8/1/2012. Introduction. Oxidative Stress. Oxidative Stress. Antioxidants. Antioxidant Mechanisms H2 O. Oxidative Stress is a hot topic:

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1 Introduction Oxidtive Stress is hot topic: ANTIOXIDANT RESEARCH & ITS APPLICATION FOR USE IN EXERCISING HORSES Crey A. Willims, Ph.D. Associte Professor Google = just under 8 million results Add horse = out 535, Try ntioxidnt = 37.8 million Tody I will highlight studies from my lortory long with other pertinent studies of ntioxidnts in exercising horses Equine Science Center Rective Oxygen Species (ROS): Oxidtive Stress Hydrogen peroxide (H 2 O 2 ), Singlet oxygen ( 1 O 2 ) Free rdicls (OH ) Oxidtive stress: ROS > cpcity of the ntioxidnt system Homeosttic environment compromised In excess, cn degrde proteins, DNA nd PUFAs Some oxidtive stress is good thing O 2 OH H 2 O 2 O e H + 2 H 2 O + CO 2 Oxidtive Stress Oxidtive stress is ssocited with: Exercise Aging Humn Diseses Dietes Alzheimer's Neurodegenertive Diseses Ctrct development Defense mechnism ginst excess ROS Vitmins - E, C, A Enzymes GPx, SOD Others GSH, LA, CoQ 1 Antioxidnt Mechnisms Antioxidnts OH H 2 O C OH H2 O C C Mesuring oxidtive stress Cn t mesure ROS Intermedites or end products LPO, TBARS/MDA Extrcellulr E E E F2-isoprostnes Redox rtios Intrcellulr E E 1

2 Introduction Plsm TBARS fter exercise Oxidtive Stress & Antioxidnt Supplementtion in the Endurnce Horse Study 1: During competition (8 km), compre Vitmin E supplementtion with Vitmin E + C Study 2: During competition (8 km), wht hppens in eliminted horses Study 3: Tredmill simulted endurnce competition (55 km), compre vitmin E with Lipoic Acid More in horses with low vitmin E (McMenimn nd Hintz, 1992) A single out of sumximl exercise does not ffect plsm vitmin E Intense conditioning my require supplementtion (Sicilino et l., 1996) Vit. C my spre vit. E y reducing rdicls nd restoring its ctivity Polo ponies t high exercise intensities hd -tocopherol nd scorte (Hoffmn et l., 21) Rnge of Vitmin E Intke Vitmin E intke: IU/d Vit. E intke, IU/d Study 1: 46 horse nd rider pirs Mterils nd Methods Pired nd rndomly divided into 2 groups E -- 5, IU vitmin E/d EC -- 5, IU vitmin E/d + 7 g vitmin C/d 3 weeks prior to rce 34 horses completed the rce With E intke: % completion rte re finishers only er CK (r = -.23; P =.2) er AST (r = -.22; P =.3) er -Tocopherol (r =.21; P =.5) Willims et l., 25 TEMP, HR, CK, AST, LPO, GSH nd GPx significnt effect of distnce (P <.1) Willims et l., 24 Ascorte Cretine Kinse Plsm ASC dj, ug/ml P <.5 Vit E Vit EC Distnce, km Willims et l., 24 CK, IU/L Vit E Vit EC c P <.5 c Distnce, km Willims et l., 24 2

3 Similr Studies These findings contrst with previous result from our lortory plsm ASC during 8 km rce Conclusions nd Implictions Horses in endurnce exercise re undergoing oxidtive stress Determined y LPO nd muscle enzymes (Hrgreves et l., 22) Plsm ASC lso during rce nd through the seson in sled dogs (Donoghue et l., 1993; Hinchcliff et l., 2) Seson ws prevented y vitmin C supplementtion t 1 g/mcl ME Compred to ~.3 g/mcl ME in the present study Antioxidnt supplementtion necessry? Adding vitmin C to E helps mintin ASC sttus Wht out horses tht do not finish the rce? Mterils & Methods Study 2: 4 horse nd rider pirs No tretments Nutritionl survey nd lood smples After ride groups were mde Finishers (n = 24) nd Non-Finishers (n = 16) log CK K, IU/L Finishers P <.5 Non-finishers Distnce, km Willims et l., 25 Willims et l., 25 Mterils & Methods Study 3: 12 Tredmill trined Arins (n=4x3) Vitmin E (5, IU/d), 3 wks Lipoic Acid (1 mg/kg/d or ~ 5 mg/kg.75 ), 2 wks CON (sl diet) 1 horse tested per dy Text ~ 5 hrs Structure: 8-crons Dithiolne ring 5-cron til Discovered in 1951 Conditionlly essentil nutrient t Antioxidnt properties: Ft solule like vitmin E Wter solule like vitmin C Neurologicl symptoms (humns) Dietes nd Alzheimer's In rts ws found to: Lipoic Acid LA GSH nd TBARS in vrious tissues (Khnn et l., 1999) 3

4 Antioxidnt Interction Mterils & Methods 7 GSH 2 Cys Cystine Vit E Vit E + DHLA HRm mx, % Averge HRmx: Loop 1 = 49.8 % Loop 2 = 51.6 % Loop 3 = 55.7 % DHAA 1 GSSG Vit C LA Distnce, km dpted from Sen nd Pcker, 2 Willims et l., E LA CON P < E LA CON P <.1 Plsm CK, IU/L Plsm -T TOC, ug/ml st 1 2 2nd 3 4 3rd h 3 h 18 h Willims et l., st 1 2 2nd 3 4 3rd h 3 h 18 h Willims et l., 24 Apoptos sis, % st E LA CON Hot dys (# 9,1,11) 1 2 2nd 3 4 3rd Smple h 3 h Willims et l., h Antioxidnt Supplementtion in the Intensely Exercised Horse Study 1: Vitmin E in vrious doses with intervl exercise Study 2: Superoxide Dismutse with repeted sprints Study 3: Nutrceuticl extrcts with grded exercise test 4

5 Mterils & Methods Study 1: CON significntly higher CK (P =.5) thn MOD & HI 12 unfit SB mres, hnd dosed dily for 4 wks, 5, or 1, IU/d dl- -tocopheryl cette 2 18 CON MOD HI c GXT to determine HRmx, performed pre-study 16 Intervl Exercise Test fter ech TRT 4 wk wshout 4 min wlk 8 min trot (6%) 2 min 4 min cnter trot (1%) 2 min cnter 5 min wlk CK, IU/L TROT CANTER WALK 1 HR 24 HR Smples Willims nd Crlucci, 26 TOC incresed with exercise; MOD nd HI groups were significntly higher thn CON t 24 hr (P <.1) TOC, ug/ml CON MOD HI BC ws significntly higher (P <.5) in MOD vs. HI groups BC, ug/ml CON LOW HI TROT CANTER WALK 1 HR 24 HR Smples Willims nd Crlucci, 26. TROT CANTER WALK 1 HR 24 HR Smples Willims nd Crlucci, 26 Mterils nd Methods Soccer Plyers Study 2: 12 helthy Stndrdred mres (6x6) SOD supplementtion for 6 wks Cross over design Repeted Sprints Exercise Test (RSET) Time 2 min 4 min 2 min 2 min 2 min 2 min 2 min 2 min 2 min 1 min TM Speed Wlk 4 m/s 7 m/s Wlk 8 m/s Wlk 9 m/s Wlk 1 m/s Wlk Mesured lood nd SF 8-Isoprostnes (pg/ml) = significnt Group x Tril interction (p<.1) EXP CON 21 d of SOD supplementtion Pre-seson trining period Rn lctte threshold test Inflmmtion, ntioxidnts nd oxidtive stress mesures Pre Post Arent et l., 21 5

6 EXP T1 EXP T2 CON T1 CON T2 Footll Plyers 8-Isoprostnes (pg/ml) = significnt Group x Tril interction (p<.1) 7 wk of SOD supplementtion Pre-seson trining period Wingte Aeroic Tests (ike) No effect of SOD supplementtion on cytokine trnscript 4 8 IL-1B IFN Ng RMT min min 2 h 24 h Dt presented s men fold chnge in trget gene expression Effect of exercise for IFNγ, IL-1, IL-1β 2 Pre post 6post Arent et l., 29 No chnges were detected for IL-6 nd TNFα Resluts No effect of supplementtion on erythrocyte SOD ctivity 35 3 A U/mg H Serum CS-846 epitope ng/ml CS-846, ng/ml SOD ctivity, 1 5 PEAK 3 min 2 h 4 h 24 h Incresed SOD ctivity with exercise my e compensting for incresed oxidtive stress 36 h Synovil fluid CS-846 epitope ng/ml P <.5 etween joints P <.1 etween joints CS-846, ng/ml min 2 h 24 h c B 3 min 2 h 24 h Crpus = white r; Hock = solid r Mterils nd Methods Study 3: 9 Stndrdred mres used in 2 experiments GXTs 1 week prt cross-over tretments Extrcts dministered 1 hr efore exercise EXP 1: Control, Ginger, Crnerry EXP 2: Control, Blck Te, Ornge Peel Extrcts developed t Rutgers Food Science Dept. No tretment differences Retinol ws in ornge peel supplemented horses thn lck te or control Determine if there would e long term effect of supplementtion (Smrsh et l., 21) Susequent studies looked t inflmmtory cytokines nd performnce Blck te nd Crnerry inflmmtory mrkers Ornge peel nd Ginger recovery time However Ginger incresed CK» Streltsov et l., 26; Liurt et l., 21 6

7 Study 1: Stndrdreds Mterils & Methods Other Exercise Studies 8 Young (ge 12 ± 2 yrs) 5 Old (ge 22 2 yrs) Exercise progrm = equine exerciser for 8 wks Sumximl work intensity etween 65-7 % HR mx Study 1: Aging nd trining on oxidtive stress, ntioxidnts nd poptosis Study 2 & 3: Oxidtive stress during 3- Dy event competition Grded exercise test (GXT) performed efore nd fter trining period Blood smples nd HR were tken Rest, 6 m/s, ftigue, 5 & 6 min post Smple (P =.3) nd Trining (P =.2) effect e, um Glutthione 16 Pre TR Post TR Age effect with OLD > YOUNG (P =.2) 7 Young Old 6 s, % Apoptosi Rest 6 m/s mx 5 post 1 hr post Rest mx 1 hr post Willims et l., 28 Willims et l., 28 Introduction Introduction Study 2 & 3: Two yers (26, 27) CCI/CCI Interntionl Competition 3-Dy Eventing: Dressge grce Cross-Country endurnce Stdium Jumping gility Over 5 % rider prticiption First report of inflmmtion nd ntioxidnt sttus of competitive event horses Other studies hve found vrying results in terms of HR, temp., plsm LAC, CK nd AST responses (White et l., 1995, Murry et l., 26) Environmentl fctors nd nutritionl sttus could ply n importnt role 7

8 Totl GSH nd GPx hd n effect of division CCI ending higher thn the CCI (P <.5) Willims nd Burk, 212 Plsm NO nd TNF over time (P <.1) Both negtively correlted with ody weight nd dietry Vitmin E intke Willims nd Burk, 21 Horses were supplemented with n verge of 4 supplements On verge they were over-supplemented 2-4 x the recommended levels of mjor minerls (C, P, K, Mg) Burk nd Willims, 28 Tke Home Messge Overll these recent exercise studies hve shown tht oxidtive stress ws oserved Endurnce, intense, nd tredmill exercise The extent ws dependent on vrious fctors: Amient temperture, horses fitness, nd work intensity Antioxidnts eneficil to horses? Still deted There hve een positive reports in horses! We cn possily provide etter helth nd welfre to equine thletes y providing ntioxidnts efore they re sked to perform under intense conditions Future Studies: Thnk you! How young horses t the strt of rce trining re effected y oxidtive stress Looking systemiclly nd loclly (skeletl muscle) Before nd fter 8 weeks of trining on n equine exerciser Before nd fter n cute out of intense exercise on tredmill LOOKING for yerlings to prticipte in the study! If interested plese contct me tody or t cwillims@esop.rutgers.edu or Acknowledgements: Rutgers fculty, stff, nd students! Grd Students: Emily Lmprecht, Ph.D. Dnielle Smrsh, Ph.D. cndidte 8