. = .05.26Relative values of peak 11-6 per workload (wattage,relative oxygen consumption,and absolute oxygen consumption)at 30 minutes post-exercise were also comparedbetween groups using independent t-tests. Significancewas set at a. = .05.The correlational relationship between pre- versus post-exercisedifferences in il-6and CRP were analyzed in both fitnessgroups. The correlational relationship betweenfitness variables and baseline values of il-6 and CRP were also analyzed. Correlationwasassessed using the Pearson product moment calculation. Significance was set at a. = .05.27Chapter 4: Results4.1 Subject Demographic CharacteristicsAll subjects in the study were between the ages of 19 and 35,with an average ageof(25. 1 +7- 4.6 years), and prescreenedfor potential medical contraindications toparticipation. As planned, the trained and untrained groups in the studywere matchedwith respect to non-performance related demographic characteristics (Table 4.1). Nosignificant differences between the trained and untrained group were found in age, height,mass or BMI. However, the sum of skin folds SOS was greater in the untrained 36.2+7-8.5 mm compared to the trained group 22.8 +7- 4.7 mm F (1,23) 2.16,p= 0.000.In accordance with subject inclusion criteria the groups differed in traininghistory. The trained group participated in 13.5+1- 3.5 hours of general exercise per weekcompared to 2.8 +7-1.9 hours in the untrained group F (1,23) = 2.5, p = 0.000 and thetrained group cycled for 9.46 +7- 3.9 hours per week compared to 0.2 +7- 0.4 hoursin theuntrained group F (1,23) = 34.6,p0.000.4.2 Subject Physiological and Performance CharacteristicsDue to the nature of the study, subjects were assigned to groups based upontraining history and aerobic fitness on a cycle ergometer. As such the two groupsdifferedsignificantly in most of these variables (Table 4.2).The trained group had significantly higher values than the untrained groupforboth absolute maximal oxygen consumption (VO2m); 5.01+1- 0.61 L/min compared to3.29 +7- 0.65 L/min 02 F (1, 23) = 0.067,p0.000 and relative maximal oxygen28consumption (VO2max) 68.61+1- 5.57 mi/kg/mm compared to 42.42+1- 4.62 mi/kg/mmF (1, 23) = 0.233,p0.000 respectively. Absoluteand relative oxygen consumptionsatventilatory threshold (VT) werealso significantly higher in the trainedgroup compared tothe untrained group; 3.57 +1- 0.51 L/min comparedto 2.00 +/- 0.36 L/min F (1, 23) =3.18,p= 0.000 and 48.90 +1- 5.80 mI/kg/mmcompared to 25.80 +/-2.68 mi/kg/mm F (1,17.20) = 5.08,p= 0.000 respectively. Althoughthe workload assigned to subjectswas anintensity corresponding to VT,V02 at VT was a significantlygreater percentage ofmaximal oxygen consumption[% VO2max (mi/kg/mm)at VT] in the trained groupcompared to the untrained group;71.1 +1- 4.6 % comparedto 61.0 +1- 4.6 % F (1, 23)0.168,p= 0.000.Maximal heart rate (HRmax)and average heart rate duringthe exercise bout(HRavg) were not significantlydifferent between groups.However, heart rate at VTwassignificantly higher in thetrained group 161.9 +/- 9.7 BPMthan the untrained group151.6 +1- 11.6 BPM F(1, 23)=0.005, p= 0.024.Performance characteristics differedgreatly between groups. Peakaerobic poweroutput and power outputat VT were significantly higherin the trained group comparedtothe untrained group; 431+1- 49 watts compared to 267 +1- 55watts F (1, 23) = 0.042,p0.000 and 265 +1- 38 watts comparedto 139 +1- 27 watts F (1, 23) = 0.751,p= 0.000respectively.294.3 Subject Compliance4.3.1 ProtocolOf the initial 26 subjects enrolled in thestudy one failed to comply withprotocol.This subject deviated from proposedprotocol by consuming alcoholthe night before day2 of testing. This was not reportedby the subject until afterday 2 of testing. The data forthis subject was excluded fromdata analysis resulting inan N of 25 instead of 26.Otherwise all subjects complied,to the best of our knowledge, withprotocol includingappropriate fasting, avoidanceof exercise, and avoidanceof alcohol consumption.4.3.2 PerformanceAll subjects were able to completethe exercise bout on day 2. InTable 4.3assigned and completed power outputsand heart rates are presentedfor each groupdemonstrating that both groupssuccessfully completed theirassigned exercise bouts. Thepower output (P0) achievedby each group was somewhat lowerthan that assigned butboth groups achieved a P0not significantly different than thatassigned from assessmenton day 1 (Table 4.3). Both groupsalso achieved an averageheart rate during the exercisebout on day 2 similar to thatcorresponding to VT.304.4 Outcome Measures4.4.1 Interleukin-6It was hypothesized that peak post-exercise 11-6 concentrationswould besignificantly lower(p < 0.05) in the trained group compared to the untrained group.Itwas also hypothesized that baselineconcentrations of 11-6 would notbe significantlydifferent between groups. At each of threetime points, pre-exercise, 30 minutespost-exercise, and 24 hours post-exercise, mean11-6 concentrations were somewhatlower inthe trained group. However, this differencewas not statistically significant (Table4.4).It was also hypothesized that 11-6 concentrationsin both groups would increasesignificantly following the assigned 45-minuteexercise challenge. When analyzedtherewas found to be a significant differencefor the within-subjects effectover the three 11-6samples F (1.60, 21.31) = 30.49,p= 0.000 after Greenhouse Geisser correction.Furtherpost-hoc tests indicated 11-6 concentrationsin both groups increased significantlybetween pre-exercise and 30 minutes post-exercisesamples; trained 0.598 +1- 0.535pg/ml to 1.826 +1- 0.741 pg/ml(p<0.01) and untrained 0.914 +1- 0.896pg/ml to 2.027+1- 1.095 pg/mi (p < 0.01) (Table 4.5).4.4.2 C-Reactive ProteinIt was hypothesized that CRP concentrationswould be similar between groups atrest and would increase significantly 24 hours post-exercisein the untrained group butnot the trained group. When analyzed therewere no significant within-subjects effectsF(1,23) = 0.604,p= 0.445 indicating that CRP values remainedsimilar over time within31each group (Table 4.3). A significant effect was found when between subjectseffectswere analyzed F (1,23) = 7.655,p= 0.011. Further post-hoc tests indicated thatCRPconcentrations were lower in the trained group for the pre-exerciseand 24 hours post-exercise sample; pre-exercise 0.162 +1- 0.247 mg/L compared to 1.167+7- 1.427 mg!L p<0.05, 24 hours post-exercise 0.192 +1- 0.202 mg/L compared to 1.233+1- 1.235 mg/L p<0.05 (Table 4.4).4.4.3 CorrelationsIt was hypothesized that differences inpre- and post-exercise concentrations ofplasma il-6 would show a significant correlation(cL .05) with differences in pre- andpost-exercise concentrations of CRP in bothstudy groups. Using the Pearson productmoment correlation there was no significant correlationbetween pre- to 30 minutes post-exercise elevations of 11-6 and pre- to 24hours post-exercise elevations of CRP in eithergroup or both groups combined. However, when thetwo groups were analyzedseparately, baseline concentrations of 11-6 andCRP were significantly correlated in theuntrained group (r=0.69, P0.01,r2=0.48), representinga moderate relationship, but notin the trained group r=-0.13, P=0.68,r2=0.02). Peakconcentrations of 11-6 and CRP werenot significantly correlated in either group.4.4.4 Blood GlucoseBlood glucose concentrations were recorded immediatelybefore and immediatelyafter the exercise bout on day 2 for each subject to monitor potentialdifferences betweengroups. There were no significant differences in bloodglucose concentrations before or32after exercise and there were also no differences between pre- and post-exercise bloodglucose concentrations in either group (Table 4.5). All blood glucose values werewithinhealthy limits with a minimum of 4.6 mmol/L and a maximum of 6.3 mmolJL.4.4.5 Ratings of Perceived ExertionAverage ratings of perceived exertion were recorderfrom each subject during theexercise bout on day 2 to compare subjectivefeelings of workload between groups.Ratings of perceived exertion were found to be similar betweenthe trained and untrainedgroups (Table 4.4).33Figure 4.1 Subjects screened, consented, assigned, and completedDay 1-Informed Consent-Body Composition-VO2max test-Group Assignment-40 subjects enrolled-14 rejected-2 due to illness-1 due to injury-11 too fit/unfitN=26I __• IDay2 Day2(Trained) (Untrained)-Pre-exercise 11-6-Pre-exercise 11-6-Pre-exercise CRP-Pre-exercise CRP-Pre-Exercise Glucose-Pre-Exercise Glucose-Ex. Challenge (45 mins@VT)-Ex. Challenge (45 mins@VT)-Post-exercise 11-6I-Post-exercise 11-6-Post-Exercise Glucose-Post-Exercise GlucoseN=13N=13Day 3 Day 3(Trained) (Trained)-24 hrs post-exercise -24 hrs post-exerciseCRP/I1-6 CRP/II-6N=13 N13Completed Completed(Trained) (Trained)N=13 N=12*1drop out due topoor compliance34Table 4.1 Subject Demographic CharacteristicsVariables Trained (n=13) Untrained (n=12) PAge (Years) 26.7 +1- 4.9 23.4 +1- 3.8 NSRaceCaucasian 12 10NSAsian 1 1NS0 1NSBody CompositionHeight (cm) 179 +/- 5.7179 +1- 8.7 NSMass (kg) 73.0 +1- 7.8 77.9 +1-15.0 NSBMI (kg/rn2) 22.6 +1-1.4 23.9 +1- 3.0 NSSOS 22.8 +1- 4.7 36.2+1-8.5*0.000Training HabitsExercise per week (hrs) 13.4 +1- 3.5 2.8+1-1.9*0.000Cycling per week (hrs) 9.5 +1- 3.90.2 +1-0.4*0.000Pimary EventCycling 10 0NSTriathlon 3 0NSValues are given as mean +/- SD, NS = Not Significant,*indicates significant difference(pO.O5).35Table 4.2 Subject Physiological and Performance CharacteristicsVariables Trained (n13) Untrained (n=12)PRespiratoryVO2max (L) 5.01 +1- 0.613.29 +1-0.65*0.000VO2max (mi/kg/mm) 68.61 +1- 5.57 42.42+/4.62*0.000V02 (L) at VT 3.57 +1- 0.57 2.00+!-0.36*0.000V02 (mi/kg/mm) at VT 48.90 +1- 5.80 25.80+1-2.68*0.000% VO2max (mi/kg/mm) at VT (%) 71.1 +1- 4.661.0 +/4.6*0.000CardiovascularHRmax (BPM) 188.1+1- 7.1 187.9 +1- 10.1 NSHRatVT(BPM) 161.9+1-9.7 151.6+/11.6*0.024HRavg during challenge (BPM) 162.7+1- 7.1 153.0 +1- 18.5 NSPerformancePeak Aerobic P0 (Watts) 430.8+1- 49.1 266.7 +1-54•7*0.000P0 at VT (Watts) 265.4 +1- 37.5138.75 +/26.9*0.000P0 during challenge (Watts) 247.3+1- 30.8 132.5 +1-29.5*0.000Values are given as mean +1- SD, NS = Not Significant,*indicates significant difference(p 0.05).36Table 4.3 Performance OutcomeMeasures Within GroupsVariable AssignedCompletedPHeart Rate (bpm)Trained 161.9+1- 9.7 162.7+1- 7.1 NSUntrained 151.6+1- 11.6 153.0+1- 18.5NSPower Output (Watts)Trained 265.4+1- 37.5 247.3 +1-30.8 NSUntrained138.75 +1- 26.9132.5 +1- 29.5NSValues are given as mean +1- SD,NS = Not Significant,*indicates significantdifference0.05).37Table 4.4 Outcome Measures BetweenGroupsVariables Trained (n=13)Untrained (n12) P11-6Pre-Exercise (pg/mi) 0.598 +1-0.535 0.9 14 +/- 0.896NS30mm Post-Exercise (pg/mi) 1.826 +/- 0.7412.027 +/- 1.095 NS24hrs Post-Exercise (pg/mi) 0.633 +/-0.350 0.93 1 +/- 0.902NSCRPPre-Exercise (mg/L)0.16 +/- 0.25 1.17 +1-1.43*< 0.0524hrs Post-Exercise (mg/L) 0.19+/- 0.20 1.23 +/-1.23*< 0.05Blood GlucosePre-Exercise (mmoi/L)5.2 +/- 0.4 5.4 +1-0.2 NSPost-Exercise (mmol/L) 5.3+/- 0.6 5.1 +1- 0.4NSRating of Perceived Exertion 14.3 +/- 1.113.8 +/- 0.8 NSValues are given as mean +/-SD, NS = Not Significant,*indicates significant difference(p 0.05).38Table 4.5 Outcome Measures 11-6 and CRPWithin Groups30mm Post- 24hrs Post-Variable P re-Exercise ExerciseExercise P11-6 (pg/mI)Trained 0.598+1-0.535 1.826+I0.741*X <0.01Trained 0.598 +1- 0.535X 0.633 +1- 0.350 NSTrained X 1.826+1-0.741 0.633+/0.350*<0.01Untrained 0.914 +7- 0.896 2.027+1-1.095*X < 0.01Untrained 0.914 +1- 0.896X 0.93 1 +7- 0.902 NSUntrained X 2.027+1- 1.095 0.93 1 +7-0.902*< 0.01CRP(mgfL)Trained 0.16 +1- 0.25X 0.19 +7- 0.20NSUntrained 1.17 +1- 1.43X 1.23 +1- 1.23 NSValues are given as mean +7- SD,NS = Not Significant,*indicates significant difference(p 0.05).39Table 4.6 Outcome Measure Blood Glucose WithinGroupsVariable Pre-Exercise Post-ExercisePBlood Glucose (mmoLfL)Trained 5.2 +1- 0.4 5.3+1- 0.6 NSUntrained 5.4 +1- 0.2 5.1 +1- 0.4NSValues are given as mean +1- SD, NS = Not Significant,*indicates significant difference(p 0.05).40*Figure 4.2 Interleukin-6 ConcentrationsSerum 11-6 Concentrations in Trained andUntrained Males*Significant increase from pre-exercisewithin group3.5*32.5 -11-6 (pglml)2 -1.50.5 -0D Pre-Exercise• 3Omins Post-Exercise0 24hrs Post-Exerce -TrainedGroupUntrained41Figure 4.3 C-Reactive Protein ConcentrationsSerum CRP Concentrations in Trained and UntrainedMales*Significant difference between groupsat same sample time3__*CRP (mgiL)1.50.50[eExercise• 24hrs Post-Exercise*TrainedGroup Untrained42Figure 4.4 Blood Glucose Concentrations5.85.6Blood Glucose (mmol!L)5.45.254.84.6Blood GlucoseD Pre-Exercise• Post-ExerciseTrained UntrainedGroup43Chapter 5: Discussion5.1 General FindingsThis cross-sectional study is, to the best of our knowledge, the first to compareresting and post-exercise measures of inflammatory proteins 11-6 and CRP betweentrained and untrained healthy males following a similar exercise challenge. This analysisprovides new information on the effect of training on inflammation and the relationshipbetween 11-6 and CRP. The main findings of this investigation are the following:1) Systemic concentrations of 11-6 increased following exercise in both trained anduntrained subject groups but concentrations between groups were similar at all testingpoints. 2) Neither the trained or untrained group showed a significant change in CRPconcentrations in response to the exercise challenge. However, at rest and 24 hours post-exercise CRP concentrations were significantly lower in the trained compared to theuntrained group. 3) Pre- to post-exercise changes in 11-6 and CRP concentrations were notsignificantly correlated. However, resting pre-exercise concentrations of 11-6 and CRPwere significantly correlated in the untrained group but not in the trained group.5.2 11-6The pattern, variability, and magnitude of 11-6 concentrations found in this studyare in agreement with similar studies incorporating endurance cycle ergometer exercisewith trained athletes; peaking at 1.826 +1- 0.741 pg/ml at 30 minutes post-exercise andreturning to baseline values 24 hours post-exercise (60, 65, 89). The variability of il-6,measured as standard deviation, at peak values was 0.74 1 pg/ml in the trained group and1.095 pg/ml in the untrained group. Although these standard deviation values are high44relative to the group means they are similar to or less than that found at peak values insimilar previous studies: 8.0 +1- 2.0 pg/mi after 1 hour of treadmill running at 75% ofVO2max (60) and 7.3 +/- 1.5 pg/mi after 45 minutes of cycle ergometry at 70% VO2max(89).Only one other study, to the best of our knowledge, has examined resting andpost-exercise 11-6 values in sedentary subjects using a similar exercise mode and bloodsampling schedule (89) to that in our study. In this study sedentary males rode on a cycleergometer at 70% of maximal oxygen consumption for 45 minutes with 11-6 valuespeaking 30 minute post-exercise with a mean of 7.3 +/- 1.5 pg/ml. Comparatively the Ii-6 values demonstrated in our untrained group are not similar, reaching a mean peakconcentration of only 2.027 +/- 1.095 pg/mi. When comparing the exercise protocols ofthese studies, our untrained group was assigned a workload at ventilatory threshold,corresponding to 61.0 +/- 4.6% ofVO2m, while subjects in the other study maintained aworkload corresponding to 70% ofVO2max regardless of ventilatory threshold. This 9%difference in oxygen consumption may account for the peak 11-6 discrepancy between ouruntrained group and those of the other study (89) considering systemic peak post-exerciseil-6 has been shown to increase linearly with exercise intensity (calculated asV02/VOmax) (63).Although the specific stimulus or stimuli for 11-6 production during exercise havenot been elucidated, it is has been demonstrated that systemic lactate is not a significantstimulus (85). Furthermore, oxidative stress has been implicated as a potential stimulusfor 11-6 production in skeletal muscle (41). Considering that free radical productionincreases as a function of oxygen consumption (68) it seems that exercise challenges maybe better matched for relative oxygen consumption as opposed to ventilatory threshold or45lactate threshold when examining 11-6 production. From a practical perspective, it isappealing to assign workloads based upon a measure closely linked to performance suchas ventilatory threshold. However, in order to understand the physiological effect oftraining upon skeletal muscle production of 11-6 it seems workload assignment should bebased upon oxygen consumption.As hypothesized, we found that 11-6 concentrations increased significantly in bothtrained and untrained groups, p < 0.01. Contrary to our hypothesis we found that therewere no significant differences in 11-6 concentration between the two groups at any timepoint. This in itself is remarkable as the trained group was working at a significantlyhigher wattage (87% higher), relative oxygen consumption (90% higher), and absoluteoxygen consumption (79% higher) than the untrained group. Furthermore, as was theinstance comparing the results of Vassilakopoulos’ study (89) to our untrained group, theworkload assigned was not based upon relative oxygen consumption. When compared byrelative oxygen consumption our trained group worked at an intensity corresponding to71.1 +1- 4.6% of maximal oxygen consumption (VO2/VO2max) whereas our untrainedgroup worked at an intensity corresponding to 61.0 +1- 4.6%; significantly different,p <0.01. In a study by Ostrowski (63) it was demonstrated that peak 11-6 concentrations insimilarly trained athletes formed a significantly correlated (p < 0.01) linear relationshipwith exercise intensity (calculated asVO2NOm). It can be extrapolated then that thepeak post-exercise 11-6 concentration difference between groups would likely have beenlarger if the workloads for each group were assigned based upon relative oxygenconsumption.It should also be noted that our blood sampling times were assigned based on thebest estimate of when peak il-6 concentrations would occur as found by previous research46(40, 41, 89). These studies indicated that il-6concentrations in response to concentricexercise between 0 and 30 minutespost-exercise. As such we chose 30 minutes post-exercise to draw our peak il-6 blood sample. Someresearchers have indicated that peakil-6 values may even occur before the end of the exercise bout;in which case the truepeak il-6 values in our subjects may have been missed. Althoughthis is possible, themajority of studies using a concentric exercise stimulus of similar duration to oursfoundpeak il-6 values to occur within 30 minutes after exercise (40, 41, 89).5.3 CRPAs hypothesized, CRP concentrations in the trained group remained similarbetween rest and 24 hours post-exercise. It was also hypothesized that CRP values wouldincrease significantly in the untrained group from rest to 24 hours post-exercise.Surprisingly, our untrained group showed no such increase. This hypothesiswas basedupon an earlier study (80) that showed a significant increase (p = 0.04) in CRP 24 hourspost-exercise in untrained subjects after riding on a cycle ergometer for 60 minutes at anintensity corresponding to 60% ofVO2m. The discrepancy in findings may be due toinvestigation methodology. In the earlier study (80) training status was not quantifiedleaving the possibility that these subjects were less active than our untrained group; 2.8+1- 1.9 hours of exercise per week and a mean relative VO2max of 42.42 +1- 4.62ml/kg/min. Health factors such as recent injury, recent illness and chronic illness areallfactors that may affect CRP values and were not accounted for (80). Also, the exerciseintensity is difficult to compare between studies as Smith (80), indirectly estimated aworkload of 60% VO2m based upon maximal heart rate alone whereas our workload was47based on direct respiratory measures of ventilatory threshold. Although surprising ourresults are similar to those found in a study run concurrently to ours in which highlytrained and moderately trained subjects both showed no significant increase in CRP in thedays following a similar moderate endurance exercise bout (66). These results indicatethat the exercise stimulus used was not great enough to examine an 11-6 induced increasein CRP. Sorichter (81) monitored the CRP response to endurance exercise in trainedrunners (VO2max 60.02 mi/kg/mm) after a one hour run at 80% on VO2max. The resultsfrom this showed significantly increased CRP values at 6 and 24 hours post-exercise withvalues at 24 hours post-exercise being the greatest. Our untrained group worked at a V02corresponding to 61% of VO2max for 45 minutes and our trained group at a V02corresponding to 70% of VO2max for 45 minutes. Considering il-6 is the chief stimulusfor CRP production and il-6 increases after exercise as a function of exercise duration andintensity our exercise bout should have lasted at least one hour and the intensity,measured as % V02, should have been increased by 19% and 10% for our untrained andtrained group respectively.Other studies that have examined the CRP response to various forms of exercisehave used different sampling timelines. In a study by Czarkowska-Paczek (18) trainedcyclists with a relative VO2max of 65.7 ml/kg/min cycled to exhaustion. The totalworkload completed in this study was comparable to that in our study. CRP was recordedpre-exercise, immediately post-exercise, and two hours post-exercise. CRP valuesshowed no significant change over time. CRP was not recorded 24 hours post-exercise.Researchers in this study assumed that if CRP was to increase following exercise it woulddo so within two hours of the exercise bout. To our knowledge few studies havemonitored systemic CRP pre-exercise, immediately post-exercise, and at subsequent time48points up to 48 hours after a discrete enduranceevent. Taylor (87) recorded CRP beforeand after an ultradistance triathlon (160 km).Measures of CRP indicated no significantchanges immediately after or within five hoursof exercise. The next measure was taken24 hours post-exercise and at that time was significantlyelevated. CRP then returned tonear baseline by 48 hours. This study suggests that CRPdoes not increase immediatelyafter endurance exercise; even with exercise of duration found in anultraenduranceevent. In a running study in which trained runners, VO2max60.02 ml/kg/min, ran on flatground for one hour at 80% of VO2max CRP concentrations were recorded pre-exercise,immediately post-exercise, and 1, 6, 24, and 48 hours post-exercise (81). CRPresultsshowed a significant increase 6 hours after exercise (increase from 0.3 mg/L atbaselineto 0.5 mg/L 6 hours post-exercise) and a greater increase 24 hours post-exercise(increasefrom 0.3 mgIL at baseline to 1.2 mg/L 24 hours post-exercise). At 48 hours afterexerciseCRP values returned to baseline. Although these studies outline the time course of CRPaccumulation, likely peaking at approximately 24 hours post-exercise, there is a lack ofresearch reporting CRP values between 6 and 24 hours post-exercise. From the availableliterature our sampling schedule seems appropriate to assess a significant elevation inCRP and the addition of a measure immediately post-exercise or up to 6 hours post-exercise would seem unnecessary. As mentioned, no research to our knowledge hasrecorded CRP values between 6 and 24 hours after endurance exercise possibly causingus and other researchers to miss the true peak post-exercise CRP elevation. This lack ofblood sample collection is likely due to inconvenience as time points falling within the 6-24 hours pos-exercise often during sleep.Another hypothesis we made a priori was that CRP concentrations would besimilar between groups pre-exercise but significantly higher in the untrained group 2449hours post-exercise. Wehypothesized that CRP concentrations atrest would be similarbetween groups since both groupswere healthy, young, of similar BMI, and free of anymedications. Also we felt that,although not highly active, our untrained groupwas notsedentary. We found that CRP wasmarkedly higher in the untrained group pre-exercise[0.16 +1- 0.25 mg/L comparedto 1.17 +1- 1.43 mg/L (p <0.05)] and 24 hourspost-exercise [0.19 +1- 0.20 mg/L comparedto 1.23 +1- 1.23 mg/L (p <0.05)]. Our hypothesisthat CRP values would be similarbetween groups was based upon values found byCzarkowska (18) in combination withlarge scale studies reporting normal CRP values ininactive individuals. Czarkowska recorded CRP beforeand after exercise in elite roadcyclists of similar fitness to our study; mean VO2max65.7 mi/kg/mm. Resting CRPconcentration in this group was reported as 3.25 mg!Lindicating no reduction comparedto inactive individuals. In retrospect, pre-samplingprotocol in this study was notdiscussed indicating that samples could havebeen taken during a heavy training period.Considering CRP peaks 24 hours post-exercise, if participantshad trained the day prior tosampling CRP may have been artificially elevated. Anecdotallyit seems unlikely thatathletes of this caliber would take 48 hours off oftraining without being prompted. Thiscould account for the massive discrepancy in CRPvalues compared to our findings. In astudy published during the course of our study aresearch team showed a non-significantdifference in CRP concentration between physicallyactive and inactive subjects (86).Although the difference was not significant the resultsare in line with our findings. Inthis study young physically active and inactivesubjects aged 18-35 years were comparedfor resting CRP. The inactive group had a restingCRP of 1.2 mg/L and the physicallyactive group had a resting CRP of 0.7 mg/L (P>0.05). This shows a similar comparisonto our results with the physically active groupmaintaining a reduced CRP concentration.50In Stewart’s study physical fitness was assessed as aVO2max estimate from the Balketest; physically active subjects having a mean of 45 mi/kg/mmand inactive subjectshaving a mean of 35 ml/kg/min. In our study relative VO2maxhad a mean difference ofmore than 20 ml!kg/min between groups; 42.4 mi/kg/mm in the untrained groupand 68.6mi/kg/mm in the untrained group. This larger difference in primary fitness measure likelyaccounts for our finding of such a vast difference in CRP in comparison to previousresearch.In large scale population studies it has generally been assumed that reductions inCRP in more active individuals are due to reduced underlying CRP stimuli; mainlysystemic il-6. Considering 11-6 concentrations were similar between groups at all timepoints it seems unlikely that the difference in CRP values can be attributed directly to Il-6. This was surprising as ii-6 has been defined as the chief regulator and stimulus forCRP production (12).One potential explanation for these findings is that secondary stimuli of hepaticCRP production, il-i and TNF-a, were greater in the untrained group. This seemsunlikely for two reasons. First, il-i and TNF-cL, when administered individually, showeda 1-fold increase in CRP production from human hepatocytes (12). In contrast, asimilaraddition of il-6 to human hepatocytes showed a 23-fold increase in CRP production.Second, it has been demonstrated that physically active and inactive groups of healthymales aged 18-35 years with similar BMI measures have shown no differences in restingconcentrations of il-i or TNF-cL suggesting that training status should not play a role inresting il-i or TNF-cL values (86).Another possible explanation is that leptin levels were higher in our untrainedgroup than our trained group. Leptin is a hormone produced in adipocytes that istypically51associated with hunger and energy balance. In relation to our work, it is of note thatsystemic leptin concentrations are significantly correlated with systemic CRP (78).Previously it was assumed that this correlation was confounded by obesity and thesubsequent increase in adipocytes and other inflammatory mediators. Later findings byShamsuzzaman (78) showed a significant correlation between the two regardless of BMI,and other associated factors. The understanding of the relationship between leptin andCRP is not fully understood; however, it has been demonstrated in vitro that leptinstimulates CRP expression in human coronary artery endothelial cells (78). Interestinglyleptin was found to be a more potent CRP stimulus in these cells than il-6, TNF-cL, or il113.Also, the leptin receptor has been shown to have signaling capabilities similar to il-6receptors indicating that leptin may act directly to induce CRP production in the absenceof il-6, il-i or TNF-a (6). Furthermore, a number of studies have found reduced leptinlevels in trained individuals compared to untrained as well as within individuals aftertraining for some weeks (9). The results typically were attributed to differences in fatmass but two groups that ran prospective endurance training studies found reductions inleptin with training without a concomitant reduction in fat mass (33, 37). With all of thisin mind it seems reasonable that our trained group may have had diminished systemicleptin due to increased endurance training and a significantly lower sum of skinfoldsreflecting reduced fat mass. Applying the mechanisms and information discussed thispotential reduction in leptin may have influenced the significant reduction in CRP in ourtrained group.It also seems possible that the il-6 cell surface receptor expression in the liver maybe desensitized in trained individuals. Evidence of this comes from a study byMackiewicz (52) that examined the acute phase response to il-6 in human hepatoma cells.52When repeatedly exposed to high levels of il-6 in vitro, the cell surface il-6 receptorsbecame desensitized resulting in a limited acute phase response to an identical il-6stimulus. By this same mechanism the constant peaks in systemic ii-6 associated withstrenuous exercise training may desensitize hepatocyte ii-6 receptors in trained subjects.This may lead to lower CRP concentrations in trained subjects when compared tountrained subjects even while maintaining similar resting il-6 concentrations. Correlationresults in our study lend some support to this mechanism. At baseline il-6 and CRPconcentrations were significantly correlated in the untrained group (r=0.69,p = 0.01),representing a moderate relationship in which 48 percent of the change in CRP can beattributed to il-6, but not in the trained group (r=-O.13, p=O.68). This lack of correlationindicates that the il-6 to CRP relationship is dissimilar in the trained and untrained group.Furthermore, the mean post-exercise increase in CRP (mg/L) per increase in il-6 (pg/mi)was 0.04 16 mg/L/pg/ml in the trained group and 1.2112 mg/L/pg/ml in the untrainedgroup. This ratio measure objectively represents the il-6 to CRP relationship. Incombination our results support this notion of a blunted CRP response to il-6 in trainedsubjects.5.4 Training AdaptationsThe results collected from our study indicate that trained subjects are able to dofar more work and turn over far more oxygen than untrained subjects while showing asimilar absolute elevation in il-6. Also, when corrected for work or oxygen consumptionthe trained group exhibited a blunted il-6 peak after exercise. With this information itseems as though training does likely affect skeletal muscle il-6 production. Upon53reviewing the research to date thereare a number of skeletal muscle adaptationsdue toendurance training that may reduceil-6 production during exercise.In exercise physiology it iswell established that skeletal muscleundergoesvarious adaptations with endurancetraining. These adaptationsinclude increasedglycogen stores in both type1 and 2 fibers, glycogen sparingdue to preferential fatty-acid oxidation, and preferential type1 fiber recruitment (1,35, 28, 29). All of theseadaptations may lead to a reductionin contraction-induced skeletalmuscle il-6production. It has beena consistent finding that intramuscularil-6 mRNA expressionandprotein release in response toconcentric-based exerciseare elevated with reduced preexercise intramuscular glycogencontent (41, 84). Currentlyit is hypothesized thatphosphorylation of nuclearp38 MAPK (mitogen activatedprotein kinase) activates il-6transcription factors in skeletalmuscle (14). It also appears thatintramuscular glycogenbinds upstream signaling moleculesfor p38 MAPK (MKK3, MKK6,AMPK) (14).Through this mechanism increasedintramuscular glycogen storageinhibits p38 MAPKphosphorylation resultingin reduced il-6 transcriptionand subsequent protein production.Assuming this mechanism iscorrect training-induced increasesin glycogen storage andglycogen sparing could reducecontraction-induced il-6 production.Through other findingsit also seems that training-induced preferentialtype 1 fiberrecruitment during enduranceexercise may reduce post-exerciseil-6 production. In vitrowork has demonstrated thatmammalian muscle tissue showsan increase in il-6 mRNAexpression and protein releasewhen incubated with calciuminophore ionomycinidentifjing Ca2 as astimulus for il-6 production (36). Thesignificance of this lies inthefinding that type 2 fibers showa near 20-fold greater Ca2release duringcontractioncompared to type 1 fibers. Whenfiber types were compared aftera bout of concentric54contractions biopsy analyses showedgreater iI-6 mRNA content aswell as proteinproduction in the type 2 fiberscompared to type 1 (34). Therefore,training-inducedalterations in fiber type recruitmentfavoring type 1 fibers would likelyalso reducecontraction-induced il-6 production.Another adaptation in musclethat may reduce contraction-inducedil-6 productionis enhanced antioxidant capacity.During heavy exercise oxygenconsumption in theactive muscle may increase as muchas 100-fold (79). Withthis comes a subsequentincrease in the productionof free radicals; harmfulmolecules or ions containing reactiveunpaired electrons. Thechief source of free radicalproduction during exercise isatcomplex 1 and 3 of the electron transportchain (15). Another sourceof free radicalproduction during heavy exercisemay be the xanthine oxidase pathwayactivated duringtissue ischemia and reperfusion(32). Regardless of the source, freeradical production hasbeen directly identifiedas a potent stimulus for il-6 productionin myocytes (41). Supportof this finding comes froma subsequent study that demonstrateda reduction in post-exercise il-6 concentrationswhen subjects were administeredantioxidant supplements(89). This research implicates oxidativestress as a primary stimulus for il-6production.To quench free radicals and preventtissue damage from oxidativestress, humans haveinnate antioxidant enzymeswithin skeletal muscle; primarily superoxidedismutase,glutathione peroxidase, andcatalase. Much evidence has beenreported suggesting thatskeletal muscle antioxidant enzymessuperoxide dismutase (SOD)and glutathioneperoxidase (GPX) are enhancedin response to regular endurance training(69, 38, 45,79). With this information it seemslikely that training, leading to enhancedinnateantioxidant capacity, couldalso likely reduce post-exerciseil-6 elevations.55Although it has been demonstratedby many groups that antioxidantsSOD andGPX are found in greater concentrationin skeletal muscle (69,38, 45, 79), athletestraining heavily may be at increasedrisk of oxidative damage. The reasonfor this is thatincreases in skeletal muscle oxidativeenzymes and antioxidantenzymes do not increasein parallel and in fact oxidative enzymecapacity is enhanced moreso than systemicantioxidant enzyme capacity(16, 67). Such unparalleledimprovements in oxidativeandantioxidant capacity allow the bodyto overshoot its innate abilityto quench free radicalsproduced during intense exercise.Intense endurance trainingthen may serve to beprotective against oxidativestress from ambient free radicalattack (eg. diet, cigarettes,infection) and submaximal exercisebut may be detrimental withrespect to intenseexercise.The result may be that extreme endurancetraining can leadto oxidative stressrelated illness. Oxidative stresshas been implicated asa cause in many illnesses; mostnotably cancer and heart disease.Free radicals are unstable productscapable of reactingwith lipids, proteins, and DNAin tissue. The cancer causing effectsof oxidative stressare rooted in the ability of free radicalsto alter DNA and cell replication.The role of freeradicals in cardiovascular illnessis due to low density lipoproteinoxidation whichcontributes to endothelial dysfunctionand subsequent atherosclerosis (11,3). It has alsobeen hypothesized that oxidativestress may inhibit cardiac functionacutely by causingintracellular calcium overload(40). In this study it was foundthat cardiac myocytesexposed to free radicals causedmyocyte injury resulting in excesscalcium influx andsubsequent contractile abnormalities.The results of a large scale epidemiologicalscale (71) potentially demonstratethesignificance of this oxidative stressimbalance with ultraendurancetraining. In this study56it was found that ultraenduranceathletes training heavily,defined as> 10500 kilojoulesof energy expended perweek through activity, wereat greater risk of cardiovasculardisease than any othergroup even the mostsedentary group expending0 kilojoules perweek through activity (71).Also surprisingwas that subjects in the secondmost activegroup, expending 6300-10499kilojoules perweek through activity, wereat greater riskof cardiovascular diseasethan those in less activegroups, 1680-3779 kilojoulesper weekand 3780-6299 kilojoulesper week. These twogroups, expending1680-3779 and 3780-6299 kilojoules per weekwere at the lowest riskfor cardiovasculardisease in this study.Although a causative relationshipis not possible to analyzein such a study this reportdisplays the potentialharmful effectsof excessive exercise andsubsequent oxidativestress.5.5 OvertrainingImplicationsIn the past five years il-6has received attentionas a cause of overtrainingorunderperformance syndrome.Underperformance syndrome(UPS) is defined asapersistent decrementin athletic performance capacitydespite 2 weeksof relative rest(72). UPS affects many athleteseach year in a varietyof sports with symptomsincludingfatigue, loss of appetite, irritability,and poor sleep quality(25). However, the definingsymptom and most commonreason for seeking treatmentis poor performance.Thepotential causes of UPS havebeen studied for yearsbut no theorized mechanismhas beenable to account for the onsetor symptoms. It is clearthat those affected byUPS not onlyhave impaired performancebut in severe cases also exhibitirregular hypothalamicpituitary function (5). Thechallenge for past researchershas been linking peripheraldamage and fatigue withthe central nervous system.In 2000 Lakier-Smith (43)57hypothesizedthat cytokines,producedin responseto constantmusculoskeletaltrauma,were likelycandidatesas a causeof UPS andcoined theterm “cytokinesickness”.Morerecently,Robson (72)suggesteda more specificand plausiblemechanismby whichil-6may accountfor UPS andassociatedsymptoms.As demonstratedin our studyand others(23, 60) enduranceexercise leadsto anincrease insystemicil-6. 11-6exerts itseffects throughoutthe bodyincludingthe centralnervous systemvia passagethroughthe blood brainbarrier (4).When administeredtohealthyhuman subjectsin dosagesequivalentto those foundduring prolongedenduranceexercise il-6induced significantincreasesin systemiccortisol andadrenocorticotropichormone (58).Subjects alsoreported increasedoverall fatigue,depressedmood, poorconcentration,and sleep disturbances(measuredas reductionin REM sleep)(58).Further, il-6has beenimplicatedas the chiefsourceof debilitatingfatiguein cancerpatients(42). To ourknowledgeonly one studyhas monitoredexerciseperformanceduring il-6administration(73). In thisstudy athletescompleteda 10 km runningtimetrial withor withoutil-6 injections.When injectedwith il-6, indosages designedto matchplasma levelsduring prolongedexercise,subjects experiencedmore fatigueandperformedsignificantlypoorerthan with placebo(73). Theseresults areintriguingas theeffects ofsystemic il-6on the centralnervous systemcan accountfor the majorityofUPS.The basisof the modelproposedby Robson(72) is thatexcessiverepetitiveexercise boutsand/or concomitantincreases inil-6 lead toa time-dependantsensitization(TDS). A TDSis a “progressiveand persistentamplificationof behavioural,endocrineand immunologicalresponsesto repeatedintermittentstimuli overtime”(72,7). Robsondraws similaritiesbetweenUPS andTDS as athletesoften endup in an overtrainedstate58following a period of probable repeated il-6 stimuli: infection, injury, heavy training orheavy competition (72). Also, UPS resembles TDS in that once UPS develops aworsening of symptoms is seen with subsequent exposure to training indicative ofsensitization (72). Robson indicates that the sensitization may manifest as enhanced il-6production, enhanced sensitivity to the il-6 protein or a combination of the two. There area number of mechanisms which may result in elevated systemic il-6 in the heavilytraining athlete. Most commonly discussed is underlying musculoskeletal injury duringwhich a recent or chronic injury may be causing an increase in il-6 regardless of exercise.A similar pattern may occur following a recent infection. For either reason il-6 in thesystem may be elevated leading to sensitization. As mentioned previously, low preexercise muscle glycogen content results in enhanced post-exercise il-6 values after asimilar exercise bout (14, 21). With heavy training loads including multiple workoutseach day, athletes may be unable to replenish glycogen stores between workouts againpotentially resulting in greater il-6 concentrations. In addition, although antioxidantenzyme capacity is elevated in the skeletal muscle of endurance trained athletes, theoxidative capacity of muscle increases to a greater extent with training (67, 68, 69). Theresult is that an athlete may have a reduced ability to quench free radicals after high levelendurance training; subsequently resulting in greater post-exercise il-6 elevations. Also,il-6 is found to be elevated in individuals after high stress situations (65) potentiallysimilar to those during times of heavy competition. These potential causes of elevated il-6seem likely to occur individually or in combination for the highly trained athlete.A recent study showed that repetitive endurance training with insufficientrecovery over three weeks resulted in elevated plasma il-6, worsened performance, andincreased fatigue (74). In this study triathletes had extra run training sessions added to59their current schedules to induceoverreaching; a state of training prior to UPS in whichone has continued to increase trainingintensity but performance begins to decline.Results from this study support Robson’s proposed il-6 hypothesis of UPS asresting il-6production increased with repetitive training bouts. The elevation in il-6 inresponse toexcessive training represented a TDS (74).The results of our study suggest that endurance trainedindividuals can completean exercise bout of much greater intensity than untrainedindividuals while maintainingsimilar il-6 values. This represents a training adaptation opposite to TDS in which thebody successfully adapts to an enhanced stimulus. Considering that individuals in anoverreached state develop a maladapted il-6 response to exercise training, and that thisresponse coincides with early performance decrements, regular measures of systemic il-6in heavily training athletes may serve as an early indicator of overtraining syndrome.5.6 Health ImplicationsFor years CRP has been seen as an indicator of underlying inflammation due toillness or injury. It was first linked to cardiovascular disease in 1985 whenresearchersextracted CRP from human atherosclerotic aortic intima (76).Until recently CRP hadbeen thought of as a bystander in cardiovascular disease; not a cause. Withfurtherresearch it has become clear that CRP is causative at various stagesof atherosclerosis.The following summary is not exhaustive but attempts to cover the primarymechanismsof CRP in atherosclerosis progression. First, CRP concentrationspredictive of vascularevents(>3 mg/L) directly upregulate endothelial cell adhesion molecules (6-1 1)thatassist in leukocyte-endothelial cell interaction. Once this interactionhas occurred CRPaids in leukocyte transmigration through the endotheliumby stimulating the release of60monocyte chemoattractant protein- 1(46, 8). CRP further inhibits vascular function bystimulating endothelin from endothelial cells; a potent vasoconstrictor anda furtherstimulus for CRP upregulation of cell adhesion molecules (8). In addition, CRP inhibitsthe production of endothelial nitric oxide; a vasodilator maintaining vascular toneandfunction (29, 75). Further, nitric oxide inhibition leads to endothelial cellapoptosisserving to enhance the role of CRP in atherosclerosis (75). Althoughpro-inflammatorycytokines il-6, il-i, and TNF-ct are the primary stimuli forCRP, CRP also provides apositive feedback mechanism by upregulating transcriptionfactor-kappa B (NF-KB); atranscription pathway required by most endothelialpro-inflammatory cytokine genes(47). As a result, CRP itself elevates pro-inflammatorycytokine production. Finally, CRPalso plays a role in accumulation of atherosclerotic plaque.Researchers have indicatedthat CRP encourages macrophage uptake of low densitylipoproteins further enhancingthe atherosclerosis process (8, 91).As mentioned in chapter 5.3 il-6 is the chief stimulatorof hepatic CRP production(12). Our results indicated that trained and untrained subjects had similaril-6concentrations at rest and CRP values were significantlyhigher at rest in the untrainedgroup. We also found that resting concentrations of CRP and il-6 were significantlycorrelated in the untrained but not the trained group. With this information it seemsthatthe relationship between il-6 and CRP is different in trained than untrained individuals.Possibilities for this are described in chapter 5.3. Regardlessof the mechanism for thelower concentrations of CRP pre- and post-exercise in the trained groupthis differencerepresents a reduction in cardiovascular risk in the trained group. These resultsareespecially surprising as both groups were matched for age, BMI, health status,medication, and smoking status; all factors linked to systemic inflammation.Even more61surprising was that two subjects in the untrainedgroup were considered to be in the highrisk factor range for CRP (>3 mg/L),two were considered to be in the moderateriskrange (2-3 mg/L), and the remaining8 were considered low risk(<1 mg/L). In thetrained group all subjects were considered lowcardiovascular risk with respectto CRP(<1 mg/L). Althoughour study was a cross-sectionaldesign our results indicate thatendurance training aside from age,BMI, health status, medication,and smoking mayreduce cardiovascular risk withrespect to CRP.5.7 LimitationsDue to the cross-sectional design of thisstudy we are limited with respecttoattributing differences in outcome measuresto training alone. Although thisstudyprovides a relatively simple wayto evaluate the effects of trainingon both 11-6 and CRPvalues, there are many variablesbetween individuals that cannotbe controlled for in across-sectional design. As such alongitudinal study, measuring theeffects of trainingover time, must be done toeliminate such variables.Another inherent limitationof this study came with attemptingto match groups.Although groups were matchedwell for health, age, height, weight,and BMI, we wereunable to match groups for sums of skinfolds.When recruiting both sedentaryindividualsand elite endurance cyclists, matchinggroups for sums of skinfolds was not possiblewiththe resources available. This is a concernas it has been reported that obese individualsshow elevated values of both il-6and CRP (9, 24, 47). Even though the sumsof skinfoldswere significantly greater in the untrainedgroup the values reported werestill well withinnormal values; likely not affectinginflammatory status.62The main outcome measures reported in this studywere systemic concentrationsof 11-6 and CRP. From the available literatureit is clear that the increases in systemic il-6occurring soon after endurance exercise canbe attributed to il-6 production and releasefrom the active muscle (65). This has beenfound by comparing arterio-venous 11-6differences across the active muscleto systemic il-6 concentrations (85).For our study noarterio-venous measures were recorded leavingthe possibility that the source of il-6production, although unlikely, maynot have been the active skeletal muscle.Also nostudies, including ours, have monitoredchanges in il-6 degradation compared toproduction while exercising. It has beenshown that a significant amountof il-6 is takenup in the hepatosplanchnic viscera duringexercise but there may be othertissues that doas well (20). With this in mind, itmay be that differences in post-exerciseil-6 betweenindividuals are due to il-6 uptake differencesrather than production differences or acombination of both. Again, with a simplevenous measure we cannot determinewhereil-6 was produced and removed;only the change in systemic concentrations. Similarly,the chief source of CRP is thoughtto be from hepatocytes and the main stimulus il-6butCRP is also produced in endothelial cellsin response to il-6. As is the case withdetermining the source of il-6, the locationof production of CRP and the ratio ofproduction to degradation cannot be determinedwith a systemic venous blood.The goal of this study was to determineif there was a difference in resting andpost-exercise inflammatory markersbetween aerobically trained and untrainedhealthymales. Generally the training effects that havebeen hypothesized to reduce theinflammatory response following exerciseare enhanced glycogen storage in type Imuscle fibers, resulting in increased endurance capacityfor those fibers and reducedreliance on type II fibers and enhanced innateantioxidant defenses (23, 89). With subject63selection and protocolwe tried to eliminate factors externalto training that may haveinfluenced these training effectsby excluding subjects who had takenantioxidantsupplements within sixmonths and having subjectsfast from the night beforetheexercise challenge. Withouttaking muscle biopsiesto analyze antioxidant enzymecapacity and glycogenstores we were unableto evaluate these trainingadaptations ineach group. As such wewere unable to attributeany differences inthe trained oruntrained group directly toparticular trainingadaptations.In order to remove as manyvariables as possible inthis study, subjects wereallmales aged 18-35 years.There is evidence ofa blunted mononuyclearil-6 response totypical stimuli suchas lypopolysaccharide exposure(77) which may affect bothbaselineand post-exercise il-6 concentrationsin a study such as ours.As a result, the resultsof ourstudy, including baselinesystemic inflammatoryvalues, cannot be transferredto femalesuntil a similar study is conductedin females. Similarly, thereis little research comparingthe inflammatory responseto exercise in old and youngindividuals. One studyto date(88) has evaluated the trainingeffect of resting il-6and CRP in both young (aged18-3 5)and older (aged 65-85)previously sedentary individuals.Baseline values ofboth proteinsshow similar responses betweengroups in responseto endurance exercise training;il-6remained similar and CRPdecreased with training.5.8 Future DirectionsThis study was a cross-sectionalstudy comparing two groupsdiffering only infitness. The results,in combination with previousfindings, indicate that endurancetraining does alter the post-exerciseincrease in il-6. Our resultsalso clearly demonstrate a64difference in CRP values at rest and post-exercise between trainedand untrained subjects.With respect to study design the next step is tocarry out a prospective study in whichuntrained subjects are trained in a concentric-basedactivity and challenged before andafter training with an identical work bout to directlydetermine the effect of training on anindividual. This would result in a statisticallymore powerful study that would have fewerconfounding variables than a cross-sectionaldesign.There are also a number of other variablesthat would be useful to examine in thefuture if resources were available.With our results that trained individualscan work at afar greater wattage and oxygen consumptionwith a similar inflammatory response itwould be useful to have objectiveevidence of what is different in the trainedmuscle andthe untrained muscle. For example, lowglycogen stores have been shown asa stimulusfor il-6 production in active skeletal muscle(14) and it has also been found that trainedmuscle can store more glycogen(35). In future work it would be beneficialto take amuscle biopsy and analyze glycogen contactto determine if glycogen was infactdifferent between trained and untrained.Considering oxidative stress is alsoa stimulus for skeletal muscle il-6 production (41)it would be beneficial to examine differencesin antioxidant enzyme capacity and markersof oxidative stress concurrent to il-6 valuesin a trained and untrained state after exercise.By evaluating the relationship between thetwo, the role of oxidative stress and freeradical production in the post-exerciseil-6 response could be quantified.Monitoring free radicals in tissue is challengingdue to the particularly short half-lifeof free radical species. As a result,free radical production is usually expressedby indirectindicators of lipid, protein or DNA oxidation; oxidativedamage. Most studies involvinghuman subjects have employed byproductsof lipid peroxidation such as conjugated65dienes, lipid hydrocarbons, andthiobarbituric acid-reactive substances(TBARS) asmeasures of oxidative stress.Conjugated dienes areinitial products of the peroxidationofunsaturated fatty acids, and areconsidered to be accurateand repeatable measures of lipidperoxidation measures (57,67-69, 79). Unfortunately,conjugated dienesmay be presentin dieters, resulting in mis-representedwhole-tissue oxidation.Other studies have usedexhaled hydrocarbon productsof lipid peroxide splitting,such as ethane and pentane,toindicate lipid peroxidation.Exhaled pentane isa particularly useful measuresince bothfatty acid types from whichpentane is produced arefound mostly within thecellmembrane (57, 67-69,79). The noninvasivenessof this measure is attractive;however, itshould only be used supplementaryto other oxidative stressmeasures.More recently, electronspin resonance andparamagnetic resonancespectrometryhave been used to directlymeasure superoxide radicalsin animals. These techniquesarethe most powerful as theyboth directly measure transitionstates of the free radicals(79).To date, these measures havenot been used as indicatorsof free radical productioninhumans.Another technique, usingchemiluminescenceto detect lipid peroxidation, hasbeen described by (57, 67-69,79), in which biological samplesassays are employed.Inthe sampled assay, the antioxidantcapacity is estimated by a decreaseinchemiluminescence whenan oxyradical is coupled withthe production of light. Thismethod is reported tobe rapid, sensitive, reproducible,and simple (57, 67-69,79). It alsoallows for total antioxidantcapacity measures from smallvolumes of fluid (57, 67-69,79Blood glutathione disulphide(GSSG) measures may alsobe a useful indicator ofoxidative stress. In the presenceofH20 and hydroperoxides, intracellularglutathionequickly oxidizes to GSSG,but is quickly reduced back toglutathione if the oxidative66stress is minimal. If the oxidative stressis beyond the cell’s ability to reduce GSSGtoglutathione, an accumulation ofblood GSSG may serve as an indicatorof oxidative stress(57, 67-69, 79).The most commonly employedmethod to evaluate lipid peroxidationis themeasure of TBARS, most commonly Malandialdehyde(MDA), which is created inperoxidizing systems (57, 67-69,79). During oxidation of polyunsaturatedfatty acids,MDA is generated as a secondaryproduct. Altjough simpleto measure, a variety of otherfactors such as side products ofthromboxane or prostaglandin synthesismay alsoincrease MDA levels inblood and tissues (57, 67-69,79).Measures of oxidative stressin conjunction with il-6 would aid indetermining theeffect of free radicals on post-exerciseil-6. However, it should benoted that current lipidperoxidation and oxidative assessmentmethods should be used carefullydue to lack ofaccuracy, validity, or both.It has been suggested that two or more techniquesshould beused to provide improved measuresof oxidative stress.It would also be of value to studythe effects of different exercise workloadson ii-6 production within individuals. This wouldprovide a better understandingof theinfluence of both oxygen consumptionand metabolic system use on il-6 production.Ostrowski (63) examinedrun intensity and il-6 production in marathonersduring amarathon with results indicatingthat run intensity, measuredas VO2max!run time, wassignificantly correlated with elevationsin il-6. However, no researchers toour knowledgehave designed a study to compareexercise bouts requiring differentoxygenconsumptions within the same individual.This could be done by having subjectsgothrough a protocol identicalto ours with initial aerobic fitness assessmentand then anexercise challenge except with repeated exercisebouts days apart; each bout at an67intensity correspondingto a differentpercentage of maximaloxygen consumption.Forexample, eachparticipant couldcomplete three exercisebouts in randomorder at 50, 60,and 70 percent ofmaximal oxygenconsumption. By analyzingil-6 peaks after eachbouta powerful relationshipbetween intensity,measured as percentageof maximal oxygenconsumption, andil-6 production couldbe reported.Another future directionfrom this research isin the area of overtraining.Although a longitudinalstudy is required tofully determine howan individual’sinflammatory responsewill adapt to training, ourwork established arelationship for thepost-exercise il-6 responsein trained and untrained groups.With these results it seemsthat training would atleast result in a similar post-exerciseil-6 increaseif not a bluntedincrease compared to pre-training.That said, an elevatedpost-exercise increase inil-6with training may indicatea skeletal muscle maladaptationthat could lead toovertraining. The mechanismby which il-6 may belinked to overtrainingis discussed inchapter 5.5. In addition,elevated il-6 values atrest may be indicative ofan underlyingmuscle maladaptationor unrecovered microtrauma.Regardless of theunderlying causeelevated systemic il-6leads to symptomsof and potentially fullblown underperformancesyndrome itself. Tobest study the link betweentraining, overtraining, andil-6 subjectswould take part in a trainingprogram aimed at inducingoverreaching or evenovertraining. This couldbe done in a similar fashionto a report by Robson-Ansley(74) inwhich triathletes hadintense run interval trainingbouts added to an alreadytaxingtraining schedule. Bymonitoring athletes,using an exercise challengeand bloodsampling schedule similarto ours, at regular intervalsthroughout a strenuoustrainingschedule it would bepossible to evaluate eachathlete’s training adaptationwith respectto resting and post-exerciseil-6. Performance andsymptoms of overtrainingcould then68be compared to changes in individual resting and post-exercise il-6values to determinewhether il-6 is a good indicator or predictor of overtraining.69Chapter 6: ConclusionIn summary, our results have shown that systemic il-6 concentrations are similarin trained and untrained subjects at rest and following a relatively similar exercise bout.However, when corrected for both work completed and oxygen consumed during theexercise bout, trained subjects demonstrated a blunted il-6 response comparedto trainedsubjects. These findings bring to the forefront the need fora training study to monitor theinflammatory response to an identical exercise bout inthe same individual before andafter successful training to fully elucidate the effect of training.Our findings alsoindicated significantly lower concentrations of CRP in trainedcompared to untrainedsubjects. 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Cuff Opin Lipidol.9: 47 1-474.(92) Yip, U.K., Wu, C.J., Chang, H.W., Yang, C.H.,Yeh, K.H., and Chua, S. (2004).Levels and values of serum high-sensitivityC-reactive protein within 6h after theonset of acute myocardial infarction. Chest. 126: 1417-1422.79Appendices80Appendix A - 11-6 concentrations FollowingCycle Exercise in Different FitnessGroupsE0014PIn II-6id cycling ExeitiseComparison of Fitness Status and ExerciseChallenge Response811210864Febbraioetal., (O3)Startdeetai, (O1in(ninutes)1W 15JzoAppendix B - ParticipantQuestionnaireAerobic Fitness Statusand Pos-ExerciseInflammatory Markersin 18 to 35 year-oldmalesParticipant QuestionnaireParticipant name:__________________________1.) Do you currentlyparticipate in anyform of exercisetraining? Yes/No(Ifnoskip to question 5)2.) If you respondedYes to question 1,approximately howmany hours per weekdoyou train? and What typeof training do you participatein? (include a breakdownoftime spent at each typeoftraining)3.) Do you currentlyparticipate in cycleracing or training,Yes/No? If so, atwhatlevel do you participateat? (ie. CAT], 2,3, other?)4.) How long haveyou participated in thislevel of cycle training?(weeks/months/years?)5.) Have yourecently undergone anaerobic fitness assessment(VO2max test)? Ifso,do you recall the resultsof your assessment?6.) To the bestof your knowledge doall of the following inclusioncriteria apply toyou?82Yes/No- Male aged 18-35 years old?Yes/No- Current and expected Vancouver Resident for duration of study?Yes/No- No antioxidant/Vitamin supplementation < 6 weeks prior to study?Yes/No- No use of steroidal anti-inflammatory medication<6 weeks prior to study?Yes/No- Healthy,o No history of chronic disease (CVD, endocrine, hepatic, inflammatory,etc...)?Yes/Noo No traumatic or chronic injuries present 6 months prior to study?Yes/Noo No febrile illness/infection 6 weeks prior to beginning of study?Yes/No- Non-smoker?Yes/No- No current or planned caloric intake restriction?Yes/No- Non shift worker?Yes/No83Appendix C — EthicalApproval Certificate84UI3CThe University ofBritish ColumbiaOffice of Research Services,Clinical Research Ethics Board — Room210, 828 West10thAvenue, Vancouver, BC V5Z 1L8Certificate ofFull Board ApprovalClinical Research Ethics BoardOfficial NotificationPRINCIPAL INVESTIGATORDEPARTMENT NUMERTaunton, J.E.Family PracticeC064J088INSTITUTION(S) WHERE RESEARCH WILL BE CARRIEDOUTProvidence Health Care, UBCCampusCO-INVESTIGATORS:Maclntyre, Donna, RehabilitationSciences; Rhodes, Edward, Human KineticsSPONSORING AGENCIESBritish Columbia Sports MedicineResearch Foundation28 March 2006 - ‘1 -,Protocol version 1 dated 02 February 2006; SubjectConsentForm version 1 dated 02 February 2006;AdvertisementCERTIFICATION:In respect of clinical trials:1. The membership of this ResearchEthics Board complies with the membership requirementsfor Research EthicsBoards defined in Division 5 of the Foodand Drug Regulations.2. The Research Ethics Board carries outits functions in a manner consistent with GoodClinical Practices.3. This Research Ethics Board has reviewedand approved the clinical trial protocol and informed consentform for the trialwhich is to be conducted by the qualified investigatornamed above at the specified clinical trial site.This approval and theviews of this Research Ethics Board have beendocumented in writing.The documentation includedfor the above-named project hasbeen reviewed by the UBC CREB, and theresearch study, as presented in the documentation,was found to be acceptable on ethical groundsforresearch involving human subjects andwas approved by the UBC CREB.The CREB approval for this studyexpires one year from the approval date.Approval ofthe Clinical ResearchEthics Board by one ofDr. Gail Beliward, ChairDr. James McCormack, AssociateChairTITLEAerobic Fitness Status and Post-ExerciseInflammatory Markers in 1 8 to 35Year-Old MalesAPPIRtDVAI flATI TERM !VFARS1 flIIMFNTS IMflI I IflFf IN THIS APPRflVA85

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