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Effects of Vitamin E Supplementation on Oxidative Stress Parameters Measured in Exercising Horses

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PAGE 1

EFFECTS OF VITAMIN E SUPPLEM ENTATION ON OXIDATIVE STRESS PARAMETERS MEASURED IN EXERCISING HORSES By KYLEE JOHNSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Kylee Johnson

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This document is dedicated to my fam ily for their support and encouragement.

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iv ACKNOWLEDGMENTS I would like to acknowledge the help a nd guidance I received from my committee members throughout my graduate program: Dr Ed Ott, Dr. Lee McDowell, Dr. Richard Hill, and Dr. Alfred Merrit. They have constantly provided advice and direction and resources to complete my work on this proj ect. I would also like to thank Dr. Sally Johnson, who was added to my committee, for allowing me to work in her lab and for her enthusiasm for my project. Without her guidan ce, I would not have been able to finish my laboratory analyses or the writing of this dissertation. She offered new and innovative ways to examine my project. I would also like to thank Ms. Karen Sco tt and Mrs. Jan Kivipelto for help in running lab assays and analyzing statistical data. They were constantly available to help with problems, both big and small, and without them this project would never have been completed. Finally, I would like to thank all of the students who worked on this project, both graduates and undergraduates. This was a ve ry labor intensive project which required immense amounts of physical effort. The st udents who helped on this project were absolutely essential and deserve special recognition.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES...........................................................................................................ix LIST OF COMMONLY USED ABBREVIATIONS..........................................................x ABSTRACT.......................................................................................................................xi CHAPTER 1 REVIEW OF THE LITERATURE..............................................................................1 Exercise Metabolism....................................................................................................1 Oxidative Stress............................................................................................................3 Exercise and Oxidative Stress......................................................................................9 The Antioxidant System.............................................................................................12 Vitamin E....................................................................................................................14 Vitamin E and Oxidative Stress..........................................................................16 Vitamin E and Exercise.......................................................................................17 Muscle Structure and Composition............................................................................20 Protein Carbonyls................................................................................................25 Muscle Soreness and Oxidative Stress................................................................29 Oxidative Stress and Genetic Pathways in Muscles............................................34 MAP Kinases.......................................................................................................36 2 INTRODUCTION......................................................................................................39 3 MATERIALS AND METHODS...............................................................................41 Animals.......................................................................................................................41 Diets and Adaptation..................................................................................................41 Washout and Cross-Over Period................................................................................43 Training Period...........................................................................................................43 Tissue Sampling..........................................................................................................44 TBARS Analysis........................................................................................................45 TEAC Analysis...........................................................................................................45

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vi Glutathione (reduced, oxidi zed, and total) Analysis..................................................46 Vitamin E Assay.........................................................................................................47 Muscle Analyses.........................................................................................................47 Myofibril Preparation..........................................................................................47 Dystrophin Immunochemistry and Fiber Morphometrics...................................48 Statistical Analysis......................................................................................................49 4 RESULTS AND DISCUSSION.................................................................................52 Vitamin E....................................................................................................................52 Lipid Peroxidation......................................................................................................54 Antioxidant Capacity..................................................................................................56 Carbonylation and Ubiquitination..............................................................................59 Muscle Morphometrics...............................................................................................62 5 IMPLICATIONS........................................................................................................75 APPENDIX A SUPPLEMENTAL PROTOCOLS.............................................................................77 B ADDITIONAL TBARS DATA.................................................................................82 C ADDITIONAL TEAC DATA....................................................................................87 D PERIOD EFFECTS FOR GLUTATHIONE DATA AND MUSCLE MORPHOMETRICS..................................................................................................90 E UBIQUITIN DATA AND MA P KINASE PATHWAYS.........................................93 Procedures for MAP Kinase Blots..............................................................................95 F HEART RATE DATA...............................................................................................97 LITERATURE CITED......................................................................................................99 BIOGRAPHICAL SKETCH...........................................................................................104

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vii LIST OF TABLES Table page 3.1 Nutrient composition of hay sampled at selected times during the study................50 3.2 Nutrient composition of grain sampled at selected times during study...................50 4.1 Effect of vitamin E supplementation on resting plasma vitamin E concentration of horses in a training program................................................................................64 4.2 Effect of vitamin E supplementation on plasma TBARS concentration in horses following strenuous exercise....................................................................................64 4.3 Plasma TEAC concentrations in contro l and vitamin E supplemented exercised horses........................................................................................................................65 4.4 Plasma TEAC concentrations befo re and after strenuous exercise..........................65 4.5 Effect of vitamin E supplementation on glutathione levels (mg/dl/%RBC) in horses undergoing moderate exerci se (Means and SEMs reported)........................66 4.6 Effect of vitamin E supplementation on glutathione measures taken at time points during a standard exercise test (Means and SEMs reported)........................67 4.7 Effect of vitamin E supplementa tion on time to fatigue in a SET...........................67 4.8 The effect of vitamin E supplementation on parameters of mu scle fiber sizes........68 4.9 Effects of exercise on mu scle cross morphometrics................................................68 A.1 Weights of horses at the start of each time period...................................................77 A.2 Daily grain intake of horses on each phase of trial..................................................78 A.3 Treadmill training schedule for period 1..................................................................79 A.4 Treadmill training schedule for period 2..................................................................80 A.5 Plasma proteins values of training phase and SET used for adjusting for plasma volume (Values reported SEM)............................................................................81

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viii B.1 Effect of vitamin E supplementati on on plasma thiobarbituric acid reactive substances (TBARS) concentr ation in exercising horses.........................................83 B.2 TBARS values for control and vi tamin E supplemented horses over time..............84 B.3 Thiobarbituric acid reactive substance (TBARS) concentration for pre exercise time points in control and vitamin E supplemented horses by period.....................85 B.4 Thiobarbituric acid reactive substance (TBARS) concentration for post exercise time points in control and vitamin E supplemented horses by period.....................86 C.1 TEAC values pre and post exercise for control and vitamin E supplemented horses over time.......................................................................................................88 C.2 TEAC values by period for pre and post exercise....................................................89 D.1 Glutathione data by period for pre ex ercise samples. SEMs for oxidized, reduced and total glutathione <0.26 mg/dl/%RBC..................................................91 D.2 Glutathione data by period for post exercise samples. SEMs for oxidized, reduced and total glutathione <0.26 mg/dl/%RBC..................................................92 D.3 Effect of period on muscle morphometrics..............................................................92 F.1 Heart rate data during a standard exercise test.........................................................98

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ix LIST OF FIGURES Figure page 3.1 Summary of experi mental timetable........................................................................51 3.2 Representative image of -dystrophin immunostaining..........................................51 4.1 Plasma TBARS concentration is not effected by vitamin E supplementation. ......69 4.2 Plasma TBARS values after exerci se are not affected by vitamin E supplementation.......................................................................................................70 4.3 TEAC values for pre exercise samples show no difference between control and vitamin E supplemented horses but increase with improved fitness........................71 4.4 Purified gluteus medius myofib rillar proteins are carbonylated..............................72 4.5 Dystrophin immunostained gluteus medius muscle fiber cryosections obtained from horses before and after SET exercise..............................................................73 4.6 Distribution of muscle fibers sizes from horses before training/supplementation, and before and after a standard exercise test............................................................74 E.1 Ubiquitin expression in cytoso lic component of muscle tissue...............................94 E.2 Summary of stress pathways in cy tosolic component of muscle tissue...................96

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x LIST OF COMMONLY USED ABBREVIATIONS ADP Adenosine diphosphate ATP Adenosine triphosphate CAIII Carbonic anhydrase III CK Creatine kinase COX Cyclooxygenase CSA Cross sectional area DI Deionized DM Dry matter 8-OHdG 8-hydroxydeoxyguanosine ECL Enhanced chemilumenescence FADH flavin adenine dinucleotide FAHP Fatty acid hydroperoxides GSH Reduced glutathione GSSG Glutathione disulf ide (oxidized glutathione) H2O2 Hydrogen peroxide IU International unit MAPK Mitogen-activa ted protein kinase MDA Malondialdehyde MyHC Myosin heavy chain NADH Nicotinamide adenine dinucleotide PBS Phosphate buffered saline PC Phosphocreatine PG(I,F2 ,E2) Prostaglandin (I,F2 ,E2) PLA2 Phospholipase A2 PMSF Phenylmethylsulfonyl fluoride PUFA Polyunsaturated fatty acid RBC Red blood cell ROS Reactive oxygen species SEM Standard error of means SET Standard exercise test TBA Thiobarbituric acid TBARS Thiobarbituric acid reactive substances TCA Tricarboxylic acid cycle TEAC Trolox equivalent antioxidant capacity TTP Tocopherol transfer protein

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xi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF VITAMIN E SUPPLEM ENTATION ON OXIDATIVE STRESS PARAMETERS MEASURED IN EXERCISING HORSES By Kylee Johnson December 2006 Chair: E. A. Ott Major Department: Animal Sciences Exercise places an increased demand on th e bodys systems, both to provide fuel for working musculature and to neutralize a nd dispose of toxic bu ildup. Byproducts of demanding performance are reactiv e free radicals. Consumption of vitamin E, a dietary antioxidant, may be a plausible way to redu ce free radical damage. The present study examined the effects of excess vitamin E on the presence of oxidation products in blood and tissue in exercising horses. Eight thor oughbred horses were used in a cross-over design with one group being fed vitamin E at the level recommended for horses in moderate to intense work (80 IU/kg DM ) (NRC 1989), and the second group being fed the control diet plus 3000 IU/day d, l -tocopherol acetate. The horses underwent an eight week training program and a final sta ndard exercise test. Blood samples were collected at specific points be fore and after exercise duri ng the training period. At the end of the eight week traini ng period, a standard exercise test (SET) was performed during which the horses ran on a 6 incline to exhaustion. Blood and muscle were

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xii collected before and after performing the S ET. Neither plasma vitamin E nor TBARS concentration were influenced by supplemental vitamin E. Blood TEAC values increased (P<0.05) following five weeks of trai ning in both groups, indicating improved antioxidant capacity as horses became fitter. Vitamin E supplementation did not alter plasma reduced, oxidized, or total glut athione levels. However, elevated reduced:oxidized glutathione ratio s were present prior to traini ng-level exercise and at all time points during the SET in the treated an imals (P<0.05). Myof ibril carbonylation, a product of free radical damage, was lowe r in vitamin E supplemented horses post exercise (P<0.05). There was, however, no difference between treatment groups in time to fatigue during a strenuous bout of exer cise. Vitamin E did not influence area, diameter, or number of nuclei per fiber in cross-sections of gluteus medius muscle. Therefore, this study demonstrates that traini ng influences antioxidant capacity of horses, and vitamin E influences some measures of oxidative stress in exercising horses, particularly following a st renuous bout of exercise.

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1 CHAPTER 1 REVIEW OF THE LITERATURE Exercise Metabolism Energy for cellular function, both at rest and of work, comes from the body reserves of carbohydrate, fat, and proteins. The primary energy-carrying molecule in the body is adenosine triphosphate (ATP). Th is compound contains a high energy phosphate bond that, when cleaved by ATPase, liberates energy which can be utilized by the cells for work. ATP is derived from the breakdown of foodstuffs in the body. However, cells store only limited amounts of ATP. Excess en ergy consumed from foodstuffs is stored by the body in the carbohydrate form as glycogen (muscle and liver tissue), or in adipose tissue as triglycerides. When the cell is faced with increased energy demands, such as muscle cell contraction, metabolic pathways are required to produce ATP rapidly. The formation of ATP by phosphocreatine breakdown is the fi rst pathway mobilized. Glucose and glycogen are metabolized by glycolysis to produce ATP. Neither the phosphocreatine (PC) pathway nor the glycolytic pathways re quire oxygen and are classified as anaerobic pathways. The PC pathway involves the dona tion of a phosphate group and its associated bond energy from phosphocreatine to ADP (a denosine diphosphate) to form ATP. Creatine kinase catalyzes this reaction. When ATP stores are liberated at the initiation of exercise, the molecule is refo rmed quickly by the PC reaction. However, PC stores in muscle are limited and the pathway only provi des energy for the initia tion of exercise and for high intensity exercise lasting less than five seconds. Phosphocreatine is reformed

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2 during exercise recovery and requires ATP. Thus, the PC system provides energy rapidly but is depleted quickly. ATP also is produced fairly rapidly by gl ycolysis. In this pathway, glucose or glycogen is degraded to pyruvi c acid with the release of ATP. Pyruvic acid, as it accumulates, is converted to lactic acid wh ich accumulates in the muscle. In this pathway, bond energy from glucose is used to rejoin inorganic phosphate to ADP. The process occurs in the sa rcoplasm of muscle cells. Under oxygen sufficient conditions, aerobic production of ATP occurs. This mitochondrial process utilizes the Krebs cycle (TCA cycle) and the electron transport chain. The TCA cycle involves the comple te removal of hydrogen atoms (oxidation) from its substrate using the hydrogen car riers NAD and FAD. The hydrogen atom possesses an unpaired electron a nd therefore contains potential energy. These electrons create an electric gradient which is used to generate ATP. Ox ygen is not a direct component of the TCA cycle but is the fi nal hydrogen acceptor for the electron transport chain (2H2 + O2 2H2O). Oxidative phosphorylation al lows for aerobic production of ATP by utilizing potential energy in NAD H and FADH (hydrogen carriers) to rephosphorylate ADP to ATP. There is no direct interaction between the hydrogen carriers and oxygen. Through -oxidation, fatty acids form ed by the breakdown of body triglyceride stores are metabolized to acetyl Co A, which enters the TCA cycle. Protein is not a major fuel source during exercise (2-15% of total fuel contri bution), but different amino acids metabolites are converted to gluc ose, pyruvic acid, acetyl CoA, or to TCA cycle intermediates when necessary. This b ecomes a more important source of energy as exercise duration increases and glucose is limiting.

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3 The electron transport chain is ultimate ly responsible for the generation of ATP from aerobic metabolism. Potential ener gy from reduced electron carriers (NADH, FADH) is used to convert ADP to ATP. This is done by passing electrons from NADH and FADH down a series of electron carriers (cytochromes) releasing enough energy at three points in the chain to rephosphorylate ADP to ATP. Oxygen acts as the final electron acceptor and combines with hydrogen to form wate r. Therefore, oxidative phosphorylation (through the elect ron transport chain) is not possible if oxygen is not present; energy must be generated solely from anaerobic pathways. Intensity and duration of exercise are th e primary determinants of which pathway contributes the most to energy producti on. Most athletic endeavors require a combination of both aerobic and anaerobic me tabolism to meet energy demands. Short term, high intensity exercise relies more h eavily on anaerobic metabolism (PC system or glycolysis). Extremely short term, high in tensity muscular contraction is powered primarily by the PC system. As the length of the exercise increases, the body relies more on glycolysis. Exercise lasting longer th an 45 seconds uses a combination of both anaerobic and aerobic energy systems. High in tensity exercise lasting for approximately 60 seconds is powered by approximately 70 % anaerobic and 30% aer obic metabolism. High intensity exercise lasti ng two minutes relies equall y on anaerobic and aerobic energy production. Long term submaximal ex ercise (>10 minutes) relies primarily on aerobic metabolism. Oxidative Stress Oxidative stress refers to a condition in wh ich there is an elev ation of steady state free radicals due to an imbalance in free radi cal generation versus cellular antioxidant defenses (Tiidus and Houston, 1995). The term oxidative stress coincides with a large

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4 increase in oxygen utilization (Rokitzki et al., 1994). The increased oxygen consumption allows for ATP production through oxidative phosphorylation to occur at a more rapid rate. However, this can lead to the incomp lete reduction of O2 as the electron transport chain becomes overloaded leading to the production of free radicals. A free radical is a molecule containing one or more unpaired electrons in the outer orbit which can exist independently (Clarkson and Thom pson, 2000). These substances are highly reactive with other molecules in an effort to gain an electron to stabilize the unpaired electron. Free radicals always exist in th e body but are increased as oxygen consumption is increased and leakage of the electron transport chain o ccurs. Normally, 2-5% of mitochondrial oxygen consumption results in generation of the oxygen free radical (superoxide) due to electron leakage at intermed iary steps in the elec tron transport chain. Subsequent reactions produce hydrogen per oxide and hydroxyl ra dicals (Tiidus and Houston, 1995). Molecular oxygen is a diradical containi ng two unpaired electrons with parallel spin configurations. Electrons must have opposite spins to occupy the same orbital. Therefore, electrons added to molecular oxygen must be transferred one at a time during its reduction. This results in several highly reactive intermediates (Clarkson and Thompson, 2000). The complete reduction of o xygen to water requires four steps and the generation of several free radicals and hydroge n peroxide. Hydrogen peroxide is not a free radical but is a reactive oxygen species because it can generate the highly reactive hydroxyl free radical through interactions with transition metals (Clarkson and Thompson, 2000).

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5 The complete reduction of molecula r oxygen involves the following ROS producing steps: O2 + electron O2 (superoxide radical) O2 + H2O HO2* + OH (hydroperoxyl radical) HO2* + electron + H H2O2 (hydrogen peroxide) H2O2 + electron *OH + OH (hydroxyl radical) Each intermediate is highly reactive because each has an unstable electron configuration which allows for interactions with electrons from other molecules. Ubiquinone and NADH oxida se are two sites of production for the superoxide radical along the mitochondrial electron transport chai n (Moslen, 1994). The superoxide radical is unlike other oxygen-derived intermediates b ecause it can lead to the formation of additional reactive species. It can be protonated to form a hydroperoxyl radical (HO2 ) which is a much stronger radical than superoxi de. Superoxide also can act as a Bronsted base in aqueous solutions to shift acid-b ase balance to form hydroperoxyl radical, thereby, forming hydrogen peroxide in acid environments. Superoxide dismutase, an enzyme in the body, catalyzes dismutation of s uperoxide radical at ne utral or acidic pH (Clarkson and Thompson, 2000). Hydrogen per oxide is nonionized and in a low charged state and therefore can diffuse through hydrophobic membranes thereby allowing it to leak from mitochondrion. It can form a hydroxyl radical by reduction, by interaction with superoxide, or by interaction with redu ced forms of metal ions such as copper and iron, which can act as electron donors. Hydroge n peroxide has the ability to remove or add hydrogen molecules to unsaturated hydroge n bonds of organic lipids. However, it has a very short half life (1 X 10-9 sec @ 37C), and therefore has limited diffusion

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6 capability (Clarkson and Thompson, 2000). On the other hand, lipid peroxyl radicals can be formed by lipid interaction with ROS and ha ve half lives of seconds. Therefore, they can travel in the bloods tream to distant locations to pr opagate oxidative damage (Moslen, 1994, Sen, 1995). ROS that are formed from the reduction of O2 can attract an electron from other molecules and result in another free radical. This creates a chain that contributes to lipid peroxi dation, DNA damage, and protein degradation (Clarkson and Thompson, 2000). Damage by ROS occurs in practically ev ery component of the cell, including peroxidation of proteins, nucleic acids, and lipids. However, lipid peroxidation levels are a primary common measure of tissue damage b ecause of their ease of measurement. One of the main measurable end products of lipid peroxidation is malondialdehyde (MDA) which is often measured by the thiobarbituric acid reactive substances assay (TBARS). Lipid peroxidation is thought to cause ex ercise induced myopat hies and hemolysis (Chiaradia et al., 1998). There are three described steps of lipid peroxidation. First is initiation: conjugated dienes are formed through the removal of a hydrogen atom from a backbone methylene group (-CH2) of a polyunsaturated fatty acid. This allows for the interaction of molecular oxyge n with carbon centered free ra dicals to form a peroxyl radical which is highly reactive. This per oxyl radical will attack other compounds in the body to form hydroperoxides and a new carbon centered radical, thus causing chain propagation, the second step of lipid peroxidation. Polyuns aturated fatty acids (i.e., biological membranes) are particularly vul nerable because of multiple unsaturation points. In the following di agrams of lipid peroxidation adapted from Surai (2002),

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7 Moslen (1994), and Sen (1995), LH represents part of a stable lipid molecule and denotes a radical. LH L* (car bon centered radical) L* + O2 LOO* (lipid peroxyl radical) LOO* + LH LOOH + L* (lipid hydroperoxi de and new carbon centered radical) This chain will continue to propagate until the final step of lipid peroxidation: termination (Clarkson and Thompson, 2000). Termination requires another molecule to donate or remove an electron to stabilize the exis ting free radical thus becoming a radical itself. The chain is terminated when the mo lecule becoming the radical is less reactive and is able to be handled and disposed of by the body. Peroxidative reactions are essential to th e body; they regulate important functions such as defense against microorganisms, cell signaling, vascular co ntrol, cell generation and degradation, and control of cellular homeostasis. However, when excess production of biologically active oxidative compounds overwhelms the capacity of the bodys cellular antioxidant defense mechanisms, these products may cause cell and organ damage by disrupting normal physiology whic h can begin and/or accelerate disease processes. Disease itself can initiate formation of these products (Basu, 2003). Peroxidation products can be formed both enzymatically and non-enzymatically. Nonenzymatic free radical induced lipid per oxidation was described in the previous paragraph. It requires a compound such as a polyunsaturated fatty acid (PUFA) to react with an oxidant inducer to form a free radical intermediate which then reacts with oxygen to form the peroxyl radical (LOO ). These radicals also may attract membrane proteins.

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8 Lipid peroxidation can occur also through enzymatic processes. Phospholipase A2 is activated by oxygen radicals or hydrogen peroxide and re sults in the release of arachidonic acid through hydrolysis of esterified arachidonic acid from the sn-2 position of phospholipids in cell membranes (Basu, 2003). This generally occurs as the first step to repair peroxidized phospholipids. Li pid peroxidation activates phospholipase A2, and alterations in the molecular conformation of oxidized phospho lipids are thought to facilitate access of the phos pholipase to the cleavage site (Moslen, 1994). The result is that arachidonic acid is rele ased and acts as a substrat e of the cyclooxygenase (COX2) enzyme or the lipoxygenase enzyme, both of which produce inflammatory products and free radical intermediates (Moslen, 1994). The enzymatic oxidation of arachidonic acid may cause COX to be deactivated due to attack on the enzyme by release of ROS during enzymatic reduction of hydroperoxides (Basu, 2003). Besides damaging lipid components of membranes and potentially increasing production of inflammatory products, free radi cals can cause significa nt damage to DNA and proteins. It is thought that oxidative damage is responsible for approximately 10,000 DNA base modifications per cell per day. Oxidation, methylation, deamination and depurination are endogenous processes that lead to significant DNA damage with oxidation representing the most significant (Surai, 2002). Oxidative damage to DNA causes base damage, sugar lesions, singl e strand DNA breaks, and DNA-nucleoprotein crosslinks. For example, the 8-hydr oxydeoxyguanosine (8-OHdG), a guanine base modification, induces G-C to T-A transver sion during DNA replication. This type of cytotoxic effect could lead to concomitant ch ange in DNA genotype (Radak et al., 1999). Proteins also are susceptible to oxidative da mage. Oxidation of proteins can lead to

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9 formation of reversible disulfide bridges or in more severe cases, the formation of chemically modified derivatives such as Schi ffs bases (Surai, 2002). If protein receptors on the cell membrane are att acked and modified, functionality of the membrane is compromised. Exercise and Oxidative Stress During oxidative metabolism, much of the oxygen consumed is bound to hydrogen during oxidative phosphorylation, thus form ing water (Clarkson and Thompson, 2000). As oxidative phosphorylation (ATP production) increases, so does formation of free radicals. Extreme aerobic stress causes an increased energy requirement, making increased oxygen utilization necessary (Rok itzki et al., 1994). Exercise induced oxidative stress refers to a condition in which oxygen free radicals are released in muscles through overload of th e mitochondrial oxidative phosphorylation system or from inflammatory cells. Activated oxygen species pl ay a role in exercise induced injury to muscle membrane components and in a ssociated alteration of lysozomal and mitochondrial enzyme activity (i.e., breakdown of membranes and leakage of enzymes) (Jacob and Burri, 1996). Therefore, leakage of mitochondrial and lysozomal enzymes is sometimes used as a measure of oxidative stre ss. Lipid peroxidation products are used most frequently as a measure of oxidative st ress. Lipid peroxidation probably occurs primarily at the sight of energy formation, the working musculature. Increased oxygen utilization, increased lipid mobilization for en ergy, and hypoxia all lead to increased lipid peroxidation (Rokitzki et al., 1994). Bi ological membranes are composed of polyunsaturated fatty acids and are vulnerab le to oxidative damage due to multiple unsaturation points. Oxidative damage to me mbranes can result in a change in membrane fluidity, compromised integrity, and inact ivation of membrane bound receptors and

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10 enzymes causing loss of membrane function (Clarkson and Thompson, 2000). Oxidative damage to proteins leads to physical cha nges such as fragmentation and aggregation which makes them more susceptible to degradation (Clarkson and Thompson, 2000). Exercise can generate free radicals also by (1) increases in ep inephrine and other catecholamines that can produce oxygen ra dicals when they are metabolically inactivated, (2) producti on of lactic acid which can convert weakly damaging free radical superoxide to strongly damagi ng radical hydroxyl, and (3) inflammatory responses to secondary muscle damage incurred with overexertion (Clarkson and Thompson, 2000). Other sources of free radicals include pros tanoid metabolism, xanthine oxidase, NAD(P)H oxidase, and radicals released by macrophages (Urso and Clarkson, 2003). The Jenkins theory states that free ra dical mediated mechanism involving lipid peroxidation and loss of membrane integrity co uld possibly be the cause of delayed onset muscle soreness (Rokitzki et al., 1994). Matsuki and colleagues (1991) proposed that in exercised horses phospholipid hydroperoxides are the primary products of pe roxidation and disrupt cellular homeostasis by activating phospholipase A. This causes an accumulation of lysophospholipids in muscle membranes. End products of peroxi dation, particularly malondialdehyde (MDA), are associated with polymerization and aggregation of membrane compounds and reduction of cellular function. Exercised thoroughbreds dem onstrated peroxidation of phosphatidylethanolamine (PE) was increased before exercise and post exercise as compared to resting controls, and peroxidi zed PE showed an increasing tendency from pre-exercise to 10 minutes post exercise to 24 hours post exercise. Total MDA (both free and protein bound) increased in all 4 horse s at 10 minutes and 24 hours post-exercise

PAGE 23

11 (Matsuki et al., 1991). The authors concluded that musc ular PE was particularly susceptible to oxidative stre ss due to exercise, that pero xidized phospholipids were able to activate phospholipase, and that total MDA in creased largely because of an increase in protein bound MDA, supporting previous reports that MDA is highly reactive to proteins (Matsuki et al., 1991). Chiardia et al. (1998) report ed MDA content increased in response to exercise. In the experiment, ten stallions were trained over a period of three months and then underwent a series of physical exercise bouts of increasing intensity. The horses were sampled before exercise, immediately after limbering up, after the exercise bouts, and 18 hours after exercise. MDA increased dramatical ly after strenuous exercise as compared to MDA levels during a limbering up period, sugge sting that lipid peroxidation is related to intensity of exercise. Also, the elimin ation of lipid peroxidation products was shown to be a slow process (Chiaradia et al., 1998). Exhaustive exercise also results in a marked increase in TBARS (a lipid peroxidation product thought to be indicative of cell memb rane breakdown) in both the muscle and liver with a decreasing tendenc y within 24 hours in non-endurance trained rats (Brady, 1979). Intracellula r glutathione rapidly oxidizes to glutathione disulfide (GSSG) in the presence of hydrogen peroxide and hydroperoxides but rapidly reduces back to glutathione if oxidative stress is not severe. Therefore, GSSG is used as a marker of oxidative stress. Sastre and associat es reported that trained men exercised to exhaustion on a treadmill had increased amounts of GSSG (oxidized glutathione) immediately after exercise but values retu rned to resting stat e well within one hour (1992).

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12 The Antioxidant System The antioxidant system consists of both enzymatic and non-enzymatic components which work coordinately to detoxify the body of ROS. Most antioxidants also act as prooxidants under certain condi tions (Jacob and Burri, 1996). The enzymatic component of the bodys antioxidant system is comprise d of the enzymes glutathione peroxidase, superoxide dismutase, and catalase which each detoxify ROS. Glutathione peroxidase is located in mitochondria and cytosol. Increases in oxyge n consumption activate the enzyme to remove hydrogen peroxide and organic hyperperoxides from the cell. Reduced glutathione (GSH) is used by gl utathione peroxidase to detoxify hydrogen peroxide with oxidized glut athione (GSSG) being formed as a result. Glutathione reductase is necessary to convert GSSG to GSH which also contribu tes to detoxification of hydrogen peroxide (Urso a nd Clarkson, 2003). Regeneration of glutathione ultimately derives from glucose. The glutathione system is under enzymatic regulation via glutathione reductase an d glutathione peroxidase and it re generates other antioxidants in vivo (Jacob and Burri, 1996). The reaction 2 H2O2 2 H2O + O2 is catalyzed by the enzyme catalase which is widely distributed in cells but concentrated in mitochondrion and peroxisomes. Glutathione peroxidase is an enzyme with greater affinity for hydrogen peroxide than catalase. Ther efore, it is thought that catala se increases when oxidative stress overwhelms the glutathione peroxi dase system (Urso and Clarkson, 2003). Superoxide dismutase acts on s uperoxide radicals. It catal yzes the addition of hydrogen ions to convert two superoxide an ions into hydrogen peroxide and O2 (2O2 + 2H+ H2O2 + O2) (Surai, 2002). The non-enzymatic components of the an tioxidant system involve dietary constituents which act as less specific antio xidants. These include both fat soluble and

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13 water soluble antioxidants. Vitamins A a nd E, carotenoids, and ubiquinones comprise the fat soluble group, while ascorbic acid (v itamin C) and uric acid act as aqueous antioxidants. Vitamins E and C are the most commonly studied and supplemented antioxidants. Vitamin C acts primarily as an aqueous phase peroxyl and radical scavenger. It is concentrated in tissue a nd fluids with highest potential for radical generation (i.e., eye, brain, liver, lung, heart, semen, leukocytes). The oxidized form is dehydroascorbic acid. It is converted back to its reduced form by glutathione, NADPH, or both. It can regenerate the reduced form of vitamin E (recycling). It also inhibits the formation of carcinogenic nitrosamines, espe cially in the stomach (Jacob and Burri, 1996). Vitamin C is very high in neutrophils and is necessary for immune function (Urso and Clarkson, 2003). Vitamin C is synthesized by the horse and therefore not generally supplemented. Carotenes are colored pigments found in yellow and green vegetables. Some, such as -carotene, are precursors to vitamin A. Carotenes currently do not have an RDA value. eta-carotenes and lycopenes are chai n breaking antioxidants and singlet oxygen quenchers in vitro (Jacob and Burri, 199 6). Two studies (one using a metabolic unit and one free living study) showed that caro tene depletion was associated with up to a five fold increase in plasma TBARS. Also, hexanal (a compound associated with oxidative damage to low density lipoprotein s) showed a 19% decrease after carotene repletion (Jacob and Burri, 1996). At the cellular level estrogen may serve as an antioxidant as well. It has a similar structure to vitamin E. Vitamin E has a diterp enoid side chain that a llows for its insertion into membranes. It has a trimethylhydr oquinone head portion which quenches free radicals and is responsible fo r its antioxidant activity. Estradiol has two hydroxyl groups

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14 potentially capable of arre sting lipid peroxidation duri ng exercise (Clarkson and Thompson, 2000). Vitamin E Vitamin E refers to eight compounds (t ocopherols and tocotrienols). Each compound consists of a chromanol head a nd isoprenoid side chain (saturated in tocopherols and unsaturated in tocotrienols). There are , and forms of each which differ in the number and position of met hyl groups on the aromatic ring. Alpha tocopherol is the most biologically activ e form in vivo. Tocopherols exist in enantiomeric forms, designated d and l. Thei r biological activity can be expressed as international units (IU). One IU is eq uivalent to the activity of 1 mg d, l -tocopherol acetate (Surai, 2002), a form of vitamin E often supplemented due to increased stability provided by the acetate mol ecule. Humans preferentially absorb and transport tocopherol above all other forms (Tiidus and Houston, 1995). In nature, only plants can synthesize vitamin E (Surai, 2002). In animal tissue, most vitamin E is locat ed in the phospholipid membrane bilayers. The chromanol head is localized near th e hydrophilic outer region, and the isoprenoid side chain is associated with the hydr ophobic lipid inner regions. The membrane composition of vitamin E ranges from 1:1000 (vitamin E:lipid) in red blood cells (RBC) to 1:2000 in mitochondrial membranes to <1: 3000 in other tissues (Tiidus and Houston, 1995). The vitamin E content of individual membranes may be based on membrane infrastructure and not easily influenced by ex cessive vitamin E dietary intake. The level of vitamin E in the plasma is limited by th e ability of tocopherol binding protein to incorporate vitamin E into VLDL (very lo w density lipoproteins) (Tiidus and Houston, 1995). It has been shown that -tocopherol concentrations in normal subjects can only be

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15 increased three to four fold by supplementa tion (Blatt et al., 2001). Humans given 100 IU -tocopherol/kg body weight (for an average of 5 to 7 gram s total) showed increased plasma values of -tocopherol approximately 4 fold above baseline at 6 hours which decreased to 3 fold above baseline at 24 hours. Possible reasons for the inability to raise plasma levels higher include: (1) decreasing efficiency of intestinal absorption (2) saturation of tocopherol transfer protein (TTP) which limits hepatic -tocopherol metabolism and (3) redistribution of plasma -tocopherol into tissue (particularly adipose tissue) (Blatt et al., 2001). After change s in vitamin E intake, it was shown that tocopherol plasma levels reach equilibrium within 30 days. However, -tocopherol content of adipose tissue takes approximately two years to equilibrate and is released very slowly from adipocytes during experime ntal deficiency (Bla tt et al., 2001). The availability of adipose tissue vitamin E stores to the animal is unclear. Machlin et al. (1979) found that the rate of -tocopherol loss from adipose tissue was negligible in guinea pigs fed a vitamin E deficient diet, even during a four day fast. The concentration of -tocopherol in muscle, heart and liver are highly correlated to plasma values. Plasma and tissue tocopherol concentrati ons remain highly stable, suggesting that they may be tightly regulated. Alpha tocopherol can be bound by -tocopherol transfer protein, tocopherol associated protein a nd tocopherol binding protein, which may possibly serve as regulatory proteins. Blatt et al. (2001) compared distribution of vitamin E from the blood into other tissue compartmen ts to perfusion of fat soluble drugs. Tissues were subdivided into rapidly perfused central compartments (heart, lungs, brain, kidney, liver), slowly perfused pe ripheral compartments (other organs, muscle, skin), and very slowly perfused compartm ents (adipose tissue). Vita min E concentrations in rats

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16 reach equilibrium more quickly in blood and tissue which are rapidly perfused and have lower lipid content and greater tocopherol regulatory protein (TRP) function. However, this rate of equilibrium is not representative of the tissues final concentration due to tissue redistribution. Redistribut ion to adipose tissue is thou ght to be significant since approximately 90% of the bodys total vitami n E is in adipose tissue. The final equilibrium concentration of vitamin E in tissues probably depends on TRP functions, tissue lipid content, vitamin E uptake and efflux, oxidati ve stress, vitamin E metabolism and interactions between vitamin E and other antioxidants (Blatt et al., 2001). Vitamin E and Oxidative Stress It is possible that oxidative stress may affect the vitamin E binding proteins. Hepatic tocopherol transfer protein (TTP) and its mRNA are increased in diabetic rats, and plasma -tocopherol is increased in diabetic humans and rats Increased TTP levels were detected in brains from humans with a vitamin E deficiency or diseases associated with cerebral oxidative stress such as Alzheimers diseas e and Downs syndrome. Thus, upregulation of cerebral TTP during oxidati ve stress may occur. TRPs might be regulated by oxidative stress and/or the vitamin E levels of its target tissue (Blatt et al., 2001). X-ray structure determination of -tocopherol suggests that the chromanol head maintains a pair of electrons on the ring oxygen almost perpendicular to the plane of the ring. This stabilizes the formation of the vi tamin E radical that is created by quenching free radicals (Tiidus and Hous ton, 1995). Vitamin E directly scavenges most free radicals including superoxide, hydroxyl ra dical, and lipid peroxides by using the hydroxyl groups on the chroman head to either donate a proton or ac cept an electron. The resulting vitamin E radical will ultimately either react with itself or with another

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17 peroxyl radical to form non-reactive de generation byproducts (T iidus and Houston, 1995). Another important function of vitamin E is that it is a structural membrane stabilizer because it increases membra ne microviscosity and decreases passive permeability to low molecular weight substa nces (Tiidus and Houston, 1995). It was proposed that the phytyl tail of -tocopherol associates with the hydrocarbon part of the lipid bilayer. The phytyl tail positions th e chroman ring system towards the membrane interface allowing for the phytyl chain a nd the arachidonyl chains of membrane phospholipids to interact. This facilitates close packing of polyunsaturated fatty acids (PUFAs) which stabilizes the membrane and protects it from phospholipase attack. Only the RRR-tocopherol form has the appropriate c onfiguration to interact with the membrane phospholipids. The phenolic group of -tocopherol is loca ted near the polar moiety of the lipid, and in creasing concentrations of -tocopherol broaden the temperature range of the gel to liquid-crysta lline phase transition (Surai, 2002). Alphatocopherol also inhibits phospholipase A2 (PLA2) activity towards lamellar fluid membranes, thereby, protecting the membranes from attack. Alpha tocopherol decreases both the initial rate of the lipase and also the extent of hydrolysis. It is a non-competitive inhibition and is thought to be due to an effect of -tocopherol on the membrane, not on the enzyme (Surai, 2002). Vitamin E and Exercise In vitamin E deficient animals, exercise increases susceptibility to free radical damage and results in premature exhaustion, greater fragility of lysosomal membranes, and marked depression of muscle mitochondr ial control (Meydani et al., 1992). It appears that muscle or tissue vitamin E concentrations do not increase as a result of training in animals or humans. Endurance tr aining significantly in creases the number of

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18 muscle mitochondria, and therefore it is a lik ely effect that vitamin E concentration in mitochondrial membranes decreases as a resu lt of endurance training. However, most studies suggest that oxidative st ress is decreased as a result of endurance training (i.e. due to increased enzymatic antioxidant capacities). Therefore, alterations in muscle vitamin E concentration may not be cr itical to the bodys ability to adapt to oxidative stress during endurance training (T iidus and Houston, 1995). It was demonstrated that endurance performance of animals with an -tocopherol deficiency was 40% lower than normally fed animals, and oxygen radical concentration increase was 2 to 3 times higher (Rokitzki et al., 1994). Packer et al. suggested that there may be an interorgan transport of vitamin E during exercise (1990). It may involve the liver and adipose tissue as exporters and the muscle and heart (areas of increased oxidative stress) as importers. However there is little evidence to support this. Some studies have reported signifi cant elevations in plasma vitamin E post-exercise in humans suggesting possible interorgan transport. However, these studies did not account fo r exercise-induced haemoconcentration. A study using nine younger men (22-29 yrs) and twelve older men (55-74 yrs) showed increased blood levels of vitamin E after exer cise. They consumed either 800 IU vitamin E or placebo for 48 days. Subsequently, they engaged in a bout of eccentric exercise by running downhill on a treadmill at 75% maximum heart rate. After 48 days, plasma concentrations of -tocopherol increased (56% in young, 70% in older) in the supplemented group and -tocopherol decreased (99% in young, 60% in older) in the placebo group. In the placebo group, both age groups had increased urinary TBA that was significant at 12 days post exercise. In the vitamin E group, there was no change in

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19 TBA in 24 hour urine samples. Muscle le vels of linoleic and arachidonic acid were higher in supplemented men post exercise as compared to placebo (Tiidus and Houston, 1995). Variable results on the efficacy of vitamin E to reduce peroxidative stress induced by exercise exist for horses, humans, dogs, a nd rats. Variation in these tests, which measure primarily lipid peroxidation products, is likely due to differe nces in duration and intensity of exercise and fitness level of the subjects. The concentration of MDA in plasma of animals that are sufficient in vitamin E seem to vary according to intensity of exercise and fitness level of subjects. Extrem ely fit subjects have no increase and in fact sometimes a decrease in MDA when exerci sed at submaximal levels suggesting an enhanced antioxidant system in fit, traine d animals (Dekkers et al., 1996). However, when comparing vitamin E sufficient to vitamin E deficient rats, tocopherol supplementation resulted in decreased TBAR S (lipid peroxidation measurement) and hydroperoxide levels in the plasma after exer cise. Therefore, supplementation of vitamin E to vitamin E deficient animals reduces tissue peroxidation regardless of training status. Vitamin E supplementation may affect pero xidation levels in subjects performing repeated bouts of intense or exhaustive exercise. Exhaus tive exercise at submaximal workloads in rats was found to increase free radical production (determined by electroparamagnetic resonance si gnals) both in muscle hom ogenates and in portions of intact muscles. The increase in free radi cal production was associated with decreased mitochondrial respiratory cont rol, loss of sarcoplasmic and endoplasmic reticulum, and increased levels of lipid peroxidation (Singh, 1992). Humans given 600 mg dltocopherol three times daily for two weeks ha d a decrease in pentane production during

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20 graded exercise up to 75% of VO2 max. Humans given 300 mg d-tocopherol acetate daily for four weeks exhibited lower exer cise induced increases in plasma lipid peroxidation products after vitamin E suppl ementation versus before supplementation (Witt et al., 1992). Jenkins et al. (1993) show ed that MDA increased significantly after exhaustive exercise at 70% VO2 max in both trained and untrained rats (60% and 63% respectively). Lovlin et al. (1987) di d a study using human subjects on a bicycle ergometer and showed that increases in pe roxidation products may depend on intensity of exercise with exhaustive exercise causi ng a more significant increase. Vitamin E possibly counters the increase. Muscle Structure and Composition Skeletal muscle is striated because of a transverse banding pattern observed microscopically. It is a vol untary muscle. Nerve fibers and blood vessels enter and exit the muscle along the connective ti ssue that covers it. A muscle cell is the structural unit of the skeletal muscle tissue and is also know n as a muscle fiber or myofiber. Muscle volume is composed 75 to 92 % of myofibers. The rest is comprised of connective tissues, blood vessels, nerve fibers, and extr acellular fluid. The muscle fibers of mammals and birds are long, multinucleated, unbra nched cells that taper slightly at both ends. Fibers may be several centimeters l ong but do not generally extend the length of the entire muscle. They can be 10 to 100 mi crometers in diameter. They are surrounded by a membrane called the sarcolemma which is composed of protein a nd lipid and is very elastic to allow for distorti on during contraction. The cyt oplasm of muscle fibers is called the sarcoplasm and is composed 75 to 80% of water. It also consists of glycogen granules, ribosomes, protei ns, nonprotein nitrogenous com pounds and other inorganic materials. The number of nuclei per muscle fiber is not constant Nuclei are more

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21 concentrated and irregularly distributed at tendinous attachments and motor end plate units (raised area caused by structures presen t at the myoneural junction). In mammals, myofiber nuclei are located at the periphery of the fiber, just beneath the sarcolemma. Myofibrils are long thin r od-like structures within the myofibers. They are surrounded in sarcoplasm and extend the entire length of the muscle fiber. There are two types of myofilaments within the myofibrils. Thick filaments are ali gned parallel to each other and are in exact alignment across the enti re myofibril. Thin filaments are also in exact alignment across the myofibril and are parallel to each other and to the thick filaments. Thick and thin filaments overlap in certain regions along their longitudinal axes and are aligned in bands which cause the striated look of the myofibril. There are areas of different density within the bands of the muscle. The A band of the muscle is much denser than the I band but both are bi sected by thin, dense lines. The I band is bisected by a band called the Z disk. A un it of the myofibril spanning two adjacent Z disks is called the sarcomere. The sarcomer e encompasses an A band and the two half I bands located on each side of the A band. The sarcomere is the repeating structural unit of the myofibril. This is where muscle cont raction and relaxation o ccur. Thick filaments of the myofibril compose the A band of the sarcomere and are referred to as myosin filaments since the predominant protein in their structure is myosin. Actin is the predominant protein in the thin filament a nd they are therefore referred to as actin filaments. Actin filaments terminate at the Z disk and extremely thin filaments, known as Z filaments, comprise the Z disk and connect with actin filaments on either side of the disk. Each actin filament conn ects to four Z filaments. E ach of those four Z filaments then connects with an actin filament in the ad jacent sarcomere. Myofibrils are composed

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22 of more than 20 different proteins. Six of these account for more than 90% of the total myofibrillar protein and are in order of decreasing abundance: myosin, actin, titin, tropomyosin, troponin, and nebulin. They are classified by function as contractile, regulatory, or cytoskeletal. Actin and myosin are the major contractile proteins, while tropomyosin and troponin are regulatory. Re gulatory proteins re gulate actin-myosin interactions during contraction. Titin and ne bulin are cytoskeletal proteins and are the template/scaffold for the alignment of myofilaments during myofibril and sarcomere formation to form the Z disk. Actin is a globular protein. It becomes fibrous in nature when monomers of the globular protein (G-actin) polym erize to form fibrous actin (F-actin). The G-actin monomers are linked together in strands, and two strands of F-actin are spirally coiled around each other to form a supe r helix which is characteris tic of the actin filament. Myosin constitutes approximately 45% of myofib rillar protein. It is elongated in a rod shape and has a thickened end portion called the head region and a long thin portion referred to as the rod or tail region which forms the backbone of the thick filament. These two fractions are known as light chain and heavy chain meromyosin. The center of the A band contains only the rod portion of the myosin molecule. The heads of the myosin contain the functionally active sites of the thick filament which form cross bridges with actin filaments during muscle cont raction. Each myosin head attaches to a G-actin molecule of the actin filament. Skeletal muscle has mitochondria which are abundant at the periphery of the fiber near the poles of nuclei and are also abundant at motor end plates. Mitochondria are located betw een the myofibrils, adjacent to Z disks, I bands, or A-I band junctions. Lysosomes are vesicles located in the sarcoplasm that

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23 contain enzymes which together are capable of digesting the cell and its contents. These enzymes include a group of proteolytic enzymes known as cathepsins. A muscle itself is composed of several individual fibers grouped together into bundles called fasciculi. The number of fibers per muscle is not constant. The outer cell membrane of an individual muscle fiber is called the sarcolemma and is surrounded by a delicate connective tissue covering called th e endomysium which is composed of collagen fibers. Twenty to forty muscle fi bers form a group called a primary bundle. Different numbers of primary bundles are group ed together to form secondary bundles. Primary and secondary bundles are both su rrounded by a sheath of collagen connective tissue called the perimysium. Secondary bundl es group together to form the muscle which is surrounded by the connective sheath calle d the epimysium. Each muscle has at least one artery and one vein for circulat ion. Blood vessels and nerve fibers are associated with the epimysium and enter through the perimysium. Branches of each supply individual muscle fibers and are supported by the endomysium. Blood vessels cover a large portion of the muscles surface and allow for exchange of nutrients and waste products of metabolism. There are four isoforms of the myosin hea vy chain protein that have been identified in rats and some other mammals: types I, IIA, IIX(C), and IIB. Each fiber can be classified according to the predominant type of myosin isoform it contains. Muscle fibers classified as red muscle fibe rs contain primarily types I a nd IIA. White muscle fibers contain primarily types IIX(C) a nd IIB. Type I fibers are slower contracting as compared to type II fibers. Red fibers are red due to a higher myoglobin cont ent. Myoglobin stores oxygen, and therefore these fibers have a high proportion of enzymes involved in

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24 oxidative metabolism and lower levels of glycol ytic enzymes. White fibers have a high content of glycolytic enzymes and a low leve l of oxidative enzymes. Therefore, red fibers have a higher number and a greater si ze of mitochondria as compared to white fibers as well as greater capi llary density. Red fibers have a greater lipid content (source of fuel) and a lower glycogen content than wh ite fibers. White fibers primarily produce energy from glycolytic metabolism and can opera te either aerobically or anaerobically. They have a more extensively developed sa rcoplasmic reticulum and T-tubule system and have a more rapid contraction speed. Sarcoplasmic proteins include myoglobin and enzymes associated with glycolysis, the tricarboxylic acid cycle, and the electron transport chain. Their Z discs are more narrow White fibers contract rapidly in short bursts and fatigue quickly. This is called a phasic mode of action. Red fibers have a tonic mode of action: they contract more slowly but for a longer period of time. They fatigue less easily if given a constant supply of oxygen. In equine muscle, three major fiber types ha ve been identified (I, IIA, and IIB), and a minor type C. Type IIX has not been se parated from IIA and IIB by the histochemical m-ATPase staining typically used. A study by Serrano et al. (1996) used electrophoresis to separate fiber isoform at different depths of gluteu s medius and gluteus profundus muscles of five sedentary horses. They found that two of the MyHC isoforms in the gluteus medius muscle comigrated with type I and IIa MyHC isoforms of rat diaphragms (control). A third isoform had a migration more similar to th at of type IIX than to IIB MyHC of the rat diaphragm. Only types I a nd IIA were detected in the gluteus profundus muscle. The bands representing type I MyHC increased in thickness as depth of sampling increased (8cm>6cm>4cm>2cm). The band similar to IIX MyHC had a reverse

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25 pattern with the thickest bands being at the shallowest dept hs. There were no variations in the thickness of the IIa band. Type I fi bers reacted with an ti-slow MyHC antibody S58, while type IIA and IIB fibers reacted with the anti-fast MyHC antibody MY-32. Protein Carbonyls Reactive oxygen species (ROS) are produced nor mally in skeletal muscle fibers in low amounts where they function during muscle contractility (Barreir o et al., 2005). At elevated concentrations, ROS are neutrali zed by the intracellular antioxidant defense system. Accumulation of ROS disrupts mu scle cellular functi ons including action potential conduction, excitatio n-contraction coupling, co ntractile proteins, and mitochondrial respiration (Ba rreiro et al., 2005). Carbonyl s result from oxidation of arginine, lysine, threonine, or proline amino acids. Carbonylation of amino acids can occur in different ways. Hydroxyl radical s are highly reactive and thought to be generated in vivo by catalytic action of transition metals su ch as iron and copper which will bind to specific sites of proteins and m odify nearby amino acid residues (Goto et al., 1999). The amount of total ir on but not copper in the kidne y was increased by age, as was the levels of carbonylated proteins in the kidney (Goto et al., 1999). This suggests that iron could be responsible for the genera tion of oxidatively damage d proteins in this tissue. However, the distribution of non-he me iron did not match that of carbonylated proteins suggesting that carbonylation may occur by other mechanisms (Goto et al., 1999). Vitellogenins are a family of proteins stored in the yolk of developing oocytes. They also are found in abundance in the ca rbonylated form in aged wild nematodes (Caenorhabditis elegans). The biological signi ficance of this is not clear. Vitellogenin can bind metals such as zinc, cadmium, and ir on and therefore may have a biological role in body fluid later in life as a protection ag ainst oxidative damage to other cellular

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26 components by acting as a sink for metals which catalyze reactions forming active oxygen species. In a similar manner, albumi n, an abundant extracellular protein, may act as an antioxidant by binding iron leading to in hibition of lipid peroxi dation (Goto et al., 1999). Carbonyl groups also result from glyca tion/glycoxidation of ly sine, cysteines, or histidine amino acids with or unsaturated aldehydes form ed during the peroxidation of polyunsaturated fatty acids (Barreiro et al., 2005). Wh en fatty acid residues of phospholipids in cell membranes are peroxidi zed, cell membranes lose integrity and potentially harmful aldehydes and alkanes are formed (Radak et al., 1999). Oxidation of proteins causes them to lose their function a nd become targets for proteolytic degradation (Radak et al., 1999). Carbonylation of several cytosolic proteins including -tubulin, actin, and creatine kinase in brain samples of patients with Alzheimers disease occurs (Barreiro et al., 2005). Goto et al. (1999) suggested that -actin and myosin heavy chain are carbonylated in skeletal mu scle. Proteins of quadricep muscles of rats trained for 4 weeks at 4000 m altitude oxygen pressure ex hibited a significantly higher extent of carbonylation than untrained rats or rats trained at sea level (Radak et al., 1999). Proteins which showed a marked increase in signal in tensity by Western blot were actins, judged by their molecular weight and abundance. Acti ns and myosin heavy chains in the cells of human arteries and veins also are highly car bonylated, indicating that contractile proteins are susceptible to oxidation (Goto et al., 1999). However, while protein oxidation did increase due to exercise, lipid peroxida tion measured by TBARS and amount of lipid peroxides did not differ between animals exposed to sea level and high-altitude conditions. This suggests that protein oxidation occurs inde pendently of lipid oxidation. Radak et al. (2002) stated that oxidized prot eins accumulated at a much higher rate (5-

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27 10% of total cellular proteins) than lipid or DNA (<0.1% at steady state level). Goto et al. (1999) also found a 40% increase of protein car bonyls in the lung following exhaustive running of rats. Septic shock causes significant deteriorat ion of ventilatory and limb muscles to generate force and sustain workloads. Evid ence suggests that incr eased levels of ROS and nitric oxide (NO) are responsible for se psis-induced muscle dysfunction, as indicated by improvement of muscle contractility in se ptic animals treated with antioxidants and inhibitors of NO synthases (B arreiro et al., 2005). Followi ng induction of sepsis, weakly carbonylated proteins in the range of 50 a nd 29 kD in the control diaphragms were reported. After one hour of LPS injec tion, carbonyl group signal intensity rose significantly with total carbonyl OD exceedi ng 200% of that detected in control diaphragm. A more pronounced increase occurred at 12 hours post LPS injection, reaching around 300% of that detected in th e control diaphragms. The increased total carbonylation resulted from introduction of ne w carbonyls on existing proteins and also the appearance of new carbonylated protein ba nds with molecular ma sses greater than 50 kD and less than 29 kD. Carbonyl groups were detected inside diaphragmatic muscle fibers close to the sarcolemma in both the control and septic animals. Positive carbonyl immunostaining also was detected in larg e blood vessels supplying muscle fibers. Fifteen different carbonylated proteins with varying intens ities were detected in the cytosolic fraction of septic rat diaphragms. Three glycolytic enzymes were carbonylated with aldolase being strongl y carbonylated and enolase 3 and glyceraldehydes 3phosphate dehydrogenase being weakly carbony lated. Creatine kinase and carbonic anhydrase III were strongly carbonylated in the cytosolic fraction of the septic rat

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28 diaphragms. Carbonic anhydrase III (CA III) is a member of zinc metallo-enzymes that catalyze the reversible hydration of carbon diox ide. Glutathione interacts with CA III by forming a disulfide link with two of the five cysteine residues of CA III in a process called S-glutathionylation, sugges ting that CA III may have a ro le in antioxidant defenses against ROS formation in skeletal muscle fibers. In the m yofibrillar-mitochondrial fraction of the septic rat diaphragms, th e most strongly carbonylated protein was -actin. Oxidative modifications of actin involve m odification of at least seven methionine residues which is associated with extreme di sruption of actin filaments, inhibition of polymerization, and impaired in teraction with the myosin pr otein. Ubiquinol-cytochrome c reductase (complex III of the electron transport chain in the mitochondria) was the second most strongly carbonylated protein sp ot. Mitochondrial creatine kinase was carbonylated. Creatine kinases are enzymes that catalyze the reversib le transfer of a phosphoryl group from ATP to creatine to produce ADP and phosphocreatine and are localized both in the cytosol and mitochondria They are critical for energy metabolism of skeletal and cardiac muscle cells. Cr eatine kinase and aldolase activities are negatively correlated with their respec tive carbonylation level suggesting that carbonylation negatively effects enzyme activity. In summar y, this study showed that protein carbonylation involves several key enzy mes of glycolysis (enolase, aldolase, and GADPH), ATP production (complex III of th e mitochondrial respiratory chain and creatine kinases), one m yofibrillar protein ( -actin), and a regulator of CO2 hydration (carbonic anhydrase III). Only enolase a nd carbonic anhydrase III showed significant rise in carbonylation one hour after LPS injection. Carbonyl ation of the other proteins

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29 rose significantly 6 hours af ter LPS injection. Diaphrag matic contractile dysfunction occurred 12 hours after LPS inj ection (Barreiro et al., 2005). Muscle Soreness and Oxidative Stress Delayed onset muscle soreness (DOMS) that appears between 24 to 48 hours post exercise may be due to an acute inflammato ry response (MacIntyre et al., 1995). Acute phase response refers to host defense res ponses and metabolic r eactions that occur during infection. These are observed after ex ercise and contribute to the breakdown and clearance of damaged tissue after exercise. Inappropriate release of these defense mechanisms can damage host tissues and is thought to be the ba sis of noninfectious inflammatory diseases (Cannon et al., 1990). Ex ercise induces reactions that are similar to acute phase response such as the influx of neutrophils and macrophages into muscle tissue as well as activation of cytokine s following muscle damage (Sacheck and Blumberg, 2001). Eccentric exercise is par ticularly damaging because the muscle is forced to lengthen as it develops tension. Delayed muscle soreness and infiltration occur following eccentric exercise (Cannon et al., 1990). Neutrophils are released from the bone marro w and have a half lif e in circulation of approximately 10 hours. Epinephrine, increas ed blood flow, and inflammatory mediators promote release of neutrophils into circulati on. Neutrophils are draw n into specific sites by products of inflammation or infection (chemotaxis) wher e they release free radicals and degradative enzymes such as elastase and lysozyme. Neutrophils probably live only 1 to 2 days after migrating into tissue (Cannon et al., 1990). Antioxidants increase human neutrophil ch emotaxis in vitro. After damaging exercise, vitamin E may promote neutrophi l accumulation at specific sites of tissue damage (Cannon et al., 1990). During reperf usion, an increase in tissue neutrophil

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30 content was observed (Cannon et al., 1990) Oxygen metabolites, such as hydrogen peroxide, that are produced by activated macrophages depre ss lymphocyte proliferation (Blumberg, 1994). Aging is associated with an increase in PGE2 production which also inhibits lymphocyte proliferation (Blu mberg, 1994). Vitamin E decreases hydrogen peroxide formation in polymorphonuclear le ukocytes (PMN) (Blumb erg, 1994). Vitamin E decreases the rate of PGE2 synthesis. Building an immune response requires membrane bound receptor mediated communica tion between cells as well as between protein and lipid mediators. This can be a ffected directly or indirectly by vitamin E status. To further this, leukotrienes and HETE (products of the lipoxygenase pathway) inhibit lymphocyte proliferation possibly by decreasing T helper and increasing T suppressor cell proliferation (Blumber g, 1994). Lipid peroxides stimulate cyclooxygenase pathways by providing an oxygen species to enhance enzyme activity. An inverse relationship was noted be tween serum vitamin E and PGI, PGE2, and PGF2 Vitamin E bidirectionally modulates lipoxygena se activity on arachidonic acid. Normal plasma vitamin E concentration enhances lipoxygenation of arachi donic acid, but higher levels of vitamin E suppress the effect. This effect of vitamin E is probably due to its role as a hydroperoxide scavenger. Theref ore, the enhancement of immune function by vitamin E is most likely due, in part, to its reduction of reactiv e oxygen metabolites such as hydrogen peroxide and by its inhibition of cyclooxygenase and lipoxygenase pathways (Blumberg, 1994). Exercise can cause mobilization of infl ammatory agents although the specific events initiating this are unknow n. The magnitude of the in flammatory response varies with the duration and intensity of exercise although the exact role of inflammation during

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31 exercise induced muscle injury is undefine d (MacIntyre et al., 1995). Evidence suggests exercise can cause early activation of neut rophils (MacIntyre et al., 1995). Neutrophil accumulation in muscle was observed in exerci se induced muscle injuries after endurance tests on mice (MacIntyre et al., 1995). Following neutrophil accumulation, monocytes and macrophages are present and responsible for the resorption of neutrophils in necrotic tissue. Macrophages are the predominant infl ammatory cell present in exercise induced muscle injury (MacIntyre et al., 1995). Men were supplemented 800 IU/day vitamin E for 48 days that correlated with a reduction in plasma cytokine (IL-1 and IL-6) response to muscle damage inducing eccentric exercise (Cannon et al., 1990). Lower IL-1 was associated with lower 3methylhistidine excretion, a marker of proteo lysis (Cannon et al., 1990). It is uncertain whether an increase in oxidative stress that o ccurs with exercise is necessary for muscle adaptation to occur or whether it is harmfu l, causing muscle damage that impairs the ability to perform or trai n (Urso and Clarkson, 2003). In a second study, 21 untrained male volunt eers in two age ranges (22-29 and 5574) were supplemented with 800 IU -tocopherol per day for 48 days before exercise (Cannon et al., 1990). Subjects were monitored for 12 days post exercise for changes in circulating leukocytes, superoxi de release from neutrophils, lipid peroxidation, and efflux of intramuscular CK into circulation. Th e <30 year old placebo group had higher plasma creatine kinase (CK) and signi ficantly greater neutrophilia th an the >55 year old group. At the time of peak concentration in th e plasma, CK correlated significantly with superoxide release from neutrophils. This supports the concept that neutrophils are involved in the delayed increase in muscle membrane permeability after damaging

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32 exercise. There was no significant change in plasma vitamin E concentration observed during the 72 hour period post exercise. V itamin E supplementation tended to increase plasma CK values post-exerci se, particularly in the > 55 year age group. Vitamin E tended to reduce CK values in the <30 year age group and increase it in the >55 year group on days 2 and 5 post exercise. Circul ating neutrophil levels peaked in both supplemented and unsupplemented <30 age groups 6 hours post exercise. The >55 year group exhibited a much smaller increase in neutrophil count, peaking 3 hours post exercise. The neutrophil count in the vitamin E supplemented group >55 years was similar to the <30 year groups. On the da ys post exercise, the >55 year supplemented group had a higher neutrophil count compar ed to the placebo. The <30 year supplemented group had lower counts than the placebo. There was no difference in immature band cells observed at any time suppor ting the concept that vitamin E acts as a chemotaxis for neutrophils. The plasma lipid peroxide concentrations increased in 18 of the subjects within 24 hours of exercise but the time th is increase occurred varied considerably between individua ls resulting in no statistically significant increase at any particular point. Increases in CK in >55 year supplemented group appear to contradict the hypothesis that exercise induced changes in muscle membrane permeability are the result of damage by oxygen radicals (i.e. leakage of membrane due to decreased stability). The authors suggest that CK is representative of increased muscle protein turnover to clear partly damaged proteins. Po ssibly, older subjects have lower clearance mechanisms and vitamin E promotes neutrophil accumulation at specific sites of muscle damage to clear damaged proteins.

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33 Neutrophils are damaged by their own produc ts and are protected by vitamin E. Diminished neutrophilia in placebo >55 group may reflec t greater autooxidation of neutrophils that have become more vulnerable to oxidative damage with age. By protecting neutrophils, vitami n E may indirectly promote a net increase in muscle membrane damage, even though it has a direct positive effect on muscle membrane itself (Cannon et al., 1990). Therefore, mobilization a nd activation of neutrophils may contribute to an increased myocellular enzyme leakage after ec centric exercise. Age related differences in response appear to be modulated by diet ary supplementation of vitamin E, possibly due to its chemotaxic properties and its protection of neutrophils. A likely chemical stimulant of pa in sensation is prostaglandin E2 (PGE2), which causes an increased sensitivity of the pain receptors and is synthesized by macrophages and possibly neutrophils (Chan et al., 1989). Neutrophils pr ovide essential products for the formation of prostaglandins (Chan et al ., 1989). Possibly when muscle is damaged, the injured cells cause an incr ease in the synthesis of PGE2 and therefore a pain sensation. Vitamin E significantly inhibits the metabo lism of arachidonic ac id by the lipoxygenase pathway (Chan et al., 1989). Arachidoni c acid is the most abundant 20 carbon polyunsaturated fatty acid in the phospholipids of mammalian tissue and is a precursor to many biological compounds that mediate inflammation (Reddanna et al., 1989). Both the lipoxygenase and cyclooxygenase pathways exhi bit an obligatory requirement for fatty acid hydroperoxides (FAHP). Therefore, FAHP are implicated as modulators of the arachidonic acid cascade which generate s inflammatory products through the lipoxygenase and cyclooxygenase pathways. The fact that lipid peroxidation provides a

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34 source of FAHP formation makes these pathways of particular inte rest in the study of antioxidants (Reddanna et al., 1989). A review by Blumberg (1994) suggests that vitamin E supplementation reduced plasma lipid peroxides and production of PGE2 by polymorphonuclear leukocytes (PMN) in elderly subjects. Aging is a ssociated with increases in PGE2 production which inhibits lymphocyte proliferation. Vitamin E decreases PGE2 production and improves cellular immunity. Vitamin E also neutralizes H2O2 and radicals used by the immune system to kill pathogens. This diminishes in vitro ki lling capacity of the cel ls but protects the immune cell itself from autooxidation, th ereby improving phagocytosis (Blumberg, 1994). Also, the enhancement of immune func tion by vitamin E may relate to a change in membrane receptor molecules involved in the immune response as vitamin E has been shown to induce changes in cell su rface glycoconjugates (Blumberg, 1994). Oxidative Stress and Genetic Pathways in Muscles Bursts of oxidant production and extreme changes in activities of antioxidant defenses alter gene expression. Free radicals and their reactions dire ctly affect processes of cell differentiation, aging, and transformation leading to the conclusion that cells have evolved pathways to utilize ROS as biologi cal stimuli. ROS influence expression of some genes and signal transduction pathways and are thought to act as subcellular messengers for certain growth factors (Allen and Tresini, 2000). Changes in cellular redox status influence transc riptional modification of co llagen (Chojkier et al., 1989), collagenase (Brenneisen et al., 1997), post transl ational control of ferritin (Hentze et al., 1989), activation of the transcriptional f actors Myb (Myrset et al., 1993) and Egr-1 (Huang and Adamson, 1993), and also binding activity of the fos/jun (AP-1) protein conjugate (activator protein 1) (Abate et al., 1990). Exposure of normal and transformed

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35 cells to UV radiation or H2O2 causes increased expression of jun-B, jun-D, c-fos, and fosB (early response proteins). Antioxidant treatments stimulat e increases in the expression of some genes. It is possi ble that the increase is cause d by autooxidation of antioxidant compounds producing enough ROS to cause a change in gene expression through oxidizing properties rather than reducing properties (Allen an d Tresini, 2000). Choi and Moore (1993) studied several structural isomers of butylat ed hydroxytoluene and showed that only the compounds with high antioxi dant potential are capable of inducing c-fos The isomers without antioxidant properties did not induce c-fos (Choi and Moore, 1993). Others demonstrated that antioxidant en zymes modulate signal transduction and gene expression. This suggests that the redox potential oxidant/ant ioxidant treatments is at least partially, if not completely, responsible for their effect on gene expression (Allen and Tresini, 2000). Lander (1997) proposed th at cellular responses influenced by ROS and reactive nitrogen species can be grouped in to five broad categories: (1) modulation of cytokine, growth factor, or hormone action/se cretion, (2) ion transpor t, (3) transcription, (4) neuromodulation, (5) apoptosis. The effect s of redox changes on transcription factors and signal transduction are not clear but ar e possibly mediated through oxidation and reduction of protein sulfhydryls. Changes in redox state of the pr otein sulfhydryls causes conformational changes that can either impair or enha nce DNA binding activity, release inhibitory subunits, or promote protein complex formations necessary for signal transduction or transcription to proceed. There are multiple conserved cysteine residues in some protein kinase C (PKC) that are possible targets for redox regulation and may explain redox effects on pathways influen ced by PKC activation. The redox-sensitive

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36 pathways, MAP (mitogen-activated protein) kinase and NFB (nuclear factorB) signal transduction pathways, have multiple steps sensitive to ROS. MAP Kinases Growth factors and cytokines are exampl es of extracellular signaling molecules which induce changes in a cell via mechanisms that involve transmission of the signal from the plasma membrane to the nucleus wh ere gene expression is altered. To activate this signaling cascade, first a receptor is activated which either has protein kinase activity or activates a protein kinase in the cytoplas m. This signal continues to be transmitted until it reaches the nucleus where it activates transcription factors regulating gene expression. MAP kinases are one of the mo st studied groups of signal transduction pathways. There are four types of MAP ki nases: (1) ERK (extracellular regulated kinase) (2) JNK kinase ( c-jun NH2-terminal kinase)/SAP kinase (stress activated protein kinase) (3) p38 kinases (4) big MAP kinase (BMK/ERK). All subfamilies have redox sensitive sites. In the ERK and JNK path ways, Ras is activated by conversion of RasGDP to Ras-GTP by a guanine nucleotide exchange factor (GEF) such as the mammalian homologue of Sos. Sos contains SH2 domains which bind to tyrosine phosphorylated motifs in other proteins. Sos is constitu tively bound in the cytoplasm to another SH2 domain-containing protein, Grb-2. When a rece ptor is activated, i.e. by a ligand binding to it, tyrosine is au tophosphorylated and SH2 domain-containing rece ptors are recruited including Grb-2. This causes plasma membra ne targeting of Sos where it is tyrosine phosphorylated. The phosphorylated Grb-2/Sos complex binds to an adapter protein (the SH-containing protein), and the multi-protei n complex converts Ras-GDP to Ras-GTP, thereby activating it. Treatment of cells with oxidants such as H2O2 stimulates the formation of the SH-containing protein-Grb2-So s complex. Ras will eventually return to

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37 baseline levels by action of Ras-GTPase ac tivating proteins which inactivate Ras. Oxidizing treatments such as hypoxia/re oxygenation or stim ulation with H2O2 activate Raf-1 possibly through effects on Ras. Once Ra f-1 is activated, it phos phorylates serines in the catalytic sites of MKK/MEK. MKK1/MEK1 and MKK2/MEK2 activate members of the MAP kinase family (ERK-1/ERK-2) which are kinases that regulate downstream responses to many mitogenic, apoptotic, diffe rentiation-inducing stimuli. MEK1 but not MEK 2 is stimulated by exposure to H2O2, indicating that only MEK1 is redox sensitive (Allen and Tresini, 2000). MAP kinases are activated by dual phosphor ylation of tyrosine and threonine residues located on the kinase. This is ca talyzed by threonine and tyrosine kinases belonging to the MKK family. ERK, JNK, and p38 are MAP kinase subfamilies which phosphorylate the COOH-terminal transcriptio nal activation domain of ERK-1 and SAP1 which then associate with other nuclear pr oteins to form a ternary complex factor (TCF). TCF proteins cannot bind to the prom oter region but instead require interaction with other transcriptiona l factors such as SRF (serum res ponsive factor). Transcription of many genes is mediated by the binding of the multiprotein complex of an SRF homodimer and a TCF family member to the SRE (serum response element) in the gene promoter region. UV light and environmenta l stress have been s hown to activate the JNK, p38, and big MAP kinase pathway while antioxidants and growth factors induce the activation of the ERK isoforms p44/ERK1 a nd p42/ERK2. Therefore, oxidants and reductants play a role on redox sensitive genes through their actions on MAP kinase pathways. Other proteins influe nced by redox status are the NFB/Rel family of transcription factors involved in the regul ation of many genes (such as acute phase

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38 proteins, cell surface receptors, and cytokines). The effect s of redox state exerted on any given gene can vary from tissue to tissue (All en and Tresini, 2000). Collart et al. (1995) showed that H2O2 and ionizing radiation had extremel y varying results on the induction of c-jun Depending on the cell type, H2O2 caused a dramatic range of reactions from large increases to no effect at all. This emphasizes the fact that signal transduction pathways are different in diffe rent cell types, and therefore the effect of redox status will vary from tissue to tissue.

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39 CHAPTER 2 INTRODUCTION Strenuous exercise increases the produc tion of ROS (reactive oxygen species) in the body causing an increase in oxidative stress (Powers and Hamilton 1999). As oxygen consumption increases, there is the potential for the electron transport chain to become overwhelmed and electrons to leak into the mitochondrial space where they can react with components of the ce ll and give rise to ROS. These ROS can damage the mitochondria and other parts of the cell, re ndering the cell not functional. Free radical damage is associated with destruction of cell membranes and aggregation of membrane compounds (Matsuki et al. 1991). The generatio n of ROS during exer cise is likely a cause of muscular disturbances, inflammation, and pain in performance animals (Avellini et al. 1999). The extent of damage done by ROS to the exercising subject is unknown. Free radical damage leads to higher levels of oxidation products in blood and tissue (Singh 1992). These products may lead to further dama ge of muscle cells as well as generation of biologically active compounds able to pr opagate the peroxidation chain or enter inflammatory pathways (Dekkers et al. 1996). There is increased interest in providing supplementa l dietary antioxidants to exercising subjects in an attempt to reduce the levels of free ra dicals in the body and therefore reduce the damage done to the an imal by these substances. However, the benefits of these supplements are controve rsial. The purpose of this study was to determine whether exercising horses supplemented vitamin E above NRC

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40 recommendations had decreased measures of oxidative stress in blood and muscle parameters both over the course of a training program and following a single strenuous bout of exercise.

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41 CHAPTER 3 MATERIALS AND METHODS Animals Eight untrained healthy thoroughbred geldi ngs between the ages of 3 and 10 years of age were used in a complete repeated measures (crossover) design. Untrained horses were used in order to monitor the response of the animals to supplementation as fitness level improves. The horses health and soundne ss were evaluated prior to the start of the experiment. All horses were maintained between body condition score of 4.5 to 5. All animal procedures were conducted within the guidelines of and approved by the University of Florida Institutional Care and Use Committee. Diets and Adaptation Prior to being divided into treatment gr oups, all horses were put on the control diet for three weeks and familiarized with the treadmill. A control baseline standard exercise test (SET) was performed at the e nd of the adaptation period. Subsequently, the animals were divided into control and vita min E supplemented groups and acclimated to the respective diets for seven weeks to achieve a consistent level of vitamin E in both blood and tissue. The horses on the control di et were fed a 12% cr ude protein textured oat-based feed at a rate of 1.25 kg feed pe r 100 kg body weight and ad libitum coastal Bermuda grass hay. Vitamin E content of the grain was 80 IU/kg DM as recommended by the NRC (1989) for horses undergoing moderate to intense levels of work. The horses receiving vitamin E supplement ation were given the same di et as the control but were supplemented with 3000 IU vitamin E/day in the form of -tocopherol acetate. This level

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42 was chosen based on a prior work demonstr ating that a basal di et containing 44 IU vitamin E/kg DM or 124 IU vitamin E/kg DM we re not sufficient to prevent decreases in plasma and muscle -tocopherol status due to repeated exercise training (Siciliano et al., 1997). However, 344 IU vitamin E/kg DM maintained vitamin E status in the blood and muscle. Supplementation of 3000 IU/day placed all vitamin E supplemented horses above 300 IU/kg DM of concentrate. The horses were fed the concentrate mixtur e twice daily, once in the morning and once in the afternoon. Once training began, th e amount of concentrate fed was increased from 1.25% of body weight to 1.5% of body weight divided equally between the feedings. Hay (Table 3.1) and feed (Tab le 3.2) were sampled throughout the project and analyzed for nutrient values. The treatmen t group received supplementation as a daily evening top dressing (68.2 gr ams of a vitamin E premix containing 20,000 IU vitamin E/lb). The premix was composed of d, l -tocopherol acetate mixed with a rice meal carrier. The control group received 68.2 grams of a placebo (rice meal feed). In addition, all horses received ad libitum access to coastal Bermudagrass hay. The horses were housed at the University of Florida in 4X4 me ter stalls or in an adjacent dry lot turnout paddock. The use of the dry lot paddock elim inated grazing and helped control dietary intake. The horses were rotated daily in groups of four with f our being kept in stalls and four being kept in the turn out paddock. On days of blood sampling, all horses were kept in stalls beginning on the evening prior to collection to eliminate variations in temperature and climate.

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43 Washout and Cross-Over Period All horses underwent a 15 week washout pe riod during which they were turned out to pasture, fed the control diet, and left untrained. The horses were then crossed over and the experiment repeated beginning with the adaptation period. Due to mechanical problems with the treadmill, the adaptation peri od for the second half of the experiment lasted ten weeks rather than seven. During the extra three weeks, the horses were fed their respective diets and left untrained. Bl ood was collected both at the end of seven weeks and at the end of ten weeks of ad aptation, and all anal yses were run and statistically compared to ensu re that there were no baseli ne differences between weeks seven and ten due to the difference in times on treatment diets. Therefore, there was a total of 25 weeks (6 months) between the end of one traini ng phase and the beginning of the next. This should have provided adequate time for the horses to detrain (return to baseline fitness levels) and fo r blood and muscle vitamin E leve ls to equilibrate. Blood samples were taken prior to the start of each training phase to ensure that the horses had similar vitamin E levels between the two phases. The horses followed the exercise schedule performed in the first half of the experiment. Training Period The training and standard exercise test s were conducted at the University of Florida Veterinary Medicine Teaching Hospital in a climate controlled room equipped with a treadmill. The training period consisted of eight weeks of gradually increasing workloads. Treadmill exercise was performed three to four days per week, depending on availability of the treadmill. The first thr ee days the horses trotted at 4 m/s for 0.6 km on a flat surface, galloped at 8 m/s for 1.0 km on a flat surface, and cooled down by trotting

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44 at 4 m/s for 0.6 km on a flat surface. The gallop distance was increased by 0.5 km every three days so that by the end of three week s, the horses were performing the following exercise: trotting at 4 m/s for 0.6 km on a fl at surface, galloping at 8 m/s for 3.0 km, and trotting at 4 m/s for 0.4 km on a flat surface. The gallop phase was performed twice a week on a flat surface and twice a week on a 6 incline for the rema inder of the study. A standard exercise test (SET) was performed at the end of each eight week conditioning period. The protocol was as follows: 4 m/s for 2 minutes on a flat surface, 8 m/s for 1 minute at a 6 incline, 9 m/s for 1 minute, 10 m/s for 1 minute, 11 m/s for 1 minute, and 12 m/s until they would not conti nue with moderate persuasion. Tissue Sampling Baseline blood samples were taken follo wing the seven week adaptation period. Once training began, blood response variables we re measured before and after exercise at the end of weeks 2, 5, and 8. At the end of the fifth week, before exercise and after exercise samples were taken on two days, one during wh ich the horses ran on th e flat and one during which the horses ran on the incline for the fi rst time. This provided a way to compare different exercise intensity leve ls when horses were at the same fitness level. Following the eighth week, all horses performed a standard exercise test with blood and muscle tissue collected before and after ex ercise. Blood was collected by jugular puncture in EDTA tubes. Muscle tissue was taken from the middle gluteal muscle by punch biopsy following local lidocaine anesthesia. Whole blood was immediately pipetted from the tubes and put on ice until storage at -80 C. Plasma was separated and stored at -80 C. Muscle tissue was immediately divided for future analyses, plac ed in cryogenic tubes in liquid nitrogen, and stored at -80 C.

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45 TBARS Analysis Plasma samples were thawed and diluted 1:100 with deionized (DI) water. Working standards were prepared from the stoc k standard (100 nM 1,1,3,3-tetramethoxypropane) each time the procedure was run. One hundred L of sample or known amount of standard was mixed with an equal amount of 8.1% sodi um dodecyl sulfate. The denatured plasma was incubated with TBA/Buffer reagent (37 mM, pH 3.5) at 95 C for 60 minutes according to company recommendations (Zeptomatrix; Bu ffalo, NY). The tubes were covered with glass marbles to prevent evapor ation. The tubes were cooled in ice water for exactly 10 minutes and then allowed to come to room temperature. Reactions were quantified on a fluorescent spectrofluorometer at the following parameters: excitation of 535 nm, emission of 552 nm, high sensitivity, slit width of 5 nm Multiple readings of each sample were taken. A standard curve was plotted and used to calculate sample values. For post exercise blood samples, plasma proteins were analyzed using a plasma refract ometer. A ratio was calculated from pre exercise plasma proteins di vided by post exercise plasma proteins. This number was multiplied by the post exercise TBARS concentration to adjust for plasma volume. Data was reported as bo th adjusted and unadjusted values. TEAC Analysis Prior to beginning the assay, metmyoglobi n was synthesized by mixing an equal volume of 0.4 mM myog lobin and potassium ferricyanide (0.74 mM K3Fe(CN)6). Metmyoglobin reagent was applied to a Sepha dex G50 column and eluted with phosphate buffered saline (PBS). The concentration of the purified metmyoglobin was calculated using the Whitburn equation: Conc. metmyo = 146 (Abs490 Abs700) 108 (Abs560 Abs700) + 2.1 (Abs580 Abs700).

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46 The metmyoglobin was diluted with PBS to a final concentration of 70 mM. Assay stock solutions were prepared fr esh and included 5 mM 2,2-azinobis-(3ethylbenzothiazoline-6-sulphonic acid) (ABT S reagent), 2.5 mM Trolox, and 0.15% H202 in PBS. Final reactions were performed in di sposable cuvettes in the presence of 1.5 mM ABTS, 250 M metmyoglobin, and 40 l of sample. Reactions were initiated by addition of 20 l of H2O2. Cuvettes were inverted once and pl aced immediately on a spectrophotometer where they were read every 20 seconds for 4 mi nutes at 600 nm. Results were reported as a percent of inhibition. Unknown value/2.5 100 = % inhibition. TEAC concentration was adjusted by plasma protein ratio for post exercise samples as described in the above section. Data is reported both as ad justed and unadjusted values. Glutathione (reduced, oxidized, and total) Analysis For glutathione assays, whole blood was d iluted ten-fold with DI water and mixed with and equal volume of meta-phosphoric acid (MPA) precipitating re agent (2 mM MPA, 5 M NaCl, 7 mM EDTA). The sample/extrac tion solution was centrifuged at 800 X G for 15 minutes, and the supernatant was saved and frozen at -80 C. The extracted samples were diluted 1:20 with respective buffers. For reduced glutathione assays, 100 L of the extracted, diluted samples was adde d to 2.0 mL GSH buffer (100 mM NaPO4, 4 mM EDTA, pH 8.0). Subsequently, 100 L of 0.1% o-phthaldialdehyde (O PT) in methanol (protected from light) was added to the tubes and incuba ted at room temperature for at least 17 minutes. Fluorescence was measured at em ission 420 nm, excitation 350 nm, with high sensitivity and slit width of 5 nm. Results were reported in mg/dl/%RBC. For total glutathione, the same procedure was followed ex cept that the extracted, diluted sample was dissolved in 2.0 ml of GSSG buffer (100mM NaOH) and allo wed to incubate at room

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47 temperature prior to addition of OPT. Oxidi zed glutathione was determined by calculation: (Total glutathione reduced glutat hione = oxidized glutathione). Vitamin E Assay Vitamin E was analyzed in plasma samp les at the initial date (following the adaptation period but prior to the beginning of any exercise) and on week 8 pre exercise (Michigan State University Diagnostic Center for Population and Animal Health; East Lansing, MI). Vitamin E was extracted from the plasma and analyzed by high performance liquid chromatography (HPLC). Muscle Analyses Myofibril Preparation Muscle tissue was immediately divided for fu ture analyses, placed in cryogenic tubes and frozen in liquid nitrogen until storage at -80 C. To isolate myofibrils, frozen muscle was placed in a homogenization buffer on ice containing the following: 75 mM KCl, 10 mM Tris (pH 6.8), 2 mM EGTA, 2 mM MgCl2, 0.1 mM PMSF, and 0.1% Triton X-100. After one hour, the samples were homogenized with a polytron, centrifuged at 1000 X G, rinsed with homogenization buffer, and centrif uged two more times, as described (Gordillo et al. 2002). The final pelle t was resuspended in homogenization buffer, minus the Triton X-100 and EGTA, containing 50% glycerol. My ofibrils were stored frozen at -20 C. Protein concentration was determined us ing the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA). Western Blot Protein carbonyls were measured in m yofibrils by Western blot (OxiblotTM Protein Oxidation Detection Kit, Chemicon Intern ational, Temecula, CA). Briefly, 20 g of proteins were electrophoretically separa ted through 10% polyacrylamide gels and

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48 transferred to nitrocellulose membrane. Me mbranes were blocked with 5% bovine serum albumin in phosphate buffered saline (PBS ) containing 0.1% Tween 20 (PBS-T) and incubated with antibodies according to manufact urer recommendations. Blots were washed with PBS-T and incubated with goat anti-mouse peroxidase (1:5000, Vector Labs, Burlingame, CA). After a final wash with PBS-T, immune complexes were visualized by enhanced chemilumenescence (ECL) and X-ray exposure. For the detection of ubiquitin, mouse anti-ubiquitin (1:1000, Santa Cruz Bi otechnology, Sacramento, CA) was used. Dystrophin Immunochemistry and Fiber Morphometrics The integrity and size of the gluteus me dius muscle fibers were evaluated by dystrophin immunostaining. Biopsies were obtaine d before and immediately after exercise in controls and horses supplemented with vitami n E. Muscle biopsies were frozen in OCT frozen tissue embedding media and cr yosections were collected (8-12 m) on SuperFrost glass slides. Tissue was oriented to give cr oss-sections of muscle. OCT was removed and tissue sections were incubate d with 5% horse serum in PBS for 20 minutes at room temperature. Subsequently, tis sues were incubated in anti-dystrophin (1:400) for one hour at room temperature. After exhaustive washes with PBS, sections were incubated with goat anti-mouse Alexafluor 488 (1: 250, Invitrogen, Carlsbad, CA). Nuclei were detected by propidium iodide (1g/mL) counterstain. Immu nostaining was visualiz ed by epifluorescent microscopy (Figure 3.2). Representative pho tomicrographs were captured and analyzed with NIS Elements software for fiber area. Three images were taken from each of three cross-sections taken from each horse for a tota l of nine images per horse. Representative images were captured at 200X using a Nikon TE2000U inverted microscope equipped with epifluorescence and a DMI200F digital camera.

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49 Statistical Analysis Horse weights and grain intake were taken prior to the start of each adaptation and training period. Statistics were analyzed by analysis of variance (ANOVA) with repeated measures over time according to treatment. Analyses were performed with Statistical Analysis System (SAS) version 6.12 using proc GLM for the ANOVAs and proc mixed for the ANOVAs for measures repeated ove r time. All analyses with P<0.05 were considered statistically significant. The blood data for vitamin E, TBARS, oxidized, reduced, and total glutathione, as well as %re duced glutathione, and TEAC was analyzed by analysis of variance (ANO VA) with repeated measures over time according to treatments. The regression relationship between each of the blood parameters and time was generated for each treatment group over the total sampling times. Analyses were performed with SAS version 6.12 using proc GLM for the ANOVAs and proc mixed for the ANOVAs for measures repeated over ti me. All analyses with P<0.05 were considered statistically significant. The mu scle data for carbonyl content and ubiquitin content was analyzed using SAS by Fischers exact test using proc freq with a chi-square value P<0.05 being considered statistically significant.

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50 Table 3.1. Nutrient composition of hay sa mpled at selected times during the study Sample #1 Sample #2 Sample #3 Sample #4 Sample #5 Sample #6 Average Dry Matter % 90.90 91.09 89.50 90.75 91.05 89.54 90.5 Ash % 4.04 3.30 3.73 3.79 3.53 3.28 3.6 Crude Protein % 6.06 5.64 5.15 9.47 8.91 9.00 5.6 Phosphorous % 0.14 0.14 0.15 0.20 0.14 0.19 0.2 Fat % 1.26 0.78 1.64 0.89 0.89 0.82 1.1 Acid Free Detergent Fiber (ash free) % 41.82 43.63 39.21 36.98 34.02 34.94 38.4 Neutral Detergent Fiber (ash free) % 81.79 81.34 80.35 76.19 72.75 76.09 78.1 Calcium % 0.20 0.18 0.46 0.40 0.40 0.61 0.4 Copper (ppm) 5.32 4.07 2.29 2.30 2.30 7.75 4.3 Manganese (ppm) 146.00 144.00 52.00 42.00 42.00 49.00 83.7 Zinc (ppm) 31.00 26.00 23.00 11.00 11.00 30.00 23.5 Iron (ppm) 180.00 77.00 102.00 103.00 103.00 475.00 214.5 Table 3.2. Nutrient composition of grain sampled at selected times during study Sample #1 Sample #2 Sample #3 Sample #4 Sample #5 Sample #6 Average Dry Matter % 95.22 94.92 94.78 95.57 96.03 96.62 95.5 Ash % 6.62 6.38 6.45 7.64 7.08 7.05 6.9 Crude Protein % 15.15 15.18 15.70 14.53 15.11 15.27 15.3 Phosphorous % 0.66 0.62 0.62 0.67 0.62 0.59 0.6 Fat % 3.34 3.28 2.62 2.95 1.37 1.35 2.5 Acid Free Detergent Fiber (ash free) % 10.02 9.15 9.46 8.34 7.36 7.60 8.7 Neutral Detergent Fiber (ash free) % 26.55 23.84 25.01 22.33 21.91 23.26 23.8 Calcium % 0.80 1.18 1.16 1.28 1.93 2.14 1.4 Copper (ppm) 37.00 52.00 48.00 58.00 44.00 62.00 50.2 Manganese (ppm) 83.00 122.00 125.00 115.00 118.00 109.00 112.0 Zinc (ppm) 90.00 144.00 138.00 111.00 131.00 187.00 133.5 Iron (ppm) 225.00 269.00 268.00 327.00 386.00 508.00 330.5

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51 7 weeks supplementation 8 weeks training/supplementation SET 15 weeks washout/detraining Cross-over treatments 10 weeks supplementation 8 weeks training/supplementation SET Figure 3.1. Summary of e xperimental timetable. Figure 3.2. Representative image of -dystrophin immunostaining. Representative photomicrographs were captured and an alyzed with NIS Elements software for fiber area. Three images were ta ken from each of three cross-sections taken from each horse for a total of nine images per horse. Representative images were captured at 200X using a Nikon TE2000U inverted microscope equipped with epifluorescence a nd a DMI200F digital camera.

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52 CHAPTER 4 RESULTS AND DISCUSSION Vitamin E Vitamin E supplementation did not increase circulating levels of the nutrient. Vitamin E was fed to horses at a concentrati on approximately five-f old higher than the NRC recommended level for horses in modera te to intense training (80 IU/kg DM). Blood samples were monitored for circulating vitamin E content at the beginning and end of the eight week training period (Table 4.1). No differences in plasma vitamin E between control and vitamin E supplemented horses were observed. Due to mechanical problems with the treadmill, the adaptation period for the second half of the experiment was actually te n weeks rather than seven. During the extra three weeks, the horses were fed their respec tive diets and left untrained. Therefore, there was a total of 25 weeks (6 months) be tween the end of one training phase and the beginning of the next. This should have provi ded adequate time for the horses to detrain (return to baseline f itness levels) and for blood and muscle vitamin E levels to equilibrate. Tissue depleti on and repletion in adult stan dardbred horses showed that plasma vitamin E values in most supplemente d groups were stabilized within a few days of beginning supplementation and upon bei ng taken off supplementation, most horses reached their baseline value within three w eeks, although a few remained slightly higher even after seven weeks (Roneus et al., 1986). Liver samples showed that this organ rapidly accumulated vitamin E and the concentration declined following placement on a low vitamin E diet, indicating that there is no pe rmanent storage of vitamin E in the liver.

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53 By contrast, skeletal muscle vitamin E con centration was most consistent regardless of supplementation levels. Muscle tissue vitami n E concentrations increased much slower than plasma and liver and decreased slower upon being placed on a low vitamin E diet. Skeletal muscle in groups that were supplemented up to 1800 mg dl-tocopheryl acetate (1800 IU) per day returned to baseline values after being taken off the supplement for 7 weeks (Roneus et al., 1986). The gr oup that was supplemented 5400 mg dl-tocopheryl acetate (5400 IU) did not. However, maximum vitamin E levels in the skeletal muscle tissue were achieved with 1800 mg dl-tocopheryl acetate per day. Therefore, the horses in our study were fed an adequate level of vitamin E to reach maximum tissue levels within 7 weeks. The blood samples taken before and afte r each training phase were not different, as describe d by others (Roneus et al., 1986). Throughout the duration of the study, a significant period effect was detected in several measures of redox potential. This may be explained partia lly by differences in tissue vitamin E concentration and retention. After several years of deficient diets, vitamin E stores can still remain in the body (Blatt et al., 2001). Our plasma vitamin E levels showed no period effect. However, circulating vitamin E reaches a saturation point and can only be increased at most three to four fold by supplementation (Blatt et al., 2001). It is possible that our control horses were at a suffi cient level of supplementation such that additional vitamin E could not further elevate plasma levels. This does not, however, mean that tissue levels of vitamin E could not have been significantly higher in period two as compared to period one.

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54 Lipid Peroxidation Vitamin E supplementation did not alter lipid oxi dation levels. The total amount of oxidized lipids in the plasma was measured befo re and after a bout of mild exercise over the duration of the 9 week training period. Plasma thiobarbituric acid reactive substances (TBARS) is a measure of lip id peroxidation in the body. The TBARS concentrations remained relatively constant throughout the feeding trial in both control and vitamin E supplemented horses (Figur e 4.1). A decline in plasma TBARS concentration at week 8 was observed for bot h groups of horses. The cause of this reduction is unknown but may reflect a sampli ng error. Following a training episode, plasma TBARS concentrations remain una ffected by treatment (Figure 4.2). The concentration of TBARS when adjusted for plasma volume using a plasma protein ratio was, however, lower post exercise as comp ared to pre exercise for both control and vitamin E supplemented horses (P<0.0001). Fr om these results, it is concluded that circulating oxidized lipid content in pl asma was altered by exercise but was not influenced by vitamin E supplementation. Feeding an excess of vitamin E does not reduce plasma TBARS suggesting no benefici al effect at this level of athletic performance. The effect of vitamin E supplementation on lipid peroxidation was examined following a single bout of strenuous exercise (Table 4.2). Horses were exercised to exhaustion and blood TBARS c ontent was measured during the recovery period. Immediately after ex ercise, TBARS content declined in control and vitamin E supplemented horses. These values returned to baseline within 24 hours and remained unchanged at 48 hours. No differences be tween control and vitamin E supplemented horses were observed at any time point. Therefore, vitamin E supplementation does not improve the antioxidant capacity of exer cising horses as measured by TBARS.

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55 The TBARS assay primarily measures malondialdehyde (MDA), a short chain fat group which is formed from damaged cell membra nes. It has been criticized in previous studies for assay variability a nd its reflection of other parame ters in the body independent of oxidative stress. In this study, there was no significant difference between treatment and control groups at any sample point over the training period. There was a significant decrease in week eight pre TBARS values (F igure 4.1). This was an expected result since as the horses fitness leve l increased there should have been less peroxidation in the body. The exercise performed at week eight was a simple run on a flat surface over the ascribed training distance. Therefore, mi nimal stress was placed on these horses. However, the pre SET blood samples for horses were the highest of all samples drawn. This is possibly due to stress associated with preparing the horses to run the exercise test (number of people, equipment used, etc.). An unexpected finding was that TBARS values actually decreased after exercise when compared to before exercise values if post exercise concentrations were adjusted for plas ma volume (P<0.05). It is possible that this is due to increased clearance in the body di rectly after exercise. MDA was observed to have a short half-life and is therefore cleared quickly from the body (Siu and Draper, 1982). It is also possibl e that longer duration of exer cise yields greater TBARS concentrations. It has been shown that horses undergoing enduran ce exercise (140 km race) have elevated levels of TBARS post exercise, and these concentrations remain elevated 16 hours post exercise (Marlin et al., 2002). Possibly an increase in TBARS concentrations is more common in low intens ity but long duration ex ercise which relies more on free fatty acids as an energy source as opposed to the spri nt-type training that our animals performed. It is also possible th at the adjustment for plasma volume using

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56 plasma proteins was not appropriate. Van Beaumont and associates in 1973 concluded that there may be a net loss of plasma proteins as a result of exercise, and thus plasma protein concentration may not be a reliable way to adjust for plasma volume. Contrary to this, a more recent study by Lindinger and collea gues showed that total plasma proteins in exercising horses are direc tly correlated to plasma vol ume (Lindinger et al., 2000). Plasma concentration of TBARS following a SET was significantly lower than pre exercise concentrations when adjusted for plasma volume but not when left unadjusted. However, during the training phase, both una djusted concentrations and adjusted concentrations were significantly lower post exercise when compared to pre exercise concentrations. Antioxidant Capacity Plasma antioxidant capacity was unaffect ed by vitamin E supplementation. Plasma was analyzed for its ability to scavenge pe roxide free radicals (Trolox equivalent antioxidant capacity, TEAC). The TEAC values increased in both control and vitamin E supplemented horses after five weeks of modera te exercise (P<0.05) (Figure 4.3). These results posit that antioxidant defenses are improved as the horses transition through the fitness period. However, supplementation of excess vitamin E did not improve global antioxidant capacity (Table 4.4). Exercise di d, however, cause antioxidant status of the blood to decrease. Post exercise TEAC va lues were lower than pre exercise TEAC values for both treatment groups when values were adjusted for plasma volume (P<0.001). To examine the effects of vitamin E on recovery from strenuous exercise, horses were exercised to exhaustion and plas ma TEAC concentrations were measured (Table 4.5). Immediately upon completion of the exhaustive bout of work, TEAC values were low in both control and supplemented an imals (P<0.05). These values returned to

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57 baseline within 24 hours and remained constant at 48 hours. Thus, c ontrol diets sufficient in vitamin E likely meet the antioxidant dema nds of horses undergoing mild exercise as well as a stressful performance regiment. An interesting finding is that the modera te exercise performe d during the training phase resulted in unadjusted post exercise TEAC values being slightly elevated as compared to pre exercise samples. This is potentially due to mild exercise causing an activation of the bodys antioxidant system. However, following a strenuous exercise bout (SET exercise) the TEAC values were sign ificantly lower than pre exercise values but returned to resting leve ls by 24 hours post. This po ssibly indicates that strenuous exercise taxes the antioxidant system enough to decrease its radical s cavenging abilities. A study on similarly exercised horses showed that horses exercising at 8 m/s on a 6 incline (similar to our highest intensity training exercise ) had oxygen consumption of 63 mL/kg/min whereas horses running at 12 m/s on a 6 incline (maximum effort in our SET prior to fatigue) had oxygen consumption of 150 mL/kg/min (Kav azis et al., 2004). Therefore, oxygen consumption increased dramat ically between our training exercise and SET exercise, and this possibly explains the horses having a different response post training exercise versus post SET exercise. Th erefore, intensity of ex ercise must be taken into account when examining antioxida nt capacity of exercising subjects. Furthermore, training was shown to in crease the antioxidant capacity of the animals regardless of treatment group (P<0.05) Following five weeks of training, horses had significantly elevated TEAC values pre exercise as compared to week 0 (initial value). This indicates that fitness level must be taken into consideration when comparing antioxidant capacities of exerci sing subjects. Our results are supported by another study

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58 using exercising horses which showed significant increases in citrate synthase activity in middle gluteus muscle following and intense ex ercise regime (27% higher activity after week 1 and 42% higher after week 2). This was accompanied by an increase in capillary density following five weeks of training (E ssen-Gustavsson et al. 1989). Therefore, training is shown to cause adaptations which may improve the antioxidant status of the exercising subject. G lutathione Data Glutathione participates in the bodys antioxidant defe nse by serving as a sink for electrons during th e reduction of H2O2 to water. More glutat hione in the reduced form may allow for efficient quenching of newly form ed free radicals. Therefore, high levels of reduced and % reduced glutathione are indi cative of improved antioxidant potential. Total, reduced, and oxidized glutathione were measured in the blood in horses fed control and excess vitamin E and maintained on a modera te exercise regime. Consistent with our prior results, vitamin E supplementation did not appear to affect oxidized, reduced, or total glutathione status in th e blood of moderately exercising horses, nor did it affect the percent of total glutathione which was in th e reduced form (Table 4.5). Vitamin E did, however, cause a significant elev ation of % reduced glutathione in response to strenuous exercise (SET) as compared to the control di et (P<0.006). This effect is attributed to lower plasma oxidized glutathi one levels in vitamin E tr eated horses (P<0.03). These results suggest that vitamin E may enhance the glutathione redox system, particularly during intense exercise. Exercise training also influenced glut athione parameters. Reduced and total glutathione decreased in pre exercise blood samples after tw o weeks of exercise training

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59 (P<0.05). This decrease persis ted throughout the remainder of the study. Exercise also caused an immediate drop in reduced, total, a nd % reduced glutathione with post exercise values being lower than pre exercise values (P<0.01). Exercise did not affect oxidized glutathione concentrations. This pattern persisted following the SET. Post exercise values for reduced, total, and % reduced glutat hione were lower than pre exercise values (P<0.01). Oxidized glutathione was not aff ected by exercise. At week 5, when the horses underwent a more rigorous form of exercise (exercise on a 6 incline as opposed to flat), post exercise concentra tions of reduced and total gl utathione were significantly lower as compared to week 2 values (P<0.01) This was also the case for week 9 (SET) data. This finding indicates that strenuous exercise caused the horses to utilize their reduced glutathione stores to a greater extent as compared to milder exercise. Marlin and colleagues (2002) found a similar trend in e ndurance horses. When blood was collected from 40 competitive endurance horses both before and after a 140 km race, all glutathione measures (reduced, total, oxidized ) decreased significantly post exercise (Marlin et al. 2002). Results of the pr esent study agree with their findings. Carbonylation and Ubiquitination Strenuous exercise can lead to formation of carbonylated proteins in muscle fibers. Carbonyl groups are formed as a consequen ce of ROS accumulation and may contribute to loss of contractile function. The presence and extent of carbonylat ion was examined in purified myofibrillar proteins isolated from the gluteus medi us of control and vitamin E supplemented horses before and after a singl e bout of strenuous exercise. Carbonylation was measured by Western blot (Figure 4.4). Statistical si gnificance was based on either presence or absence of carbonyl groups in all horses undergoing the standard exercise test. Three major carbonylated pr oteins were apparent prior to exercise that corresponded

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60 to the approximate sizes of myosin heavy chain, -actinin, and -actin. The presence of carbonylation was lower in vitamin E suppl emented horses (P<0.07). Following the SET, the relative amount of carbonylated m yosin heavy chain and actin remained substantially lower for the vitamin E suppl emented group (P<0.02). The lower amounts of carbonylated myofibrillar proteins does not equate with improved performance. Times to exhaustion did not differ between contro l and vitamin E supplemented horses (Table 4.7). It is unclear why carbonylation of pre exer cise samples was more extensive than post exercise samples. Carbonylated proteins are present in the gluteus medius four hours after exercise (Kinnunen et al., 2005a ; Kinnunen et al., 2005b). Carbonyls in plasma were shown to increase post exercise, were the highest at 4 hours post exercise, and had not returned to baseline values at 24 hours post exercise. In the muscle tissue, 4HNE-modified proteins (marker of lipid pe roxidation) did not increase between pre exercise samples and at 4 hours post exercise. This demonstrates that measures of lipid peroxidation do not always follow the same tr end as markers of protein oxidation and helps to explain the results of our study in which TBARS wa s not affected in the same way as protein carbonyls by either exercise or treatment. Protein carbonyl levels remained high at 24 hours post exercise as compared to pre exercise samples (Kinnunen et al., 2005a). It is possible, therefore, th at the more extensive carbonylated proteins in our pre exercise samples actually represent car bonylation from chroni c exercise training. These modified proteins may be rapidly eliminated upon commencement of exercise. The means by which muscle protein car bonyls are degraded is unknown but likely does not involve the ubiquitin system.

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61 Ubiquitin was not detected in purified myofibrils at any point in time indicating clearance of damaged musc le fibers by another mech anism (data not shown). Ubiquitinated proteins were, however, present in the supernatant collected from isolate myofibrils, demonstrating that ubiquitin path ways are active (data in appendix). It is possible that by taking biopsies immediately following exercise, the ubiquitin levels detected were not actually reflective of that particular exercise bout. Radak et al. (2000) showed that reactive carbonyl derivatives were detectable in the mitochondrial fraction of rat muscle tissue post exercise but not in the cytosolic fraction. The peptidase-like activity of the proteosome complex (20S a nd 26S proteosomes) was increased in the cytosol following exercise. The 26S proteosome is known to degrade ubiquitinated proteins (Radak et al., 2000), and theref ore, cytosolic expression of ubiquitin may indicate proteins marked for degradati on of the proteosome complex. Since the myofibrillar fraction of our muscle samples did not express ubiquitination, this may explain why they are more su sceptible to carbonylation. How damaged myofibrils are degraded and replaced is still unknown. Sangorrin et al. (2002) states that non-lysosomal pathwa ys are the main regulators of myofibrillar protein breakdown in the early stages of damage but are replaced by lysosomal proteolytic enzymes later. Three non-lysos omal pathways suggested in this study are ubiquitin-proteolytic system, Ca2+-activated proteases ( -calpain and m-calpain), and ATP-independent proteolysis. Our study indi cated that ubiquitin is not present in myofibers. The calpains are unable to degrad e actin and myosin in myofibers (Sangorrin et al., 2002). Therefore, a nother system must be pres ent to degrade damaged and carbonylated myofibers. Sangorri n et al. (2000) reported the presence of a serine-type

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62 protease bound to myofibrils in mouse skeletal muscle. This protease was called protease M and was able to degrade protein constituents from whole myofibrils in vitro. They suggest that this protease could be respons ible for skeletal muscle myofibrillar ATPindependent proteolysis (Sangorrin et al., 2002). Muscle Morphometrics Exercise training, but not diet, caused a cha nge in muscle fiber size. The integrity and size of the gluteus medius mu scle fibers were evaluated by -dystrophin immunostaining (Figure 4.5). No differences in diameter, area, or number of nuclei per fiber were observed between control and vi tamin E supplemented horses (Table 4.8). Exercise training did, however, decrease fiber diameter size (Table 4.9). The distribution of fiber cross-sectional area (CSA) was plotted for control and vitamin E supplemented horses following the SET. As shown in Figure 4.6, very little difference in the distribution of fiber CSA be tween the groups was observed. However, the vitamin E supplemented horses appeared to possess a high er percentage of small muscle fibers. This suggests a shift toward the smaller di ameter, type I oxidative metabolism muscle fiber. The CSAs of muscle fibers in our study were in a similar range to other studies documenting fiber characteristics. Serrano et al. (1996) detected thr ee fiber types in the gluteus medius of horses: (1) Type 1 whic h had a cross sectiona l area (CSA) between 20 and 23 m2 X 100, (2) Type IIA which had a CSA between 22 and 25 m2 X 100, and (3) Type IIB which had a CSA between 26 and 36 m2 X 100. Essen-Gustavsson et al. (1989) showed that five weeks of intense exer cise training caused an increase in type IIA fibers and a decrease in type IIB fibers and also a decrease in fiber area of both types IIA and IIB fibers. Since our study did not fiber ty pe, it is difficult to determine if our horses had the same fiber type shifting. However, our horses did appear to have a decrease in

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63 total fiber diameter following training, a nd our vitamin E supplemented horses had a higher percentage of predicted type I fibers post exercise as compared to controls.

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64 Table 4.1. Effect of vitamin E supple mentation on resting plasma vitamin E concentration of horses in a training program Effect Variable Vitamin E Level (g/mL) Treatment Controla 2.52.17 Vitamin Eb 2.70.15 Time 1 2.48.15 2 2.74.17 Period 1 2.60.12 2 2.61.19 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E Time 1 = initial value; before commencement of 8 wk exercise program Time 2 = pre exercise value at the conclusion of 8 wk exercise program Period 1 = Adaptation phase, training pha se, and SET before diet cross-over Period 2 = Adaptation phase, training phase, an d SET after washout, detraining, and diet cross-over Table 4.2. Effect of vitamin E supplement ation on plasma TBARS concentration in horses following strenuous exercise Time Treatment TBARS Concentration (mmol/L) Pre Controla 143.9.3 Vitamin Eb 141.6.2 Post Control 132.15.9 Vitamin E 140.58.3 Adj Post Control 110.0.8* Vitamin E 118.6.0* 24 Post Control 138.7.4 Vitamin E 138.9.2 48 Post Control 135.1.9 Vitamin E 150.3.9 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E = significantly lower values as compar ed to pre exercise values (P<0.05)

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65 Table 4.3. Plasma TEAC concentrations in control and vitamin E supplemented exercised horses Main Effect Item Treatment TEAC Values^ Treatment Time Treatment X Time Pre Exercise Controla 1.07.02 Vitamin Eb 1.07.02 P= 0.995 P<0.001 P=0.999 Post Exercise Control 1.10.02 Vitamin E 1.10.02 P=0.985 P<0.001 P=0.999 Adj Post Ex# Control 1.00.03 P=0.867 P<0.001 P=0.972 Vitamin E 1.01.04 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E ^ = TEAC values reported in arbitrary TEAC units # = Post exercise values adjusted for pl asma volume by total plasma protein ratio Table 4.4. Plasma TEAC concentrations be fore and after strenuous exercise Time Treatment TEAC Value Pre Controla 1.15.01 Vitamin Eb 1.14.02 Post Control 1.13.01* Vitamin E 1.13.01* Post adj# Control 0.94.01* Vitamin E 0.95.04* 24 Post Control 1.13.04 Vitamin E 1.13.04 48 Post Control 1.16.00 Vitamin E 1.16.01 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E = Values lower than pre exercise values (P<0.05) # = Post exercise values adjusted for plasma volume by total plasma protein ratio

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66 Table 4.5. Effect of vitamin E supplement ation on glutathione levels (mg/dl/%RBC) in horses undergoing moderate exercise (Mean s and SEMs reported) PreExercise PostExercise Week Diet Oxid# Reduced# Total# %Red^ Oxid# Reduced# Total# %Red^ 0 CTLa 0.710.17 1.550.14 2.250.13 69.636.6 Vit Eb 0.620.26 1.710.20 2.260.17 75.889.1 2 CTL 0.470.09 1.190.09 1.960.07* 76.264.4 0.470.16 0.890.17 1.330.12 65.179.3 Vit E 0.410.09 1.430.09 1.850.13* 78.834.2 0.340.09 1.030.07 1.370.07 76.025.8 5 CTL 0.460.14 1.220.12* 1.680.10* 73.817.3 0.410.18 0.920.10 1.270.14 72.018.4 Vit E 0.680.16 1.080.13* 1.730.21* 62.706.5 0.630.08 0.830.13 1.450.13 54.337.5 52 CTL 0.420.12* 1.220.19* 1.610.11* 72.058.5 0.570.08 0.500.08* 1.070.11* 47.397.3 Vit E 0.320.13* 1.360.19* 1.630.18* 81.004.6 0.450.10 0.560.10* 1.000.12* 65.937.8 8 CTL 0.780.11 1.040.11* 1.820.12* 57.915.5* 0.750.10* 0.750.10 1.500.18 50.552.8* Vit E 0.800.12 1.100.23* 1.900.21* 54.676.9* 0.680.12* 0.750.12 1.420.21 55.368.7* 9 CTL 0.720.14 1.240.11* 1.950.12* 64.535.7 0.560.11 0.360.11* 0.890.08* 50.277.4* Vit E 0.570.17 1.250.09* 1.810.18* 72.797.3 0.580.06 0.430.06* 0.960.13* 48.6714.0* Avg CTL 0.590.13 1.240.13* 1.890.11* 69.036.3 0.550.13 0.680.11 1.210.13 57.087.0 Vit E 0.570.16 1.320.16* 1.860.18* 70.986.4 0.540.09 0.720.10 1.240.13 60.068.8 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E Time reported in weeks # reported in mg.dl-1.%rbc-1 ^ Calculated as % of total glutathi one that is in the reduced form 2 horses trained on an incline significant differences in time points (P<0.05) as compar ed to time 0 (pre exercise) or time 2 (post exercise)

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67 Table 4.6. Effect of vitamin E supplementa tion on glutathione measures taken at time points during a standard exercise test (Means and SEMs reported) Time Treatment Oxidized#* Reduced# Total# %Reduced^* Pre Controla 0.72.14 1.24.11 1.95.12 62.90.7 Vit Eb 0.57.17 1.25.09 1.81.18 72.80.3 Post Control 0.56.12 0.37.11 0.89.08 45.76.5 Vit E 0.58.20 0.43.06 0.96.13 48.66.0 24 Post Control 0.81.23 1.16.09 1.96.16 63.15.9 Vit E 0.64.17 1.20.10 1.82.17 68.04.2 48 Post Control 0.89.32 0.97.51 1.85.11 54.58.5 Vit E 0.65.24 0.91.52 1.51.18 68.67.8 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E # reported in mg.dl-1.%rbc-1 ^ Calculated as % of total glutathi one that is in the reduced form significant differences between tr eatments (P<0.05) over time points significant differences between time points (P<0.05) as comp ared to pre exercise value Table 4.7. Effect of vitamin E supple mentation on time to fatigue in a SET Treatment Horse NumberTime to Fatigue (minutes) Average (minutes) Controla 1 5.08 5.22.23 2 5.25 3 4.70 4 5.83 Vitamin Eb 5 5.02 5.09.10 6 4.83 7 5.25 8 5.25 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E n = 4 No statistical differences between treatment groups

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68 Table 4.8. The effect of vitamin E supplemen tation on parameters of muscle fiber sizes Variable Measured Controla Vitamin Eb Area ( m2) 39883676 Diameter ( m) 62 59 #Nuclei/Fiber 4.63.364.45.36 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E Table 4.9. Effects of exercise on muscle cross morphometrics Variable Measured Initial Pre Post Area (m2) 405437363691 Diameter (m) 69* 57 56.07 #Nuclei/fiber 4.51.724.30.690.78.76 = significant difference (P<0.05)

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69 05689 0 50 100 150 200TBARS Concentration (Mmol/L)Week Control Vitamin E 2 * 05689 0 50 100 150 200TBARS Concentration (Mmol/L)Week Control Vitamin E 2 * Figure 4.1. Plasma TBARS concentration is not effected by vitamin E supplementation. Prior to initiation of treadmill exer cise, blood was collected and plasma harvested. TBARS assays were perfo rmed on duplicate samples. Values were calculated based on known concentra tions. Means and SEMs are shown. Asterics indicate concentrations lower than initial concentration (week 0) (P<0.05).

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70 25689 0 50 100 150 200 Week Control Vitamin ETBARS Concentration (Mmol/L) 25689 0 50 100 150 200 Week Control Vitamin ETBARS Concentration (Mmol/L) Figure 4.2. Plasma TBARS values after exercise are not affected by vitamin E supplementation. Plasma was isolated after treadmill exercise. TBARS concentrations were calculated us ing known amounts and values were corrected for plasma volume using plasma protein ratio. Means and SEMs of duplicate samples are shown.

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71 0 Control Vitamin E 025689 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 WeekTEAC Units****** 0 Control Vitamin E 025689 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 WeekTEAC Units 0 Control Vitamin E 025689 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 WeekTEAC Units****** Figure 4.3. TEAC values for pre exercise samples show no difference between control and vitamin E supplemented horses but in crease with improved fitness. TEAC values are reported in arbitrary TEAC units and increase over time in horses undergoing an exercise trai ning program. indicates significantly different time points as compared to week 0 (P<0.05).

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72 MyHC a -actinin actinCTLE CTLE Pre SETPost SET MyHC a -actinin actinCTLE CTLE Pre SETPost SET Figure 4.4. Purified gluteus medius myof ibrillar proteins are carbonylated. Myofibril proteins were purified from muscle biopsies obtained from the gluteus medius prior to SET from control and vitami n E treated horses. Equal amounts of protein were analyzed by Western blot using mouse anti-carbonyl. Immune complexes were visualized by ECL. Representative blots are shown. Predicted myosin heavy chain (MyHC), -actinin, and -actin protein sizes are shown.

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73 Control Pre Control Post VitE Pre VitE Post Control Pre Control Post VitE Pre VitE Post Figure 4.5. Dystrophin imm unostained gluteus medius muscle fiber cryosections obtained from horses before and after SE T exercise. Biopsies were harvested before and after exercise and stored fr ozen. Twelve micron cryosections were collected and incubated with mouse anti -dystrophin. Immune complexes were detected with goat anti-mouse Al exafluor 488. Representative photomicrographs at 200X are depicte

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74 Vitamin E Post Control Post Initial 10001500200025003000350040004500500055006000 0.00 5.00 10.00 15.00 20.00 25.00 30.00Size ( m)Percentage Vitamin E Post Control Post Initial Vitamin E Post Control Post Initial 10001500200025003000350040004500500055006000 0.00 5.00 10.00 15.00 20.00 25.00 30.00Size ( m)Percentage Figure 4.6. Distributi on of muscle fibers sizes from horses before training/supplementation, and before a nd after a standard exercise test. Biopsies were obtained prior to the be ginning of the study and from control and vitamin E supplemented horses upon completion of each 9 week training phase. Cross sectional area (CSA) was m easured and the percentage of fibers in each 1000m2 quartile was calculated.

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75 CHAPTER 5 IMPLICATIONS When all of our results are taken togeth er, this study clearly supports the premise that training enhances the antio xidant status of the animal, and therefore future studies trying to compare antioxidant s upplementation benefits should take into consideration the fitness level and expected workload of the s ubjects. It appears from analyzing the data collected on this study that vitamin E may ha ve a significant effect on the bodys ability to handle oxidative stress. Blood glutathione data and muscle carbonyl data both showed favorable effects of vitamin E supplementation. It is possible that training somewhat mutes many of the effects of antioxidant supplementation simply because increasing fitness will increase the natural antioxidant capacity of the body, as shown by our TEAC data. However, a single strenuous bout of exer cise, such as our SET, is sufficient to show beneficial effects of vitamin E supplementa tion on glutathione and carbonyl parameters measured. It is however, worth mentioning that v itamin E has not been shown to enhance performance and in some cases has been shown to decrease performance. Coombes et al. (2001) showed that extremely high levels of vitamin E supplementation (10,000 IU of additional vitamin E/kg diet over control diet) combined with -lipoic acid actually caused a decrease in maximal twitch tension a nd titanic force producti on in the skeletal muscle of rats when artificially stimulated. In further studies, they used varying levels of vitamin E and added dihydrolipoic acid at di fferent concentrations to baths containing diaphragm muscle strips. The results from this study implicate high levels of vitamin E

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76 in causing depressed skeletal fo rce production at low stimulati on frequencies. Thus, it is possible that feeding too high a level of vitamin E might be detrimental to some measurements of performance. Our study di d not show any differences in indices of performance. It is likely that the level of vitamin E, if suppl emented, must be adjusted to reach potential maximum benefits in the prev ention of muscle damage and reduction of peroxidation without causing impaired pe rformance due to decreased muscle contractility.

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APPENDIX A SUPPLEMENTAL PROTOCOLS Table A.1. Weights of horses at the start of each time period Horse Number Adaptation Period 1 Training Period 1 Adaptation Period 2 Training Period 2 1 509 501 508 510 2 505 495 516 528 3 503 492 505 514 4 494 500 510 513 5 576 587 610 606 6 525 529 531 559 7 604 581 574 596 8 502 500 517 518 Avg 527 523 534 543 Weights are reported in kg. Horses 1-4 were on vitamin E diet for pe riod 1 and control di et for period 2. Horses 5-8 were on control diet for peri od 1 and vitamin E diet for period 2. Period 1 = Adaptation phase, training pha se, and SET before diet cross-over Period 2 = Adaptation phase, training phase, an d SET after washout, de training, and diet Statistical differences not observe d between treatments or periods.

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78 Table A.2. Daily grain intake of horses on each phase of trial Horse Number Adaptation Period 1a Training Period 1b Adaptation Period 2a Training Period 2b 1 6.36 7.52 6.35 7.65 2 6.31 7.42 6.45 7.92 3 6.29 7.38 6.31 7.71 4 6.18 7.50 6.37 7.70 5 7.20 8.80 7.63 9.09 6 6.56 7.94 6.64 8.39 7 7.55 8.72 7.18 8.94 8 6.28 7.50 6.46 7.77 Avg 6.59.18 7.85.21 6.67.17 8.15.21 a = Grain fed at 1.25% of body weight b = Grain fed at 1.5% of body weight Intakes are reported in kg/day. Horses 1-4 were on vitamin E diet for pe riod 1 and control di et for period 2. Horses 5-8 were on control diet for peri od 1 and vitamin E diet for period 2. Period 1 = Adaptation phase, training pha se, and SET before diet cross-over Period 2 = Adaptation phase, training phase, an d SET after washout, de training, and diet Statistical differences not observe d between treatments or periods.

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79 Table A.3. Treadmill training schedule for period 1 Week # Monday TuesdayWednesdayThursdayFriday 1 DNT 1.0 DNT 1.0 1.0 2 1.5 1.5 DNT 1.5 2.0* 3 DNT DNT 2.0 2.0 2.5 4 2.5 2.5 DNT 3.0 3.0 5 3.0 3.0 DNT 3.0* DNT 6 3.0i* 3.0 3.0 3.0i DNT 7 DNT 3.0 3.0i 3.0 3.0i 8 3.0 3.0i DNT 3.0* 3.0i Distance reported in kilometers. i indicates exercise on 6 incline. denotes blood collection day DNT = did not train

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80 Table A.4. Treadmill training schedule for period 2 Week # Monday TuesdayWednesdayThursdayFriday 1 1.0 DNT 1.0 1.0 1.5 2 DNT 1.5 1.5 DNT DNT 3 1.5 2.0* 2.0 2.0 2.5 4 2.5 2.5 DNT 3.0 3.0 5 3.0 3.0 DNT 3.0* 3.0 6 3.0i* 3.0 3.0i DNT DNT 7 3.0i* 3.0 DNT 3.0i DNT 8 3.0 3.0i DNT 3.0* 3.0i Distance reported in kilometers. i indicates exercise on 6 incline. denotes blood collection day DNT = did not train

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81 Table A.5. Plasma proteins values of tr aining phase and SET used for adjusting for plasma volume (Values reported SEM) Week Time Treatment Plasma ProteinsPlasma Proteins Period 1 Period 2 0 Controla 6.53.07 Vit Eb 6.50.06 6.43.03 2 Pre Control 6.25.09 6.35.06 Vit E 6.45.01 6.43.02 Post Control 6.85.07 6.63.09 Vit E 6.98.05 7.05.06 5 Pre Control 6.45.10 6.85.11 Vit E 6.63.02 6.60.02 Post Control 6.83.14 6.50.11 Vit E 6.88.02 6.95.04 52 Pre Control 6.63.19 6.93.07 Vit E 6.33.04 6.38.04 Post Control 6.98.12 6.48.11 Vit E 7.20.03 7.18.13 8 Pre Control 6.35.10 7.23.12 Vit E 6.48.02 6.33.02 Post Control 6.85.11 6.63.07 Vit E 6.65.04 6.78.05 9 Pre Control 6.67.20 6.88.10 Vit E 6.58.16 6.45.06 Post Control 7.87.23 6.60.10 Vit E 7.70.16 7.83.06 24 Post Control 6.43.17 7.93.11 Vit E 6.50.11 6.43.04 48 Post Control 6.33.12 6.48.10 Vit E 6.48.03 6.30.04 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E Period 1 = Adaptation phase, training pha se, and SET before diet cross-over Period 2 = Adaptation phase, training phase, an d SET after washout, de training, and diet cross-over 2 horses trained on an incline

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APPENDIX B ADDITIONAL TBARS DATA

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83 Table B.1. Effect of vitamin E suppleme ntation on plasma thiobarbituric acid reactive substances (TBARS) concentr ation in exercising horses Main Effect Item Treatment TBARS concentration (Mmol/L) Treatment Time Treatment X Time Pre Exercise Controla 128.58.71 Vitamin Eb 132.13.51 p=0.494 p<0.001 p=0.343 Post Exercise* Control 104.27.51 Vitamin E 104.48.79 p=0.972 p=0.072 p=0.20 Difference Control 24.31.56 Vitamin E 27.66.02 p=0.613 p<0.006 p<0.05 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E Difference = pre post Exercise = average of pre or post exercise samples at 5 time points during trial Post exercise values are significantly lower than pre exercise values (P<0.01)

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84 Table B.2. TBARS values for control a nd vitamin E supplemented horses over time Time Treatment Pre TBARS Concentr ation^Post TBARS Concentration^ Initial Controla 126.93 N/A Vitamin Eb 130.64 N/A Week 2 Control 138.84 111.13 Vitamin E 126.39 111.50 Week 5 Control 141.09 88.40 Vitamin E 137.69 104.54 Week 5@ Control 131.00 110.30 Vitamin E 139.51 91.09 Week 8 Control 89.73 101.91 Vitamin E 116.93 98.11 Week 9 (SET) Control 143.91 111.26 Vitamin E 141.60 118.59 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E ^ indicates concentrations reported in Mmol/L and adjusted for plasma volume @ indicates run on incline

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85 Table B.3. Thiobarbituric acid reactiv e substance (TBARS) concentration for pre exercise time points in control and vitamin E supplemented horses by period Treatment Period TBARS concentration^ Controla 1 119.01.53 Controla 2 138.15.10 Vitamin Eb 1 123.53.43 Vitamin Eb 2 140.75.73 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E ^ indicates concentrations reported in Mmo l/L SEM and adjusted for plasma volume

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86 Table B.4. Thiobarbituric acid reactive substance (TBARS) concentration for post exercise time points in control and vitamin E supplemented horses by period Treatment Period TBARS concentration^ Controla 1 100.77.83 Controla 2 108.43.78 Vitamin Eb 1 99.65.50 Vitamin Eb 2 109.88.91 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E ^ indicates concentrations reported in Mmol/L and adjusted for plasma volume

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APPENDIX C ADDITIONAL TEAC DATA

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88 Table C.1. TEAC values pre and post exer cise for control and vitamin E supplemented horses over time Pre Post Time Treatment TEAC values^ TEAC values^ Initial Controla 1.02 N/A Vitamin Eb 1.02 N/A Week 2 Control 1.02 0.93 Vitamin E 1.02 0.97 Week 5 Control 1 1.11 Vitamin E 0.99 1.11 Week 5 incline Control 1.12 0.99 Vitamin E 1.12 0.95 Week 8 Control 1.14 1.09 Vitamin E 1.14 1.1 Week 9 (SET) Control 1.15 0.94 Vitamin E 1.14 0.95 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E ^ indicates TEAC data reported in arbitrary TEAC units and adjusted for plasma volume

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89 Table C.2. TEAC values by period for pre and post exercise Treatment Period Pre TEAC values^Post TEAC values^ Controla 1 1.05.02 1.02.03 2 1.10.01 1.00.02 Vitamin Eb 1 1.04.02 1.01.03 2 1.11.01 1.03.02 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E ^ indicates TEAC data reported in arbitrary TEAC units and adjusted for plasma volume

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APPENDIX D PERIOD EFFECTS FOR GLUTATHIONE DATA AND MUSCLE MORPHOMETRICS

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91 Table D.1. Glutathione data by period for pre exercise samples. SEMs for oxidized, reduced and total glutathione <0.26 mg/dl/%RBC Treatment Period Oxidized#Reduced#Total#%Reduced^ Controla 1 0.739 1.153 1.943 59.94.8 Control 2 0.393 1.433 1.817 78.13.9 Vitamin Eb 1 0.812 1.388 2.184 62.97.1 Vitamin E 2 0.323 1.256 1.540 78.99.7 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E Period 1 = Adaptation phase, training pha se, and SET before diet cross-over Period 2 = Adaptation phase, training phase, an d SET after washout, detraining, and diet # reported in mg.dl-1.%rbc-1 ^ Calculated as % of total glutathione that is in the reduced form; reported SEM

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92 Table D.2. Glutathione data by period for post exercise samples. SEMs for oxidized, reduced and total glutathione <0.26 mg/dl/%RBC. Treatment Period Oxidized#Reduced#Total#%Reduced^ Controla 1 0.761 0.707 1.420 50.07.9 Control 2 0.342 0.659 1.001 64.08.3 Vitamin Eb 1 0.780 0.750 1.531 47.30.4 Vitamin E 2 0.291 0.687 0.953 72.82.0 a = 80 IU vitamin E / kg DM b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E Period 1 = Adaptation phase, training pha se, and SET before diet cross-over Period 2 = Adaptation phase, training phase, an d SET after washout, detraining, and diet # reported in mg.dl-1.%rbc-1 ^ Calculated as % of total glutathione that is in the reduced form; reported SEM Table D.3. Effect of period on muscle morphometrics Variable Measured Period 1 Period 2 Area (m2) 4241.93.463384.84.13 Diameter (m) 59.15.22 61.37.18 #Nuclei/fiber 4.31.91 4.68.57 Period 1 = Adaptation phase, training pha se, and SET before diet cross-over Period 2 = Adaptation phase, training phase, an d SET after washout, de training, and diet Values reported SEM

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93 APPENDIX E UBIQUITIN DATA AND MA P KINASE PATHWAYS

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94 Ubiquitinexpression pre post Figure E.1. Ubiquitin expression in cytosolic component of muscle tissue. Ubiquitin was able to be detected in cytosolic comp onent of gluteus medius muscle samples both pre an post exercise indicati ng the ubiquitin antibody was active.

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95 Procedures for MAP Kinase Blots MAPK, cjun, and total phosphorylation were measured by Western blot. A sample of 150 mg was loaded onto 12% gels, electrophoresed and transfe rred as described in text. Nonfat dry milk (5%) in PBS was used as a blocking solution for one hour. Rabbit antiMAPK was used at a diluti on of 1:1000 overnight at 4 C on a rocker. The secondary antibodies (goat anti-rabbit) were used at a 1:2000 dilution for 45 minutes at room temperature. The blots were exposed to EC L reagents for 1 minute and developed in a darkroom for 1 hour. Total and phospho c-jun were detected in a similar manner except primary antibodies were diluted in 3% BSA rather than nonfat dry milk.

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96 (+) (-) Vit. E(+) Vit. E(+) (-) Vit. E(+) Vit. E P p38 p38 c-jun P c-jun P MAPK MAPK Figure E.2. Summary of stress pathways in cytosolic component of muscle tissue. Pre exercise samples are on the left; pos t exercise samples are on the right.

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APPENDIX F HEART RATE DATA

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98 Table F.1. Heart rate data duri ng a standard exercise test Speed Treatment Heart Rate (beats per minute)^ Pre Control 59.50.42 Vitamin E 77.25.17 Trot Control 141.75.65 Vitamin E 144.25.18 8 m/s Control 173.50.96 Vitamin E 182.25.86 9 m/s Control 200.00.74 Vitamin E 203.50.66 10 m/s Control 207.00.58 Vitamin E 208.50.85 11 m/s Control 213.00.58 Vitamin E 215.75.80 12 m/s Control 220.33.57 Vitamin E 220.67.04 5 min post Control 100.25.54 Vitamin E 108.25.86 ^Values are reported in beat s per minute SEM. (n = 4)

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104 BIOGRAPHICAL SKETCH Kylee Johnson was born in Columbia, Missouri, on March 21, 1977. She relocated to Gainesville, Florida, in 1980 with her fa mily. She attended Buchholz High School and graduated in 1995. She pursued a degr ee in animal biology (Animal Sciences Department) and graduated from the Universi ty of Florida in May of 1999. She took a job in New Jersey working for a competitive international show jumping farm for a year after graduation. In August of 2000, she return ed to the University of Florida to pursue a graduate degree. She received an Alumni Fellowship and worked towards completing a Ph.D. in equine nutrition in the Department of Animal Sciences.


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EFFECTS OF VITAMIN E SUPPLEMENTATION ON OXIDATIVE STRESS
PARAMETERS MEASURED IN EXERCISING HORSES
















By

KYLEE JOHNSON


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Kylee Johnson
































This document is dedicated to my family for their support and encouragement.















ACKNOWLEDGMENTS

I would like to acknowledge the help and guidance I received from my committee

members throughout my graduate program: Dr. Ed Ott, Dr. Lee McDowell, Dr. Richard

Hill, and Dr. Alfred Merrit. They have constantly provided advice and direction and

resources to complete my work on this project. I would also like to thank Dr. Sally

Johnson, who was added to my committee, for allowing me to work in her lab and for her

enthusiasm for my project. Without her guidance, I would not have been able to finish

my laboratory analyses or the writing of this dissertation. She offered new and

innovative ways to examine my project.

I would also like to thank Ms. Karen Scott and Mrs. Jan Kivipelto for help in

running lab assays and analyzing statistical data. They were constantly available to help

with problems, both big and small, and without them this project would never have been

completed.

Finally, I would like to thank all of the students who worked on this project, both

graduates and undergraduates. This was a very labor intensive project which required

immense amounts of physical effort. The students who helped on this project were

absolutely essential and deserve special recognition.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .......... .. .......... .......... ...................... ............ vii

L IS T O F F IG U R E S ........ ..................... .................................................. ............ .. .. ix

LIST OF COMMONLY USED ABBREVIATIONS.........................................x

A B S T R A C T ............................................................ ............... x i

CHAPTER

1 REVIEW OF THE LITERATURE ........................... ...............

E exercise M etabolism ........................ .... ...................... .. ....... ....................... .. 1
O xidativ e Stress ......... ....... ........................................................................ 3
Exercise and Oxidative Stress .................................. ....................................... 9
T he A antioxidant System ............................................ ....................................... 12
Vitam in E ...................... ....................... ................ ............... 14
V itam in E and Oxidative Stress ........................................ ........................ 16
V itam in E and E exercise ......... ................ .................................... .............. 17
M uscle Structure and Com position ........................................ ......................... 20
Protein Carbonyls .................. .... ............ ... .......... .. ................ 25
Muscle Soreness and Oxidative Stress........ ................... ...... ............29
Oxidative Stress and Genetic Pathways in Muscles.........................................34
M A P K inases ............................................................................................36

2 IN T R O D U C T IO N ...................................... .................................... .................... ... 39

3 M ATERIALS AND M ETHOD S ........................................... .......................... 41

A nim als................................................... 4 1
D iets and A adaptation ...................... .. ....................................... ........ 41
W ashout and Cross-O ver Period ........................................... ........................... 43
Training Period ................................... ................................ .........43
Tissue Sam pling.................................................. 44
TBAR S A analysis ......................... ........... .. .. .. ......... ....... 45
TEA C A analysis ...............4......................45



v









Glutathione (reduced, oxidized, and total) Analysis ...............................................46
Vitamin E Assay .............. ... ......................................... ... ..... 47
M u scle A n aly ses ................................................. ................. 4 7
M yofibril P reparation ............... .......... .... .. .......................... .....................47
Dystrophin Immunochemistry and Fiber Morphometrics...............................48
S statistical A n aly sis......................................................................................... .. 4 9

4 RESULTS AND DISCU SSION .......................................... ........................... 52

V itam in E ................................................................5 2
L ipid Peroxidation .......................... ................ ... .... ...... .... ....... 54
Antioxidant Capacity ............. ................ .................. .... ....... 56
Carbonylation and U biquitination ........................................ ......................... 59
M uscle M orphom etrics ............................................................. ............. ............... 62

5 IM PLICA TIO N S ..................................... .. .. ... ..... .............. .. 75

APPENDIX

A SUPPLEMENTAL PROTOCOLS ................................... .................................... 77

B ADDITIONAL TBARS DATA ........................................ ........................... 82

C A D D ITION AL TEA C D A TA ........................................................................ ...... 87

D PERIOD EFFECTS FOR GLUTATHIONE DATA AND MUSCLE
M O R P H O M E T R IC S .................................................................... .... ....................90

E UBIQUITIN DATA AND MAP KINASE PATHWAYS .......................................93

Procedures for M A P K inase B lots .............................................................................95

F H E A R T R A TE D A T A ...................................................................... ...................97

L IT E R A T U R E C IT E D ............................................................................ ....................99

BIOGRAPHICAL SKETCH ............................................................. ............... 104
















LIST OF TABLES


Table pge

3.1 Nutrient composition of hay sampled at selected times during the study ...............50

3.2 Nutrient composition of grain sampled at selected times during study ...................50

4.1 Effect of vitamin E supplementation on resting plasma vitamin E concentration
of horses in a training program .............. ....................................... ....................64

4.2 Effect of vitamin E supplementation on plasma TBARS concentration in horses
follow ing strenuous exercise........................................................ ............. 64

4.3 Plasma TEAC concentrations in control and vitamin E supplemented exercised
h o rses ............... .. ...... .. ............. ............................................6 5

4.4 Plasma TEAC concentrations before and after strenuous exercise........................65

4.5 Effect of vitamin E supplementation on glutathione levels (mg/dl/%RBC) in
horses undergoing moderate exercise (Means and SEMs reported) ......................66

4.6 Effect of vitamin E supplementation on glutathione measures taken at time
points during a standard exercise test (Means and SEMs reported) ......................67

4.7 Effect of vitamin E supplementation on time to fatigue in a SET ...........................67

4.8 The effect of vitamin E supplementation on parameters of muscle fiber sizes........68

4.9 Effects of exercise on muscle cross morphometrics .............................................68

A. 1 Weights of horses at the start of each time period ............... ..... ..........77

A.2 Daily grain intake of horses on each phase of trial .................... .................78

A.3 Treadmill training schedule for period 1 ...................................... ............... 79

A.4 Treadmill training schedule for period 2...................................... ............... 80

A.5 Plasma proteins values of training phase and SET used for adjusting for plasma
volum e (V alues reported + SEM ) ........................................ ........................ 81









B. 1 Effect of vitamin E supplementation on plasma thiobarbituric acid reactive
substances (TBARS) concentration in exercising horses...................................83

B.2 TBARS values for control and vitamin E supplemented horses over time.............. 84

B.3 Thiobarbituric acid reactive substance (TBARS) concentration for pre exercise
time points in control and vitamin E supplemented horses by period ...................85

B.4 Thiobarbituric acid reactive substance (TBARS) concentration for post exercise
time points in control and vitamin E supplemented horses by period ...................86

C. 1 TEAC values pre and post exercise for control and vitamin E supplemented
h o rses ov er tim e .................................................... ................ 8 8

C.2 TEAC values by period for pre and post exercise........................................89

D. 1 Glutathione data by period for pre exercise samples. SEMs for oxidized,
reduced and total glutathione <0.26 mg/dl/%RBC ...............................................91

D.2 Glutathione data by period for post exercise samples. SEMs for oxidized,
reduced and total glutathione <0.26 mg/dl/%RBC. ............................................92

D.3 Effect of period on muscle morphometrics ............................ ..... ...........92

F.1 Heart rate data during a standard exercise test .............. ........................................98
















LIST OF FIGURES


Figure pge

3.1 Sum m ary of experim ental tim etable .......................... ........................................ 51

3.2 Representative image of a-dystrophin immunostaining ........................................51

4.1 Plasma TBARS concentration is not effected by vitamin E supplementation. ......69

4.2 Plasma TBARS values after exercise are not affected by vitamin E
su p p lem entation .................................................... ................ 7 0

4.3 TEAC values for pre exercise samples show no difference between control and
vitamin E supplemented horses but increase with improved fitness.....................71

4.4 Purified gluteus medius myofibrillar proteins are carbonylated ........................ 72

4.5 Dystrophin immunostained gluteus medius muscle fiber cryosections obtained
from horses before and after SET exercise ................................... .................73

4.6 Distribution of muscle fibers sizes from horses before training/supplementation,
and before and after a standard exercise test .........................................................74

E.1 Ubiquitin expression in cytosolic component of muscle tissue ............................94

E.2 Summary of stress pathways in cytosolic component of muscle tissue ...............96















LIST OF COMMONLY USED ABBREVIATIONS


ADP
ATP
CAIII
CK
COX
CSA
DI
DM
8-OHdG
ECL
FADH
FAHP
GSH
GSSG
H202
IU
MAPK
MDA
MyHC
NADH
PBS
PC
PG(I,F2.,E2)
PLA2
PMSF
PUFA
RBC
ROS
SEM
SET
TBA
TBARS
TCA
TEAC
TTP


Adenosine diphosphate
Adenosine triphosphate
Carbonic anhydrase III
Creatine kinase
Cyclooxygenase
Cross sectional area
Deionized
Dry matter
8-hydroxydeoxyguanosine
Enhanced chemilumenescence
flavin adenine dinucleotide
Fatty acid hydroperoxides
Reduced glutathione
Glutathione disulfide (oxidized glutathione)
Hydrogen peroxide
International unit
Mitogen-activated protein kinase
Malondialdehyde
Myosin heavy chain
Nicotinamide adenine dinucleotide
Phosphate buffered saline
Phosphocreatine
Prostaglandin (I,F2a,E2)
Phospholipase A2
Phenylmethylsulfonyl fluoride
Polyunsaturated fatty acid
Red blood cell
Reactive oxygen species
Standard error of means
Standard exercise test
Thiobarbituric acid
Thiobarbituric acid reactive substances
Tricarboxylic acid cycle
Trolox equivalent antioxidant capacity
Tocopherol transfer protein















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECTS OF VITAMIN E SUPPLEMENTATION ON OXIDATIVE STRESS
PARAMETERS MEASURED IN EXERCISING HORSES

By

Kylee Johnson

December 2006

Chair: E. A. Ott
Major Department: Animal Sciences

Exercise places an increased demand on the body's systems, both to provide fuel

for working musculature and to neutralize and dispose of toxic buildup. Byproducts of

demanding performance are reactive free radicals. Consumption of vitamin E, a dietary

antioxidant, may be a plausible way to reduce free radical damage. The present study

examined the effects of excess vitamin E on the presence of oxidation products in blood

and tissue in exercising horses. Eight thoroughbred horses were used in a cross-over

design with one group being fed vitamin E at the level recommended for horses in

moderate to intense work (80 IU/kg DM) (NRC 1989), and the second group being fed

the control diet plus 3000 IU/day d, 1 a-tocopherol acetate. The horses underwent an

eight week training program and a final standard exercise test. Blood samples were

collected at specific points before and after exercise during the training period. At the

end of the eight week training period, a standard exercise test (SET) was performed

during which the horses ran on a 6 incline to exhaustion. Blood and muscle were









collected before and after performing the SET. Neither plasma vitamin E nor TBARS

concentration were influenced by supplemental vitamin E. Blood TEAC values increased

(P<0.05) following five weeks of training in both groups, indicating improved

antioxidant capacity as horses became fitter. Vitamin E supplementation did not alter

plasma reduced, oxidized, or total glutathione levels. However, elevated

reduced:oxidized glutathione ratios were present prior to training-level exercise and at all

time points during the SET in the treated animals (P<0.05). Myofibril carbonylation, a

product of free radical damage, was lower in vitamin E supplemented horses post

exercise (P<0.05). There was, however, no difference between treatment groups in time

to fatigue during a strenuous bout of exercise. Vitamin E did not influence area,

diameter, or number of nuclei per fiber in cross-sections of gluteus medius muscle.

Therefore, this study demonstrates that training influences antioxidant capacity of horses,

and vitamin E influences some measures of oxidative stress in exercising horses,

particularly following a strenuous bout of exercise.














CHAPTER 1
REVIEW OF THE LITERATURE

Exercise Metabolism

Energy for cellular function, both at rest and of work, comes from the body

reserves of carbohydrate, fat, and proteins. The primary energy-carrying molecule in the

body is adenosine triphosphate (ATP). This compound contains a high energy phosphate

bond that, when cleaved by ATPase, liberates energy which can be utilized by the cells

for work. ATP is derived from the breakdown of foodstuffs in the body. However, cells

store only limited amounts of ATP. Excess energy consumed from foodstuffs is stored

by the body in the carbohydrate form as glycogen (muscle and liver tissue), or in adipose

tissue as triglycerides.

When the cell is faced with increased energy demands, such as muscle cell

contraction, metabolic pathways are required to produce ATP rapidly. The formation of

ATP by phosphocreatine breakdown is the first pathway mobilized. Glucose and

glycogen are metabolized by glycolysis to produce ATP. Neither the phosphocreatine

(PC) pathway nor the glycolytic pathways require oxygen and are classified as anaerobic

pathways. The PC pathway involves the donation of a phosphate group and its associated

bond energy from phosphocreatine to ADP (adenosine diphosphate) to form ATP.

Creatine kinase catalyzes this reaction. When ATP stores are liberated at the initiation of

exercise, the molecule is reformed quickly by the PC reaction. However, PC stores in

muscle are limited and the pathway only provides energy for the initiation of exercise and

for high intensity exercise lasting less than five seconds. Phosphocreatine is reformed









during exercise recovery and requires ATP. Thus, the PC system provides energy rapidly

but is depleted quickly.

ATP also is produced fairly rapidly by glycolysis. In this pathway, glucose or

glycogen is degraded to pyruvic acid with the release of ATP. Pyruvic acid, as it

accumulates, is converted to lactic acid which accumulates in the muscle. In this

pathway, bond energy from glucose is used to rejoin inorganic phosphate to ADP. The

process occurs in the sarcoplasm of muscle cells.

Under oxygen sufficient conditions, aerobic production of ATP occurs. This

mitochondrial process utilizes the Krebs cycle (TCA cycle) and the electron transport

chain. The TCA cycle involves the complete removal of hydrogen atoms (oxidation)

from its substrate using the hydrogen carriers NAD and FAD. The hydrogen atom

possesses an unpaired electron and therefore contains potential energy. These electrons

create an electric gradient which is used to generate ATP. Oxygen is not a direct

component of the TCA cycle but is the final hydrogen acceptor for the electron transport

chain (2H2 + 02 -* 2H20). Oxidative phosphorylation allows for aerobic production of

ATP by utilizing potential energy in NADH and FADH (hydrogen carriers) to

rephosphorylate ADP to ATP. There is no direct interaction between the hydrogen

carriers and oxygen. Through P-oxidation, fatty acids formed by the breakdown of body

triglyceride stores are metabolized to acetyl CoA, which enters the TCA cycle. Protein is

not a major fuel source during exercise (2-15% of total fuel contribution), but different

amino acids metabolites are converted to glucose, pyruvic acid, acetyl CoA, or to TCA

cycle intermediates when necessary. This becomes a more important source of energy as

exercise duration increases and glucose is limiting.









The electron transport chain is ultimately responsible for the generation of ATP

from aerobic metabolism. Potential energy from reduced electron carriers (NADH,

FADH) is used to convert ADP to ATP. This is done by passing electrons from NADH

and FADH down a series of electron carriers (cytochromes) releasing enough energy at

three points in the chain to rephosphorylate ADP to ATP. Oxygen acts as the final

electron acceptor and combines with hydrogen to form water. Therefore, oxidative

phosphorylation (through the electron transport chain) is not possible if oxygen is not

present; energy must be generated solely from anaerobic pathways.

Intensity and duration of exercise are the primary determinants of which pathway

contributes the most to energy production. Most athletic endeavors require a

combination of both aerobic and anaerobic metabolism to meet energy demands. Short

term, high intensity exercise relies more heavily on anaerobic metabolism (PC system or

glycolysis). Extremely short term, high intensity muscular contraction is powered

primarily by the PC system. As the length of the exercise increases, the body relies more

on glycolysis. Exercise lasting longer than 45 seconds uses a combination of both

anaerobic and aerobic energy systems. High intensity exercise lasting for approximately

60 seconds is powered by approximately 70% anaerobic and 30% aerobic metabolism.

High intensity exercise lasting two minutes relies equally on anaerobic and aerobic

energy production. Long term submaximal exercise (>10 minutes) relies primarily on

aerobic metabolism.

Oxidative Stress

Oxidative stress refers to a condition in which there is an elevation of steady state

free radicals due to an imbalance in free radical generation versus cellular antioxidant

defenses (Tiidus and Houston, 1995). The term oxidative stress coincides with a large









increase in oxygen utilization (Rokitzki et al., 1994). The increased oxygen

consumption allows for ATP production through oxidative phosphorylation to occur at a

more rapid rate. However, this can lead to the incomplete reduction of 02 as the electron

transport chain becomes overloaded leading to the production of free radicals. A free

radical is a molecule containing one or more unpaired electrons in the outer orbit which

can exist independently (Clarkson and Thompson, 2000). These substances are highly

reactive with other molecules in an effort to gain an electron to stabilize the unpaired

electron. Free radicals always exist in the body but are increased as oxygen consumption

is increased and leakage of the electron transport chain occurs. Normally, 2-5% of

mitochondrial oxygen consumption results in generation of the oxygen free radical

superoxidee) due to electron leakage at intermediary steps in the electron transport chain.

Subsequent reactions produce hydrogen peroxide and hydroxyl radicals (Tiidus and

Houston, 1995).

Molecular oxygen is a diradical containing two unpaired electrons with parallel

spin configurations. Electrons must have opposite spins to occupy the same orbital.

Therefore, electrons added to molecular oxygen must be transferred one at a time during

its reduction. This results in several highly reactive intermediates (Clarkson and

Thompson, 2000). The complete reduction of oxygen to water requires four steps and the

generation of several free radicals and hydrogen peroxide. Hydrogen peroxide is not a

free radical but is a reactive oxygen species because it can generate the highly reactive

hydroxyl free radical through interactions with transition metals (Clarkson and

Thompson, 2000).









The complete reduction of molecular oxygen involves the following ROS

producing steps:

02 + electron -* 02- superoxidee radical)

02- + H20 -- HO2* + OH- (hydroperoxyl radical)

HO2* + electron + H -* H202 (hydrogen peroxide)

H202 + electron -- *OH + OH- (hydroxyl radical)

Each intermediate is highly reactive because each has an unstable electron

configuration which allows for interactions with electrons from other molecules.

Ubiquinone and NADH oxidase are two sites of production for the superoxide radical

along the mitochondrial electron transport chain (Moslen, 1994). The superoxide radical

is unlike other oxygen-derived intermediates because it can lead to the formation of

additional reactive species. It can be protonated to form a hydroperoxyl radical (H02')

which is a much stronger radical than superoxide. Superoxide also can act as a Bronsted

base in aqueous solutions to shift acid-base balance to form hydroperoxyl radical,

thereby, forming hydrogen peroxide in acid environments. Superoxide dismutase, an

enzyme in the body, catalyzes dismutation of superoxide radical at neutral or acidic pH

(Clarkson and Thompson, 2000). Hydrogen peroxide is nonionized and in a low charged

state and therefore can diffuse through hydrophobic membranes thereby allowing it to

leak from mitochondrion. It can form a hydroxyl radical by reduction, by interaction

with superoxide, or by interaction with reduced forms of metal ions such as copper and

iron, which can act as electron donors. Hydrogen peroxide has the ability to remove or

add hydrogen molecules to unsaturated hydrogen bonds of organic lipids. However, it

has a very short half life (1 X 10-9 sec @ 37C), and therefore has limited diffusion









capability (Clarkson and Thompson, 2000). On the other hand, lipid peroxyl radicals can

be formed by lipid interaction with ROS and have half lives of seconds. Therefore, they

can travel in the bloodstream to distant locations to propagate oxidative damage (Moslen,

1994, Sen, 1995). ROS that are formed from the reduction of 02 can attract an electron

from other molecules and result in another free radical. This creates a chain that

contributes to lipid peroxidation, DNA damage, and protein degradation (Clarkson and

Thompson, 2000).

Damage by ROS occurs in practically every component of the cell, including

peroxidation of proteins, nucleic acids, and lipids. However, lipid peroxidation levels are

a primary common measure of tissue damage because of their ease of measurement. One

of the main measurable end products of lipid peroxidation is malondialdehyde (MDA)

which is often measured by the thiobarbituric acid reactive substances assay (TBARS).

Lipid peroxidation is thought to cause exercise induced myopathies and hemolysis

(Chiaradia et al., 1998). There are three described steps of lipid peroxidation. First is

initiation: conjugated dienes are formed through the removal of a hydrogen atom from a

backbone methylene group (-CH2) of a polyunsaturated fatty acid. This allows for the

interaction of molecular oxygen with carbon centered free radicals to form a peroxyl

radical which is highly reactive. This peroxyl radical will attack other compounds in the

body to form hydroperoxides and a new carbon centered radical, thus causing chain

propagation, the second step of lipid peroxidation. Polyunsaturated fatty acids (i.e.,

biological membranes) are particularly vulnerable because of multiple unsaturation

points. In the following diagrams of lipid peroxidation adapted from Surai (2002),









Moslen (1994), and Sen (1995), LH represents part of a stable lipid molecule and *

denotes a radical.

LH -- L* (carbon centered radical)

L* + 02 LOO* (lipid peroxyl radical)

LOO* + LH -* LOOH + L* (lipid hydroperoxide and new carbon centered

radical)

This chain will continue to propagate until the final step of lipid peroxidation:

termination (Clarkson and Thompson, 2000). Termination requires another molecule to

donate or remove an electron to stabilize the existing free radical thus becoming a radical

itself. The chain is terminated when the molecule becoming the radical is less reactive

and is able to be handled and disposed of by the body.

Peroxidative reactions are essential to the body; they regulate important functions

such as defense against microorganisms, cell signaling, vascular control, cell generation

and degradation, and control of cellular homeostasis. However, when excess production

of biologically active oxidative compounds overwhelms the capacity of the body's

cellular antioxidant defense mechanisms, these products may cause cell and organ

damage by disrupting normal physiology which can begin and/or accelerate disease

processes. Disease itself can initiate formation of these products (Basu, 2003).

Peroxidation products can be formed both enzymatically and non-enzymatically. Non-

enzymatic free radical induced lipid peroxidation was described in the previous

paragraph. It requires a compound such as a polyunsaturated fatty acid (PUFA) to react

with an oxidant inducer to form a free radical intermediate which then reacts with oxygen

to form the peroxyl radical (LOO-). These radicals also may attract membrane proteins.









Lipid peroxidation can occur also through enzymatic processes. Phospholipase A2 is

activated by oxygen radicals or hydrogen peroxide and results in the release of

arachidonic acid through hydrolysis of esterified arachidonic acid from the sn-2 position

of phospholipids in cell membranes (Basu, 2003). This generally occurs as the first step

to repair peroxidized phospholipids. Lipid peroxidation activates phospholipase A2, and

alterations in the molecular conformation of oxidized phospholipids are thought to

facilitate access of the phospholipase to the cleavage site (Moslen, 1994). The result is

that arachidonic acid is released and acts as a substrate of the cyclooxygenase (COX2)

enzyme or the lipoxygenase enzyme, both of which produce inflammatory products and

free radical intermediates (Moslen, 1994). The enzymatic oxidation of arachidonic acid

may cause COX to be deactivated due to attack on the enzyme by release of ROS during

enzymatic reduction of hydroperoxides (Basu, 2003).

Besides damaging lipid components of membranes and potentially increasing

production of inflammatory products, free radicals can cause significant damage to DNA

and proteins. It is thought that oxidative damage is responsible for approximately 10,000

DNA base modifications per cell per day. Oxidation, methylation, deamination and

depurination are endogenous processes that lead to significant DNA damage with

oxidation representing the most significant (Surai, 2002). Oxidative damage to DNA

causes base damage, sugar lesions, single strand DNA breaks, and DNA-nucleoprotein

crosslinks. For example, the 8-hydroxydeoxyguanosine (8-OHdG), a guanine base

modification, induces G-C to T-A transversion during DNA replication. This type of

cytotoxic effect could lead to concomitant change in DNA genotype (Radak et al., 1999).

Proteins also are susceptible to oxidative damage. Oxidation of proteins can lead to









formation of reversible disulfide bridges or in more severe cases, the formation of

chemically modified derivatives such as Schiff s bases (Surai, 2002). If protein receptors

on the cell membrane are attacked and modified, functionality of the membrane is

compromised.

Exercise and Oxidative Stress

During oxidative metabolism, much of the oxygen consumed is bound to hydrogen

during oxidative phosphorylation, thus forming water (Clarkson and Thompson, 2000).

As oxidative phosphorylation (ATP production) increases, so does formation of free

radicals. Extreme aerobic stress causes an increased energy requirement, making

increased oxygen utilization necessary (Rokitzki et al., 1994). Exercise induced

oxidative stress refers to a condition in which oxygen free radicals are released in

muscles through overload of the mitochondrial oxidative phosphorylation system or from

inflammatory cells. Activated oxygen species play a role in exercise induced injury to

muscle membrane components and in associated alteration of lysozomal and

mitochondrial enzyme activity (i.e., breakdown of membranes and leakage of enzymes)

(Jacob and Burri, 1996). Therefore, leakage of mitochondrial and lysozomal enzymes is

sometimes used as a measure of oxidative stress. Lipid peroxidation products are used

most frequently as a measure of oxidative stress. Lipid peroxidation probably occurs

primarily at the sight of energy formation, the working musculature. Increased oxygen

utilization, increased lipid mobilization for energy, and hypoxia all lead to increased lipid

peroxidation (Rokitzki et al., 1994). Biological membranes are composed of

polyunsaturated fatty acids and are vulnerable to oxidative damage due to multiple

unsaturation points. Oxidative damage to membranes can result in a change in membrane

fluidity, compromised integrity, and inactivation of membrane bound receptors and









enzymes causing loss of membrane function (Clarkson and Thompson, 2000). Oxidative

damage to proteins leads to physical changes such as fragmentation and aggregation

which makes them more susceptible to degradation (Clarkson and Thompson, 2000).

Exercise can generate free radicals also by (1) increases in epinephrine and other

catecholamines that can produce oxygen radicals when they are metabolically

inactivated, (2) production of lactic acid which can convert weakly damaging free radical

superoxide to strongly damaging radical hydroxyl, and (3) inflammatory responses to

secondary muscle damage incurred with overexertion (Clarkson and Thompson, 2000).

Other sources of free radicals include prostanoid metabolism, xanthine oxidase,

NAD(P)H oxidase, and radicals released by macrophages (Urso and Clarkson, 2003).

The Jenkins theory states that free radical mediated mechanism involving lipid

peroxidation and loss of membrane integrity could possibly be the cause of delayed onset

muscle soreness (Rokitzki et al., 1994).

Matsuki and colleagues (1991) proposed that in exercised horses phospholipid

hydroperoxides are the primary products of peroxidation and disrupt cellular homeostasis

by activating phospholipase A. This causes an accumulation of lysophospholipids in

muscle membranes. End products of peroxidation, particularly malondialdehyde (MDA),

are associated with polymerization and aggregation of membrane compounds and

reduction of cellular function. Exercised thoroughbreds demonstrated peroxidation of

phosphatidylethanolamine (PE) was increased before exercise and post exercise as

compared to resting controls, and peroxidized PE showed an increasing tendency from

pre-exercise to 10 minutes post exercise to 24 hours post exercise. Total MDA (both free

and protein bound) increased in all 4 horses at 10 minutes and 24 hours post-exercise









(Matsuki et al., 1991). The authors concluded that muscular PE was particularly

susceptible to oxidative stress due to exercise, that peroxidized phospholipids were able

to activate phospholipase, and that total MDA increased largely because of an increase in

protein bound MDA, supporting previous reports that MDA is highly reactive to proteins

(Matsuki et al., 1991).

Chiardia et al. (1998) reported MDA content increased in response to exercise. In

the experiment, ten stallions were trained over a period of three months and then

underwent a series of physical exercise bouts of increasing intensity. The horses were

sampled before exercise, immediately after limbering up, after the exercise bouts, and 18

hours after exercise. MDA increased dramatically after strenuous exercise as compared

to MDA levels during a limbering up period, suggesting that lipid peroxidation is related

to intensity of exercise. Also, the elimination of lipid peroxidation products was shown

to be a slow process (Chiaradia et al., 1998).

Exhaustive exercise also results in a marked increase in TBARS (a lipid

peroxidation product thought to be indicative of cell membrane breakdown) in both the

muscle and liver with a decreasing tendency within 24 hours in non-endurance trained

rats (Brady, 1979). Intracellular glutathione rapidly oxidizes to glutathione disulfide

(GSSG) in the presence of hydrogen peroxide and hydroperoxides but rapidly reduces

back to glutathione if oxidative stress is not severe. Therefore, GSSG is used as a marker

of oxidative stress. Sastre and associates reported that trained men exercised to

exhaustion on a treadmill had increased amounts of GSSG (oxidized glutathione)

immediately after exercise but values returned to resting state well within one hour

(1992).









The Antioxidant System

The antioxidant system consists of both enzymatic and non-enzymatic components

which work coordinately to detoxify the body of ROS. Most antioxidants also act as

prooxidants under certain conditions (Jacob and Burri, 1996). The enzymatic component

of the body's antioxidant system is comprised of the enzymes glutathione peroxidase,

superoxide dismutase, and catalase which each detoxify ROS. Glutathione peroxidase is

located in mitochondria and cytosol. Increases in oxygen consumption activate the

enzyme to remove hydrogen peroxide and organic hyperperoxides from the cell.

Reduced glutathione (GSH) is used by glutathione peroxidase to detoxify hydrogen

peroxide with oxidized glutathione (GSSG) being formed as a result. Glutathione

reductase is necessary to convert GSSG to GSH which also contributes to detoxification

of hydrogen peroxide (Urso and Clarkson, 2003). Regeneration of glutathione ultimately

derives from glucose. The glutathione system is under enzymatic regulation via

glutathione reductase and glutathione peroxidase and it regenerates other antioxidants in

vivo (Jacob and Burri, 1996). The reaction 2 H202 2 H20 + 02 is catalyzed by the

enzyme catalase which is widely distributed in cells but concentrated in mitochondrion

and peroxisomes. Glutathione peroxidase is an enzyme with greater affinity for hydrogen

peroxide than catalase. Therefore, it is thought that catalase increases when oxidative

stress overwhelms the glutathione peroxidase system (Urso and Clarkson, 2003).

Superoxide dismutase acts on superoxide radicals. It catalyzes the addition of hydrogen

ions to convert two superoxide anions into hydrogen peroxide and 02 (202* + 2H+ -

H202 + 02) (Surai, 2002).

The non-enzymatic components of the antioxidant system involve dietary

constituents which act as less specific antioxidants. These include both fat soluble and









water soluble antioxidants. Vitamins A and E, carotenoids, and ubiquinones comprise

the fat soluble group, while ascorbic acid (vitamin C) and uric acid act as aqueous

antioxidants. Vitamins E and C are the most commonly studied and supplemented

antioxidants. Vitamin C acts primarily as an aqueous phase peroxyl and radical

scavenger. It is concentrated in tissue and fluids with highest potential for radical

generation (i.e., eye, brain, liver, lung, heart, semen, leukocytes). The oxidized form is

dehydroascorbic acid. It is converted back to its reduced form by glutathione, NADPH,

or both. It can regenerate the reduced form of vitamin E (recycling). It also inhibits the

formation of carcinogenic nitrosamines, especially in the stomach (Jacob and Burri,

1996). Vitamin C is very high in neutrophils and is necessary for immune function (Urso

and Clarkson, 2003). Vitamin C is synthesized by the horse and therefore not generally

supplemented. Carotenes are colored pigments found in yellow and green vegetables.

Some, such as P-carotene, are precursors to vitamin A. Carotenes currently do not have

an RDA value. Beta-carotenes and lycopenes are chain breaking antioxidants and singlet

oxygen quenchers in vitro (Jacob and Burri, 1996). Two studies (one using a metabolic

unit and one free living study) showed that carotene depletion was associated with up to a

five fold increase in plasma TBARS. Also, hexanal (a compound associated with

oxidative damage to low density lipoproteins) showed a 19% decrease after carotene

repletion (Jacob and Burri, 1996).

At the cellular level estrogen may serve as an antioxidant as well. It has a similar

structure to vitamin E. Vitamin E has a diterpenoid side chain that allows for its insertion

into membranes. It has a trimethylhydroquinone head portion which quenches free

radicals and is responsible for its antioxidant activity. Estradiol has two hydroxyl groups









potentially capable of arresting lipid peroxidation during exercise (Clarkson and

Thompson, 2000).

Vitamin E

Vitamin E refers to eight compounds tocopherolss and tocotrienols). Each

compound consists of a chromanol head and isoprenoid side chain (saturated in

tocopherols and unsaturated in tocotrienols). There are a, 0, y, and 6 forms of each which

differ in the number and position of methyl groups on the aromatic ring. Alpha

tocopherol is the most biologically active form in vivo. Tocopherols exist in

enantiomeric forms, designated d and 1. Their biological activity can be expressed as

international units (IU). One IU is equivalent to the activity of 1 mg d, 1 a-tocopherol

acetate (Surai, 2002), a form of vitamin E often supplemented due to increased stability

provided by the acetate molecule. Humans preferentially absorb and transport a-

tocopherol above all other forms (Tiidus and Houston, 1995). In nature, only plants can

synthesize vitamin E (Surai, 2002).

In animal tissue, most vitamin E is located in the phospholipid membrane bilayers.

The chromanol head is localized near the hydrophilic outer region, and the isoprenoid

side chain is associated with the hydrophobic lipid inner regions. The membrane

composition of vitamin E ranges from 1:1000 (vitamin E:lipid) in red blood cells (RBC)

to 1:2000 in mitochondrial membranes to <1:3000 in other tissues (Tiidus and Houston,

1995). The vitamin E content of individual membranes may be based on membrane

infrastructure and not easily influenced by excessive vitamin E dietary intake. The level

of vitamin E in the plasma is limited by the ability of tocopherol binding protein to

incorporate vitamin E into VLDL (very low density lipoproteins) (Tiidus and Houston,

1995). It has been shown that a-tocopherol concentrations in normal subjects can only be









increased three to four fold by supplementation (Blatt et al., 2001). Humans given 100

IU a-tocopherol/kg body weight (for an average of 5 to 7 grams total) showed increased

plasma values of a-tocopherol approximately 4 fold above baseline at 6 hours which

decreased to 3 fold above baseline at 24 hours. Possible reasons for the inability to raise

plasma levels higher include: (1) decreasing efficiency of intestinal absorption (2)

saturation of tocopherol transfer protein (TTP) which limits hepatic a-tocopherol

metabolism and (3) redistribution of plasma a-tocopherol into tissue (particularly adipose

tissue) (Blatt et al., 2001). After changes in vitamin E intake, it was shown that a-

tocopherol plasma levels reach equilibrium within 30 days. However, a-tocopherol

content of adipose tissue takes approximately two years to equilibrate and is released

very slowly from adipocytes during experimental deficiency (Blatt et al., 2001). The

availability of adipose tissue vitamin E stores to the animal is unclear. Machlin et al.

(1979) found that the rate of a-tocopherol loss from adipose tissue was negligible in

guinea pigs fed a vitamin E deficient diet, even during a four day fast. The concentration

of a-tocopherol in muscle, heart and liver are highly correlated to plasma values.

Plasma and tissue tocopherol concentrations remain highly stable, suggesting that

they may be tightly regulated. Alpha tocopherol can be bound by a-tocopherol transfer

protein, tocopherol associated protein and tocopherol binding protein, which may

possibly serve as regulatory proteins. Blatt et al. (2001) compared distribution of vitamin

E from the blood into other tissue compartments to perfusion of fat soluble drugs.

Tissues were subdivided into rapidly perfused central compartments (heart, lungs, brain,

kidney, liver), slowly perfused peripheral compartments (other organs, muscle, skin), and

very slowly perfused compartments (adipose tissue). Vitamin E concentrations in rats









reach equilibrium more quickly in blood and tissue which are rapidly perfused and have

lower lipid content and greater tocopherol regulatory protein (TRP) function. However,

this rate of equilibrium is not representative of the tissue's final concentration due to

tissue redistribution. Redistribution to adipose tissue is thought to be significant since

approximately 90% of the body's total vitamin E is in adipose tissue. The final

equilibrium concentration of vitamin E in tissues probably depends on TRP functions,

tissue lipid content, vitamin E uptake and efflux, oxidative stress, vitamin E metabolism

and interactions between vitamin E and other antioxidants (Blatt et al., 2001).

Vitamin E and Oxidative Stress

It is possible that oxidative stress may affect the vitamin E binding proteins.

Hepatic tocopherol transfer protein (TTP) and its mRNA are increased in diabetic rats,

and plasma a-tocopherol is increased in diabetic humans and rats. Increased TTP levels

were detected in brains from humans with a vitamin E deficiency or diseases associated

with cerebral oxidative stress such as Alzheimer's disease and Down's syndrome. Thus,

upregulation of cerebral TTP during oxidative stress may occur. TRPs might be

regulated by oxidative stress and/or the vitamin E levels of its target tissue (Blatt et al.,

2001).

X-ray structure determination of a-tocopherol suggests that the chromanol head

maintains a pair of electrons on the ring oxygen almost perpendicular to the plane of the

ring. This stabilizes the formation of the vitamin E radical that is created by quenching

free radicals (Tiidus and Houston, 1995). Vitamin E directly scavenges most free

radicals including superoxide, hydroxyl radical, and lipid peroxides by using the

hydroxyl groups on the chroman head to either donate a proton or accept an electron.

The resulting vitamin E radical will ultimately either react with itself or with another









peroxyl radical to form non-reactive degeneration byproducts (Tiidus and Houston,

1995). Another important function of vitamin E is that it is a structural membrane

stabilizer because it increases membrane microviscosity and decreases passive

permeability to low molecular weight substances (Tiidus and Houston, 1995). It was

proposed that the phytyl tail of a-tocopherol associates with the hydrocarbon part of the

lipid bilayer. The phytyl tail positions the chroman ring system towards the membrane

interface allowing for the phytyl chain and the arachidonyl chains of membrane

phospholipids to interact. This facilitates close packing of polyunsaturated fatty acids

(PUFAs) which stabilizes the membrane and protects it from phospholipase attack. Only

the RRR-a-tocopherol form has the appropriate configuration to interact with the

membrane phospholipids. The phenolic group of a-tocopherol is located near the polar

moiety of the lipid, and increasing concentrations of a-tocopherol broaden the

temperature range of the gel to liquid-crystalline phase transition (Surai, 2002). Alpha-

tocopherol also inhibits phospholipase A2 (PLA2) activity towards lamellar fluid

membranes, thereby, protecting the membranes from attack. Alpha tocopherol decreases

both the initial rate of the lipase and also the extent of hydrolysis. It is a non-competitive

inhibition and is thought to be due to an effect of a-tocopherol on the membrane, not on

the enzyme (Surai, 2002).

Vitamin E and Exercise

In vitamin E deficient animals, exercise increases susceptibility to free radical

damage and results in premature exhaustion, greater fragility of lysosomal membranes,

and marked depression of muscle mitochondrial control (Meydani et al., 1992). It

appears that muscle or tissue vitamin E concentrations do not increase as a result of

training in animals or humans. Endurance training significantly increases the number of









muscle mitochondria, and therefore it is a likely effect that vitamin E concentration in

mitochondrial membranes decreases as a result of endurance training. However, most

studies suggest that oxidative stress is decreased as a result of endurance training (i.e. due

to increased enzymatic antioxidant capacities). Therefore, alterations in muscle vitamin

E concentration may not be critical to the body's ability to adapt to oxidative stress

during endurance training (Tiidus and Houston, 1995). It was demonstrated that

endurance performance of animals with an a-tocopherol deficiency was 40% lower than

normally fed animals, and oxygen radical concentration increase was 2 to 3 times higher

(Rokitzki et al., 1994).

Packer et al. suggested that there may be an interorgan transport of vitamin E

during exercise (1990). It may involve the liver and adipose tissue as exporters and the

muscle and heart (areas of increased oxidative stress) as importers. However there is

little evidence to support this. Some studies have reported significant elevations in

plasma vitamin E post-exercise in humans suggesting possible interorgan transport.

However, these studies did not account for exercise-induced haemoconcentration. A

study using nine younger men (22-29 yrs) and twelve older men (55-74 yrs) showed

increased blood levels of vitamin E after exercise. They consumed either 800 IU vitamin

E or placebo for 48 days. Subsequently, they engaged in a bout of eccentric exercise by

running downhill on a treadmill at 75% maximum heart rate. After 48 days, plasma

concentrations of a-tocopherol increased (56% in young, 70% in older) in the

supplemented group and a-tocopherol decreased (99% in young, 60% in older) in the

placebo group. In the placebo group, both age groups had increased urinary TBA that

was significant at 12 days post exercise. In the vitamin E group, there was no change in









TBA in 24 hour urine samples. Muscle levels of linoleic and arachidonic acid were

higher in supplemented men post exercise as compared to placebo (Tiidus and Houston,

1995).

Variable results on the efficacy of vitamin E to reduce peroxidative stress induced

by exercise exist for horses, humans, dogs, and rats. Variation in these tests, which

measure primarily lipid peroxidation products, is likely due to differences in duration and

intensity of exercise and fitness level of the subjects. The concentration of MDA in

plasma of animals that are sufficient in vitamin E seem to vary according to intensity of

exercise and fitness level of subjects. Extremely fit subjects have no increase and in fact

sometimes a decrease in MDA when exercised at submaximal levels suggesting an

enhanced antioxidant system in fit, trained animals (Dekkers et al., 1996). However,

when comparing vitamin E sufficient to vitamin E deficient rats, tocopherol

supplementation resulted in decreased TBARS (lipid peroxidation measurement) and

hydroperoxide levels in the plasma after exercise. Therefore, supplementation of vitamin

E to vitamin E deficient animals reduces tissue peroxidation regardless of training status.

Vitamin E supplementation may affect peroxidation levels in subjects performing

repeated bouts of intense or exhaustive exercise. Exhaustive exercise at submaximal

workloads in rats was found to increase free radical production (determined by

electroparamagnetic resonance signals) both in muscle homogenates and in portions of

intact muscles. The increase in free radical production was associated with decreased

mitochondrial respiratory control, loss of sarcoplasmic and endoplasmic reticulum, and

increased levels of lipid peroxidation (Singh, 1992). Humans given 600 mg dl-a-

tocopherol three times daily for two weeks had a decrease in pentane production during









graded exercise up to 75% of VO2 max. Humans given 300 mg d-a-tocopherol acetate

daily for four weeks exhibited lower exercise induced increases in plasma lipid

peroxidation products after vitamin E supplementation versus before supplementation

(Witt et al., 1992). Jenkins et al. (1993) showed that MDA increased significantly after

exhaustive exercise at 70% V02 max in both trained and untrained rats (60% and 63%

respectively). Lovlin et al. (1987) did a study using human subjects on a bicycle

ergometer and showed that increases in peroxidation products may depend on intensity of

exercise with exhaustive exercise causing a more significant increase. Vitamin E

possibly counters the increase.

Muscle Structure and Composition

Skeletal muscle is striated because of a transverse banding pattern observed

microscopically. It is a voluntary muscle. Nerve fibers and blood vessels enter and exit

the muscle along the connective tissue that covers it. A muscle cell is the structural unit

of the skeletal muscle tissue and is also known as a muscle fiber or myofiber. Muscle

volume is composed 75 to 92 % of myofibers. The rest is comprised of connective

tissues, blood vessels, nerve fibers, and extracellular fluid. The muscle fibers of

mammals and birds are long, multinucleated, unbranched cells that taper slightly at both

ends. Fibers may be several centimeters long but do not generally extend the length of

the entire muscle. They can be 10 to 100 micrometers in diameter. They are surrounded

by a membrane called the sarcolemma which is composed of protein and lipid and is very

elastic to allow for distortion during contraction. The cytoplasm of muscle fibers is

called the sarcoplasm and is composed 75 to 80% of water. It also consists of glycogen

granules, ribosomes, proteins, nonprotein nitrogenous compounds and other inorganic

materials. The number of nuclei per muscle fiber is not constant. Nuclei are more









concentrated and irregularly distributed at tendinous attachments and motor end plate

units (raised area caused by structures present at the myoneural junction). In mammals,

myofiber nuclei are located at the periphery of the fiber, just beneath the sarcolemma.

Myofibrils are long thin rod-like structures within the myofibers. They are

surrounded in sarcoplasm and extend the entire length of the muscle fiber. There are two

types of myofilaments within the myofibrils. Thick filaments are aligned parallel to each

other and are in exact alignment across the entire myofibril. Thin filaments are also in

exact alignment across the myofibril and are parallel to each other and to the thick

filaments. Thick and thin filaments overlap in certain regions along their longitudinal

axes and are aligned in bands which cause the striated look of the myofibril. There are

areas of different density within the bands of the muscle. The A band of the muscle is

much denser than the I band but both are bisected by thin, dense lines. The I band is

bisected by a band called the Z disk. A unit of the myofibril spanning two adjacent Z

disks is called the sarcomere. The sarcomere encompasses an A band and the two half I

bands located on each side of the A band. The sarcomere is the repeating structural unit

of the myofibril. This is where muscle contraction and relaxation occur. Thick filaments

of the myofibril compose the A band of the sarcomere and are referred to as myosin

filaments since the predominant protein in their structure is myosin. Actin is the

predominant protein in the thin filament and they are therefore referred to as actin

filaments. Actin filaments terminate at the Z disk and extremely thin filaments, known as

Z filaments, comprise the Z disk and connect with actin filaments on either side of the

disk. Each actin filament connects to four Z filaments. Each of those four Z filaments

then connects with an actin filament in the adjacent sarcomere. Myofibrils are composed









of more than 20 different proteins. Six of these account for more than 90% of the total

myofibrillar protein and are in order of decreasing abundance: myosin, actin, titin,

tropomyosin, troponin, and nebulin. They are classified by function as contractile,

regulatory, or cytoskeletal. Actin and myosin are the major contractile proteins, while

tropomyosin and troponin are regulatory. Regulatory proteins regulate actin-myosin

interactions during contraction. Titin and nebulin are cytoskeletal proteins and are the

template/scaffold for the alignment of myofilaments during myofibril and sarcomere

formation to form the Z disk.

Actin is a globular protein. It becomes fibrous in nature when monomers of the

globular protein (G-actin) polymerize to form fibrous actin (F-actin). The G-actin

monomers are linked together in strands, and two strands of F-actin are spirally coiled

around each other to form a super helix which is characteristic of the actin filament.

Myosin constitutes approximately 45% of myofibrillar protein. It is elongated in a rod

shape and has a thickened end portion called the head region and a long thin portion

referred to as the rod or tail region which forms the backbone of the thick filament.

These two fractions are known as light chain and heavy chain meromyosin. The center of

the A band contains only the rod portion of the myosin molecule. The heads of the

myosin contain the functionally active sites of the thick filament which form cross

bridges with actin filaments during muscle contraction. Each myosin head attaches to a

G-actin molecule of the actin filament. Skeletal muscle has mitochondria which are

abundant at the periphery of the fiber near the poles of nuclei and are also abundant at

motor end plates. Mitochondria are located between the myofibrils, adjacent to Z disks, I

bands, or A-I band junctions. Lysosomes are vesicles located in the sarcoplasm that









contain enzymes which together are capable of digesting the cell and its contents. These

enzymes include a group of proteolytic enzymes known as cathepsins.

A muscle itself is composed of several individual fibers grouped together into

bundles called fasciculi. The number of fibers per muscle is not constant. The outer cell

membrane of an individual muscle fiber is called the sarcolemma and is surrounded by a

delicate connective tissue covering called the endomysium which is composed of

collagen fibers. Twenty to forty muscle fibers form a group called a primary bundle.

Different numbers of primary bundles are grouped together to form secondary bundles.

Primary and secondary bundles are both surrounded by a sheath of collagen connective

tissue called the perimysium. Secondary bundles group together to form the muscle

which is surrounded by the connective sheath called the epimysium. Each muscle has at

least one artery and one vein for circulation. Blood vessels and nerve fibers are

associated with the epimysium and enter through the perimysium. Branches of each

supply individual muscle fibers and are supported by the endomysium. Blood vessels

cover a large portion of the muscle's surface and allow for exchange of nutrients and

waste products of metabolism.

There are four isoforms of the myosin heavy chain protein that have been identified

in rats and some other mammals: types I, IIA, IIX(C), and IIB. Each fiber can be

classified according to the predominant type of myosin isoform it contains. Muscle fibers

classified as red muscle fibers contain primarily types I and IIA. White muscle fibers

contain primarily types IIX(C) and IIB. Type I fibers are slower contracting as compared

to type II fibers. Red fibers are red due to a higher myoglobin content. Myoglobin stores

oxygen, and therefore these fibers have a high proportion of enzymes involved in









oxidative metabolism and lower levels of glycolytic enzymes. White fibers have a high

content of glycolytic enzymes and a low level of oxidative enzymes. Therefore, red

fibers have a higher number and a greater size of mitochondria as compared to white

fibers as well as greater capillary density. Red fibers have a greater lipid content (source

of fuel) and a lower glycogen content than white fibers. White fibers primarily produce

energy from glycolytic metabolism and can operate either aerobically or anaerobically.

They have a more extensively developed sarcoplasmic reticulum and T-tubule system

and have a more rapid contraction speed. Sarcoplasmic proteins include myoglobin and

enzymes associated with glycolysis, the tricarboxylic acid cycle, and the electron

transport chain. Their Z discs are more narrow. White fibers contract rapidly in short

bursts and fatigue quickly. This is called a phasic mode of action. Red fibers have a

tonic mode of action: they contract more slowly but for a longer period of time. They

fatigue less easily if given a constant supply of oxygen.

In equine muscle, three major fiber types have been identified (I, IIA, and IIB), and

a minor type C. Type IIX has not been separated from IIA and IIB by the histochemical

m-ATPase staining typically used. A study by Serrano et al. (1996) used electrophoresis

to separate fiber isoform at different depths of gluteus medius and gluteus profundus

muscles of five sedentary horses. They found that two of the MyHC isoforms in the

gluteus medius muscle comigrated with type I and IIa MyHC isoforms of rat diaphragms

(control). A third isoform had a migration more similar to that of type IIX than to IIB

MyHC of the rat diaphragm. Only types I and IIA were detected in the gluteus profundus

muscle. The bands representing type I MyHC increased in thickness as depth of

sampling increased (8cm>6cm>4cm>2cm). The band similar to IIX MyHC had a reverse









pattern with the thickest bands being at the shallowest depths. There were no variations

in the thickness of the IIa band. Type I fibers reacted with anti-slow MyHC antibody S-

58, while type IIA and IIB fibers reacted with the anti-fast MyHC antibody MY-32.

Protein Carbonyls

Reactive oxygen species (ROS) are produced normally in skeletal muscle fibers in

low amounts where they function during muscle contractility (Barreiro et al., 2005). At

elevated concentrations, ROS are neutralized by the intracellular antioxidant defense

system. Accumulation of ROS disrupts muscle cellular functions including action

potential conduction, excitation-contraction coupling, contractile proteins, and

mitochondrial respiration (Barreiro et al., 2005). Carbonyls result from oxidation of

arginine, lysine, threonine, or proline amino acids. Carbonylation of amino acids can

occur in different ways. Hydroxyl radicals are highly reactive and thought to be

generated in vivo by catalytic action of transition metals such as iron and copper which

will bind to specific sites of proteins and modify nearby amino acid residues (Goto et al.,

1999). The amount of total iron but not copper in the kidney was increased by age, as

was the levels of carbonylated proteins in the kidney (Goto et al., 1999). This suggests

that iron could be responsible for the generation of oxidatively damaged proteins in this

tissue. However, the distribution of non-heme iron did not match that of carbonylated

proteins suggesting that carbonylation may occur by other mechanisms (Goto et al.,

1999). Vitellogenins are a family of proteins stored in the yolk of developing oocytes.

They also are found in abundance in the carbonylated form in aged wild nematodes

(Caenorhabditis elegans). The biological significance of this is not clear. Vitellogenin

can bind metals such as zinc, cadmium, and iron and therefore may have a biological role

in body fluid later in life as a protection against oxidative damage to other cellular









components by acting as a sink for metals which catalyze reactions forming active

oxygen species. In a similar manner, albumin, an abundant extracellular protein, may act

as an antioxidant by binding iron leading to inhibition of lipid peroxidation (Goto et al.,

1999). Carbonyl groups also result from glycation/glycoxidation of lysine, cysteines, or

histidine amino acids with a or 0 unsaturated aldehydes formed during the peroxidation

of polyunsaturated fatty acids (Barreiro et al., 2005). When fatty acid residues of

phospholipids in cell membranes are peroxidized, cell membranes lose integrity and

potentially harmful aldehydes and alkanes are formed (Radak et al., 1999). Oxidation of

proteins causes them to lose their function and become targets for proteolytic degradation

(Radak et al., 1999). Carbonylation of several cytosolic proteins including 0-tubulin, 3-

actin, and creatine kinase in brain samples of patients with Alzheimer's disease occurs

(Barreiro et al., 2005). Goto et al. (1999) suggested that a-actin and myosin heavy chain

are carbonylated in skeletal muscle. Proteins of quadricep muscles of rats trained for 4

weeks at 4000 m altitude oxygen pressure exhibited a significantly higher extent of

carbonylation than untrained rats or rats trained at sea level (Radak et al., 1999). Proteins

which showed a marked increase in signal intensity by Western blot were actions, judged

by their molecular weight and abundance. Actins and myosin heavy chains in the cells of

human arteries and veins also are highly carbonylated, indicating that contractile proteins

are susceptible to oxidation (Goto et al., 1999). However, while protein oxidation did

increase due to exercise, lipid peroxidation measured by TBARS and amount of lipid

peroxides did not differ between animals exposed to sea level and high-altitude

conditions. This suggests that protein oxidation occurs independently of lipid oxidation.

Radak et al. (2002) stated that oxidized proteins accumulated at a much higher rate (5-









10% of total cellular proteins) than lipid or DNA (<0.1% at steady state level). Goto et

al. (1999) also found a 40% increase of protein carbonyls in the lung following

exhaustive running of rats.

Septic shock causes significant deterioration of ventilatory and limb muscles to

generate force and sustain workloads. Evidence suggests that increased levels of ROS

and nitric oxide (NO) are responsible for sepsis-induced muscle dysfunction, as indicated

by improvement of muscle contractility in septic animals treated with antioxidants and

inhibitors of NO synthases (Barreiro et al., 2005). Following induction of sepsis, weakly

carbonylated proteins in the range of 50 and 29 kD in the control diaphragms were

reported. After one hour of LPS injection, carbonyl group signal intensity rose

significantly with total carbonyl OD exceeding 200% of that detected in control

diaphragm. A more pronounced increase occurred at 12 hours post LPS injection,

reaching around 300% of that detected in the control diaphragms. The increased total

carbonylation resulted from introduction of new carbonyls on existing proteins and also

the appearance of new carbonylated protein bands with molecular masses greater than 50

kD and less than 29 kD. Carbonyl groups were detected inside diaphragmatic muscle

fibers close to the sarcolemma in both the control and septic animals. Positive carbonyl

immunostaining also was detected in large blood vessels supplying muscle fibers.

Fifteen different carbonylated proteins with varying intensities were detected in the

cytosolic fraction of septic rat diaphragms. Three glycolytic enzymes were carbonylated

with aldolase being strongly carbonylated and enolase 3p and glyceraldehydes 3-

phosphate dehydrogenase being weakly carbonylated. Creatine kinase and carbonic

anhydrase III were strongly carbonylated in the cytosolic fraction of the septic rat









diaphragms. Carbonic anhydrase III (CA III) is a member of zinc metallo-enzymes that

catalyze the reversible hydration of carbon dioxide. Glutathione interacts with CA III by

forming a disulfide link with two of the five cysteine residues of CA III in a process

called S-glutathionylation, suggesting that CA III may have a role in antioxidant defenses

against ROS formation in skeletal muscle fibers. In the myofibrillar-mitochondrial

fraction of the septic rat diaphragms, the most strongly carbonylated protein was a-actin.

Oxidative modifications of actin involve modification of at least seven methionine

residues which is associated with extreme disruption of actin filaments, inhibition of

polymerization, and impaired interaction with the myosin protein. Ubiquinol-cytochrome

c reductase (complex III of the electron transport chain in the mitochondria) was the

second most strongly carbonylated protein spot. Mitochondrial creatine kinase was

carbonylated. Creatine kinases are enzymes that catalyze the reversible transfer of a

phosphoryl group from ATP to creatine to produce ADP and phosphocreatine and are

localized both in the cytosol and mitochondria. They are critical for energy metabolism

of skeletal and cardiac muscle cells. Creatine kinase and aldolase activities are

negatively correlated with their respective carbonylation level suggesting that

carbonylation negatively effects enzyme activity. In summary, this study showed that

protein carbonylation involves several key enzymes of glycolysis (enolase, aldolase, and

GADPH), ATP production (complex III of the mitochondrial respiratory chain and

creatine kinases), one myofibrillar protein (a-actin), and a regulator of CO2 hydration

(carbonic anhydrase III). Only enolase and carbonic anhydrase III showed significant

rise in carbonylation one hour after LPS injection. Carbonylation of the other proteins









rose significantly 6 hours after LPS injection. Diaphragmatic contractile dysfunction

occurred 12 hours after LPS injection (Barreiro et al., 2005).

Muscle Soreness and Oxidative Stress

Delayed onset muscle soreness (DOMS) that appears between 24 to 48 hours post

exercise may be due to an acute inflammatory response (MacIntyre et al., 1995). "Acute

phase response" refers to host defense responses and metabolic reactions that occur

during infection. These are observed after exercise and contribute to the breakdown and

clearance of damaged tissue after exercise. Inappropriate release of these defense

mechanisms can damage host tissues and is thought to be the basis of noninfectious

inflammatory diseases (Cannon et al., 1990). Exercise induces reactions that are similar

to acute phase response such as the influx of neutrophils and macrophages into muscle

tissue as well as activation of cytokines following muscle damage (Sacheck and

Blumberg, 2001). Eccentric exercise is particularly damaging because the muscle is

forced to lengthen as it develops tension. Delayed muscle soreness and infiltration occur

following eccentric exercise (Cannon et al., 1990).

Neutrophils are released from the bone marrow and have a half life in circulation of

approximately 10 hours. Epinephrine, increased blood flow, and inflammatory mediators

promote release of neutrophils into circulation. Neutrophils are drawn into specific sites

by products of inflammation or infection (chemotaxis) where they release free radicals

and degradative enzymes such as elastase and lysozyme. Neutrophils probably live only

1 to 2 days after migrating into tissue (Cannon et al., 1990).

Antioxidants increase human neutrophil chemotaxis in vitro. After damaging

exercise, vitamin E may promote neutrophil accumulation at specific sites of tissue

damage (Cannon et al., 1990). During reperfusion, an increase in tissue neutrophil









content was observed (Cannon et al., 1990). Oxygen metabolites, such as hydrogen

peroxide, that are produced by activated macrophages depress lymphocyte proliferation

(Blumberg, 1994). Aging is associated with an increase in PGE2 production which also

inhibits lymphocyte proliferation (Blumberg, 1994). Vitamin E decreases hydrogen

peroxide formation in polymorphonuclear leukocytes (PMN) (Blumberg, 1994). Vitamin

E decreases the rate of PGE2 synthesis. Building an immune response requires

membrane bound receptor mediated communication between cells as well as between

protein and lipid mediators. This can be affected directly or indirectly by vitamin E

status. To further this, leukotrienes and HETE (products of the lipoxygenase pathway)

inhibit lymphocyte proliferation possibly by decreasing T helper and increasing T

suppressor cell proliferation (Blumberg, 1994). Lipid peroxides stimulate

cyclooxygenase pathways by providing an oxygen species to enhance enzyme activity.

An inverse relationship was noted between serum vitamin E and PGI, PGE2, and PGF2,.

Vitamin E bidirectionally modulates lipoxygenase activity on arachidonic acid. Normal

plasma vitamin E concentration enhances lipoxygenation of arachidonic acid, but higher

levels of vitamin E suppress the effect. This effect of vitamin E is probably due to its

role as a hydroperoxide scavenger. Therefore, the enhancement of immune function by

vitamin E is most likely due, in part, to its reduction of reactive oxygen metabolites such

as hydrogen peroxide and by its inhibition of cyclooxygenase and lipoxygenase pathways

(Blumberg, 1994).

Exercise can cause mobilization of inflammatory agents although the specific

events initiating this are unknown. The magnitude of the inflammatory response varies

with the duration and intensity of exercise although the exact role of inflammation during









exercise induced muscle injury is undefined (MacIntyre et al., 1995). Evidence suggests

exercise can cause early activation of neutrophils (MacIntyre et al., 1995). Neutrophil

accumulation in muscle was observed in exercise induced muscle injuries after endurance

tests on mice (MacIntyre et al., 1995). Following neutrophil accumulation, monocytes

and macrophages are present and responsible for the resorption of neutrophils in necrotic

tissue. Macrophages are the predominant inflammatory cell present in exercise induced

muscle injury (MacIntyre et al., 1995).

Men were supplemented 800 IU/day vitamin E for 48 days that correlated with a

reduction in plasma cytokine (IL-10 and IL-6) response to muscle damage inducing

eccentric exercise (Cannon et al., 1990). Lower IL-10 was associated with lower 3-

methylhistidine excretion, a marker of proteolysis (Cannon et al., 1990). It is uncertain

whether an increase in oxidative stress that occurs with exercise is necessary for muscle

adaptation to occur or whether it is harmful, causing muscle damage that impairs the

ability to perform or train (Urso and Clarkson, 2003).

In a second study, 21 untrained male volunteers in two age ranges (22-29 and 55-

74) were supplemented with 800 IU a-tocopherol per day for 48 days before exercise

(Cannon et al., 1990). Subjects were monitored for 12 days post exercise for changes in

circulating leukocytes, superoxide release from neutrophils, lipid peroxidation, and efflux

of intramuscular CK into circulation. The <30 year old placebo group had higher plasma

creatine kinase (CK) and significantly greater neutrophilia than the >55 year old group.

At the time of peak concentration in the plasma, CK correlated significantly with

superoxide release from neutrophils. This supports the concept that neutrophils are

involved in the delayed increase in muscle membrane permeability after damaging









exercise. There was no significant change in plasma vitamin E concentration observed

during the 72 hour period post exercise. Vitamin E supplementation tended to increase

plasma CK values post-exercise, particularly in the >55 year age group. Vitamin E

tended to reduce CK values in the <30 year age group and increase it in the >55 year

group on days 2 and 5 post exercise. Circulating neutrophil levels peaked in both

supplemented and unsupplemented <30 age groups 6 hours post exercise. The >55 year

group exhibited a much smaller increase in neutrophil count, peaking 3 hours post

exercise. The neutrophil count in the vitamin E supplemented group >55 years was

similar to the <30 year groups. On the days post exercise, the >55 year supplemented

group had a higher neutrophil count compared to the placebo. The <30 year

supplemented group had lower counts than the placebo. There was no difference in

immature band cells observed at any time supporting the concept that vitamin E acts as a

chemotaxis for neutrophils. The plasma lipid peroxide concentrations increased in 18 of

the subjects within 24 hours of exercise but the time this increase occurred varied

considerably between individuals resulting in no statistically significant increase at any

particular point. Increases in CK in >55 year supplemented group appear to contradict

the hypothesis that exercise induced changes in muscle membrane permeability are the

result of damage by oxygen radicals (i.e. leakage of membrane due to decreased

stability). The authors suggest that CK is representative of increased muscle protein turn-

over to clear partly damaged proteins. Possibly, older subjects have lower clearance

mechanisms and vitamin E promotes neutrophil accumulation at specific sites of muscle

damage to clear damaged proteins.









Neutrophils are damaged by their own products and are protected by vitamin E.

Diminished neutrophilia in placebo >55 group may reflect greater autooxidation of

neutrophils that have become more vulnerable to oxidative damage with age. By

protecting neutrophils, vitamin E may indirectly promote a net increase in muscle

membrane damage, even though it has a direct positive effect on muscle membrane itself

(Cannon et al., 1990).

Therefore, mobilization and activation of neutrophils may contribute to an

increased myocellular enzyme leakage after eccentric exercise. Age related differences

in response appear to be modulated by dietary supplementation of vitamin E, possibly

due to its chemotaxic properties and its protection of neutrophils.

A likely chemical stimulant of pain sensation is prostaglandin E2 (PGE2), which

causes an increased sensitivity of the pain receptors and is synthesized by macrophages

and possibly neutrophils (Chan et al., 1989). Neutrophils provide essential products for

the formation of prostaglandins (Chan et al., 1989). Possibly when muscle is damaged,

the injured cells cause an increase in the synthesis of PGE2 and therefore a pain sensation.

Vitamin E significantly inhibits the metabolism of arachidonic acid by the lipoxygenase

pathway (Chan et al., 1989). Arachidonic acid is the most abundant 20 carbon

polyunsaturated fatty acid in the phospholipids of mammalian tissue and is a precursor to

many biological compounds that mediate inflammation (Reddanna et al., 1989). Both the

lipoxygenase and cyclooxygenase pathways exhibit an obligatory requirement for fatty

acid hydroperoxides (FAHP). Therefore, FAHP are implicated as modulators of the

arachidonic acid cascade which generates inflammatory products through the

lipoxygenase and cyclooxygenase pathways. The fact that lipid peroxidation provides a









source of FAHP formation makes these pathways of particular interest in the study of

antioxidants (Reddanna et al., 1989).

A review by Blumberg (1994) suggests that vitamin E supplementation reduced

plasma lipid peroxides and production of PGE2 by polymorphonuclear leukocytes (PMN)

in elderly subjects. Aging is associated with increases in PGE2 production which inhibits

lymphocyte proliferation. Vitamin E decreases PGE2 production and improves cellular

immunity. Vitamin E also neutralizes H202 and radicals used by the immune system to

kill pathogens. This diminishes in vitro killing capacity of the cells but protects the

immune cell itself from autooxidation, thereby improving phagocytosis (Blumberg,

1994). Also, the enhancement of immune function by vitamin E may relate to a change

in membrane receptor molecules involved in the immune response as vitamin E has been

shown to induce changes in cell surface glycoconjugates (Blumberg, 1994).

Oxidative Stress and Genetic Pathways in Muscles

Bursts of oxidant production and extreme changes in activities of antioxidant

defenses alter gene expression. Free radicals and their reactions directly affect processes

of cell differentiation, aging, and transformation leading to the conclusion that cells have

evolved pathways to utilize ROS as biological stimuli. ROS influence expression of

some genes and signal transduction pathways and are thought to act as subcellular

messengers for certain growth factors (Allen and Tresini, 2000). Changes in cellular

redox status influence transcriptional modification of collagen (Chojkier et al., 1989),

collagenase (Brenneisen et al., 1997), post translational control of ferritin (Hentze et al.,

1989), activation of the transcriptional factors Myb (Myrset et al., 1993) and Egr-1

(Huang and Adamson, 1993), and also binding activity of the fos/jun (AP-1) protein

conjugate (activator protein 1) (Abate et al., 1990). Exposure of normal and transformed









cells to UV radiation or H202 causes increased expression ofjun-B, jun-D, c-fos, and fos-

B (early response proteins). Antioxidant treatments stimulate increases in the expression

of some genes. It is possible that the increase is caused by autooxidation of antioxidant

compounds producing enough ROS to cause a change in gene expression through

oxidizing properties rather than reducing properties (Allen and Tresini, 2000). Choi and

Moore (1993) studied several structural isomers of butylated hydroxytoluene and showed

that only the compounds with high antioxidant potential are capable of inducing c-fos.

The isomers without antioxidant properties did not induce c-fos (Choi and Moore, 1993).

Others demonstrated that antioxidant enzymes modulate signal transduction and gene

expression. This suggests that the redox potential oxidant/antioxidant treatments is at

least partially, if not completely, responsible for their effect on gene expression (Allen

and Tresini, 2000). Lander (1997) proposed that cellular responses influenced by ROS

and reactive nitrogen species can be grouped into five broad categories: (1) modulation of

cytokine, growth factor, or hormone action/secretion, (2) ion transport, (3) transcription,

(4) neuromodulation, (5) apoptosis. The effects of redox changes on transcription factors

and signal transduction are not clear but are possibly mediated through oxidation and

reduction of protein sulfhydryls. Changes in redox state of the protein sulfhydryls causes

conformational changes that can either impair or enhance DNA binding activity, release

inhibitory subunits, or promote protein complex formations necessary for signal

transduction or transcription to proceed. There are multiple conserved cysteine residues

in some protein kinase C (PKC) that are possible targets for redox regulation and may

explain redox effects on pathways influenced by PKC activation. The redox-sensitive









pathways, MAP (mitogen-activated protein) kinase and NF-xB (nuclear factor-KB) signal

transduction pathways, have multiple steps sensitive to ROS.

MAP Kinases

Growth factors and cytokines are examples of extracellular signaling molecules

which induce changes in a cell via mechanisms that involve transmission of the signal

from the plasma membrane to the nucleus where gene expression is altered. To activate

this signaling cascade, first a receptor is activated which either has protein kinase activity

or activates a protein kinase in the cytoplasm. This signal continues to be transmitted

until it reaches the nucleus where it activates transcription factors regulating gene

expression. MAP kinases are one of the most studied groups of signal transduction

pathways. There are four types of MAP kinases: (1) ERK extracellularr regulated

kinase) (2) JNK kinase (c-jun NH2-terminal kinase)/SAP kinase (stress activated protein

kinase) (3) p38 kinases (4) big MAP kinase (BMK/ERK). All subfamilies have redox

sensitive sites. In the ERK and JNK pathways, Ras is activated by conversion of Ras-

GDP to Ras-GTP by a guanine nucleotide exchange factor (GEF) such as the mammalian

homologue of Sos. Sos contains SH2 domains which bind to tyrosine phosphorylated

motifs in other proteins. Sos is constitutively bound in the cytoplasm to another SH2

domain-containing protein, Grb-2. When a receptor is activated, i.e. by a ligand binding

to it, tyrosine is autophosphorylated and SH2 domain-containing receptors are recruited

including Grb-2. This causes plasma membrane targeting of Sos where it is tyrosine

phosphorylated. The phosphorylated Grb-2/Sos complex binds to an adapter protein (the

SH-containing protein), and the multi-protein complex converts Ras-GDP to Ras-GTP,

thereby activating it. Treatment of cells with oxidants such as H202 stimulates the

formation of the SH-containing protein-Grb2-Sos complex. Ras will eventually return to









baseline levels by action of Ras-GTPase activating proteins which inactivate Ras.

Oxidizing treatments such as hypoxia/reoxygenation or stimulation with H202 activate

Raf-1 possibly through effects on Ras. Once Raf-1 is activated, it phosphorylates series

in the catalytic sites of MKK/MEK. MKK1/MEK1 and MKK2/MEK2 activate members

of the MAP kinase family (ERK-1/ERK-2) which are kinases that regulate downstream

responses to many mitogenic, apoptotic, differentiation-inducing stimuli. MEK1 but not

MEK 2 is stimulated by exposure to H202, indicating that only MEK1 is redox sensitive

(Allen and Tresini, 2000).

MAP kinases are activated by dual phosphorylation of tyrosine and threonine

residues located on the kinase. This is catalyzed by threonine and tyrosine kinases

belonging to the MKK family. ERK, JNK, and p38 are MAP kinase subfamilies which

phosphorylate the COOH-terminal transcriptional activation domain of ERK-1 and SAP-

1 which then associate with other nuclear proteins to form a ternary complex factor

(TCF). TCF proteins cannot bind to the promoter region but instead require interaction

with other transcriptional factors such as SRF (serum responsive factor). Transcription of

many genes is mediated by the binding of the multiprotein complex of an SRF

homodimer and a TCF family member to the SRE (serum response element) in the gene

promoter region. UV light and environmental stress have been shown to activate the

JNK, p38, and big MAP kinase pathway while antioxidants and growth factors induce the

activation of the ERK isoforms p44/ERK1 and p42/ERK2. Therefore, oxidants and

reductants play a role on redox sensitive genes through their actions on MAP kinase

pathways. Other proteins influenced by redox status are the NF-KB/Rel family of

transcription factors involved in the regulation of many genes (such as acute phase






38


proteins, cell surface receptors, and cytokines). The effects of redox state exerted on any

given gene can vary from tissue to tissue (Allen and Tresini, 2000). Collart et al. (1995)

showed that H202 and ionizing radiation had extremely varying results on the induction

of c-jun. Depending on the cell type, H202 caused a dramatic range of reactions from

large increases to no effect at all. This emphasizes the fact that signal transduction

pathways are different in different cell types, and therefore the effect of redox status will

vary from tissue to tissue.














CHAPTER 2
INTRODUCTION

Strenuous exercise increases the production of ROS (reactive oxygen species) in

the body causing an increase in oxidative stress (Powers and Hamilton 1999). As oxygen

consumption increases, there is the potential for the electron transport chain to become

overwhelmed and electrons to leak into the mitochondrial space where they can react

with components of the cell and give rise to ROS. These ROS can damage the

mitochondria and other parts of the cell, rendering the cell not functional. Free radical

damage is associated with destruction of cell membranes and aggregation of membrane

compounds (Matsuki et al. 1991). The generation of ROS during exercise is likely a

cause of muscular disturbances, inflammation, and pain in performance animals (Avellini

et al. 1999).

The extent of damage done by ROS to the exercising subject is unknown. Free

radical damage leads to higher levels of oxidation products in blood and tissue (Singh

1992). These products may lead to further damage of muscle cells as well as generation

of biologically active compounds able to propagate the peroxidation chain or enter

inflammatory pathways (Dekkers et al. 1996).

There is increased interest in providing supplemental dietary antioxidants to

exercising subjects in an attempt to reduce the levels of free radicals in the body and

therefore reduce the damage done to the animal by these substances. However, the

benefits of these supplements are controversial. The purpose of this study was to

determine whether exercising horses supplemented vitamin E above NRC






40


recommendations had decreased measures of oxidative stress in blood and muscle

parameters both over the course of a training program and following a single strenuous

bout of exercise.














CHAPTER 3
MATERIALS AND METHODS

Animals

Eight untrained healthy thoroughbred geldings between the ages of 3 and 10 years

of age were used in a complete repeated measures (crossover) design. Untrained horses

were used in order to monitor the response of the animals to supplementation as fitness

level improves. The horses' health and soundness were evaluated prior to the start of the

experiment. All horses were maintained between body condition score of 4.5 to 5. All

animal procedures were conducted within the guidelines of and approved by the

University of Florida Institutional Care and Use Committee.

Diets and Adaptation

Prior to being divided into treatment groups, all horses were put on the control

diet for three weeks and familiarized with the treadmill. A control baseline standard

exercise test (SET) was performed at the end of the adaptation period. Subsequently, the

animals were divided into control and vitamin E supplemented groups and acclimated to

the respective diets for seven weeks to achieve a consistent level of vitamin E in both

blood and tissue. The horses on the control diet were fed a 12% crude protein textured

oat-based feed at a rate of 1.25 kg feed per 100 kg body weight and ad libitum coastal

Bermuda grass hay. Vitamin E content of the grain was 80 IU/kg DM as recommended

by the NRC (1989) for horses undergoing moderate to intense levels of work. The horses

receiving vitamin E supplementation were given the same diet as the control but were

supplemented with 3000 IU vitamin E/day in the form of a-tocopherol acetate. This level









was chosen based on a prior work demonstrating that a basal diet containing 44 IU

vitamin E/kg DM or 124 IU vitamin E/kg DM were not sufficient to prevent decreases in

plasma and muscle ca-tocopherol status due to repeated exercise training (Siciliano et al.,

1997). However, 344 IU vitamin E/kg DM maintained vitamin E status in the blood and

muscle. Supplementation of 3000 IU/day placed all vitamin E supplemented horses

above 300 IU/kg DM of concentrate.

The horses were fed the concentrate mixture twice daily, once in the morning and

once in the afternoon. Once training began, the amount of concentrate fed was increased

from 1.25% of body weight to 1.5% of body weight divided equally between the

feedings. Hay (Table 3.1) and feed (Table 3.2) were sampled throughout the project and

analyzed for nutrient values. The treatment group received supplementation as a daily

evening top dressing (68.2 grams of a vitamin E premix containing 20,000 IU vitamin

E/lb). The premix was composed of d, 1 a-tocopherol acetate mixed with a rice meal

carrier. The control group received 68.2 grams of a placebo (rice meal feed). In addition,

all horses received ad libitum access to coastal Bermudagrass hay. The horses were

housed at the University of Florida in 4X4 meter stalls or in an adjacent dry lot turnout

paddock. The use of the dry lot paddock eliminated grazing and helped control dietary

intake. The horses were rotated daily in groups of four with four being kept in stalls and

four being kept in the turn out paddock. On days of blood sampling, all horses were kept

in stalls beginning on the evening prior to collection to eliminate variations in

temperature and climate.









Washout and Cross-Over Period

All horses underwent a 15 week washout period during which they were turned

out to pasture, fed the control diet, and left untrained. The horses were then crossed over

and the experiment repeated beginning with the adaptation period. Due to mechanical

problems with the treadmill, the adaptation period for the second half of the experiment

lasted ten weeks rather than seven. During the extra three weeks, the horses were fed

their respective diets and left untrained. Blood was collected both at the end of seven

weeks and at the end often weeks of adaptation, and all analyses were run and

statistically compared to ensure that there were no baseline differences between weeks

seven and ten due to the difference in times on treatment diets. Therefore, there was a

total of 25 weeks (6 months) between the end of one training phase and the beginning of

the next. This should have provided adequate time for the horses to detrain (return to

baseline fitness levels) and for blood and muscle vitamin E levels to equilibrate. Blood

samples were taken prior to the start of each training phase to ensure that the horses had

similar vitamin E levels between the two phases. The horses followed the exercise

schedule performed in the first half of the experiment.

Training Period

The training and standard exercise tests were conducted at the University of

Florida Veterinary Medicine Teaching Hospital in a climate controlled room equipped

with a treadmill. The training period consisted of eight weeks of gradually increasing

workloads. Treadmill exercise was performed three to four days per week, depending on

availability of the treadmill. The first three days the horses trotted at 4 m/s for 0.6 km on

a flat surface, galloped at 8 m/s for 1.0 km on a flat surface, and cooled down by trotting









at 4 m/s for 0.6 km on a flat surface. The gallop distance was increased by 0.5 km every

three days so that by the end of three weeks, the horses were performing the following

exercise: trotting at 4 m/s for 0.6 km on a flat surface, galloping at 8 m/s for 3.0 km, and

trotting at 4 m/s for 0.4 km on a flat surface. The gallop phase was performed twice a

week on a flat surface and twice a week on a 60 incline for the remainder of the study. A

standard exercise test (SET) was performed at the end of each eight week conditioning

period. The protocol was as follows: 4 m/s for 2 minutes on a flat surface, 8 m/s for 1

minute at a 60 incline, 9 m/s for 1 minute, 10 m/s for 1 minute, 11 m/s for 1 minute, and

12 m/s until they would not continue with moderate persuasion.

Tissue Sampling

Baseline blood samples were taken following the seven week adaptation period.

Once training began, blood response variables were measured before and after exercise at

the end of weeks 2, 5, and 8. At the end of the fifth week, before exercise and after exercise

samples were taken on two days, one during which the horses ran on the flat and one during

which the horses ran on the incline for the first time. This provided a way to compare

different exercise intensity levels when horses were at the same fitness level. Following the

eighth week, all horses performed a standard exercise test with blood and muscle tissue

collected before and after exercise. Blood was collected by jugular puncture in EDTA

tubes. Muscle tissue was taken from the middle gluteal muscle by punch biopsy following

local lidocaine anesthesia. Whole blood was immediately pipetted from the tubes and put on

ice until storage at -800C. Plasma was separated and stored at -800C. Muscle tissue was

immediately divided for future analyses, placed in cryogenic tubes in liquid nitrogen, and

stored at -800C.









TBARS Analysis

Plasma samples were thawed and diluted 1:100 with deionized (DI) water. Working

standards were prepared from the stock standard (100 nM 1,1,3,3-tetramethoxypropane)

each time the procedure was run. One hundred tL of sample or known amount of standard

was mixed with an equal amount of 8.1% sodium dodecyl sulfate. The denatured plasma

was incubated with TBA/Buffer reagent (37 mM, pH 3.5) at 95C for 60 minutes according

to company recommendations (Zeptomatrix; Buffalo, NY). The tubes were covered with

glass marbles to prevent evaporation. The tubes were cooled in ice water for exactly 10

minutes and then allowed to come to room temperature. Reactions were quantified on a

fluorescent spectrofluorometer at the following parameters: excitation of 535 nm, emission

of 552 nm, high sensitivity, slit width of 5 nm. Multiple readings of each sample were

taken. A standard curve was plotted and used to calculate sample values. For post exercise

blood samples, plasma proteins were analyzed using a plasma refractometer. A ratio was

calculated from pre exercise plasma proteins divided by post exercise plasma proteins. This

number was multiplied by the post exercise TBARS concentration to adjust for plasma

volume. Data was reported as both adjusted and unadjusted values.

TEAC Analysis

Prior to beginning the assay, metmyoglobin was synthesized by mixing an equal

volume of 0.4 mM myoglobin and potassium ferricyanide (0.74 mM K3Fe(CN)6).

Metmyoglobin reagent was applied to a Sephadex G50 column and eluted with phosphate

buffered saline (PBS). The concentration of the purified metmyoglobin was calculated

using the Whitburn equation:

Conc. metmyo = 146 (Abs490 Abs700) 108 (Abs560 Abs70o) + 2.1 (Abs580 Abs70o).









The metmyoglobin was diluted with PBS to a final concentration of 70 mM. Assay

stock solutions were prepared fresh and included 5 mM 2,2-azinobis-(3-

ethylbenzothiazoline-6-sulphonic acid) (ABTS reagent), 2.5 mM Trolox, and 0.15% H202 in

PBS. Final reactions were performed in disposable cuvettes in the presence of 1.5 mM

ABTS, 250 [M metmyoglobin, and 40 [l of sample. Reactions were initiated by addition of

20 [l of H202. Cuvettes were inverted once and placed immediately on a spectrophotometer

where they were read every 20 seconds for 4 minutes at 600 nm. Results were reported as a

percent of inhibition. Unknown value/2.5 100 = % inhibition. TEAC concentration was

adjusted by plasma protein ratio for post exercise samples as described in the above section.

Data is reported both as adjusted and unadjusted values.

Glutathione (reduced, oxidized, and total) Analysis

For glutathione assays, whole blood was diluted ten-fold with DI water and mixed

with and equal volume of meta-phosphoric acid (MPA) precipitating reagent (2 mM MPA, 5

M NaC1, 7 mM EDTA). The sample/extraction solution was centrifuged at 800 X G for 15

minutes, and the supernatant was saved and frozen at -80C. The extracted samples were

diluted 1:20 with respective buffers. For reduced glutathione assays, 100 tL of the

extracted, diluted samples was added to 2.0 mL GSH buffer (100 mM NaPO4, 4 mM EDTA,

pH 8.0). Subsequently, 100 tL of 0.1% o-phthaldialdehyde (OPT) in methanol (protected

from light) was added to the tubes and incubated at room temperature for at least 17

minutes. Fluorescence was measured at emission 420 nm, excitation 350 nm, with high

sensitivity and slit width of 5 nm. Results were reported in mg/dl/%RBC. For total

glutathione, the same procedure was followed except that the extracted, diluted sample was

dissolved in 2.0 ml of GSSG buffer (100mM NaOH) and allowed to incubate at room









temperature prior to addition of OPT. Oxidized glutathione was determined by calculation:

(Total glutathione reduced glutathione = oxidized glutathione).

Vitamin E Assay

Vitamin E was analyzed in plasma samples at the initial date (following the

adaptation period but prior to the beginning of any exercise) and on week 8 pre exercise

(Michigan State University Diagnostic Center for Population and Animal Health; East

Lansing, MI). Vitamin E was extracted from the plasma and analyzed by high performance

liquid chromatography (HPLC).

Muscle Analyses

Myofibril Preparation

Muscle tissue was immediately divided for future analyses, placed in cryogenic tubes

and frozen in liquid nitrogen until storage at -800C. To isolate myofibrils, frozen muscle

was placed in a homogenization buffer on ice containing the following: 75 mM KC1, 10

mM Tris (pH 6.8), 2 mM EGTA, 2 mM MgC12, 0.1 mM PMSF, and 0.1% Triton X-100.

After one hour, the samples were homogenized with a polytron, centrifuged at 1000 X G,

rinsed with homogenization buffer, and centrifuged two more times, as described (Gordillo

et al. 2002). The final pellet was resuspended in homogenization buffer, minus the Triton

X-100 and EGTA, containing 50% glycerol. Myofibrils were stored frozen at -20C.

Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad

Laboratories, Inc., Hercules, CA).

Western Blot

Protein carbonyls were measured in myofibrils by Western blot (OxiblotTM Protein

Oxidation Detection Kit, Chemicon International, Temecula, CA). Briefly, 20 [tg of

proteins were electrophoretically separated through 10% polyacrylamide gels and









transferred to nitrocellulose membrane. Membranes were blocked with 5% bovine serum

albumin in phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and

incubated with antibodies according to manufacturer recommendations. Blots were washed

with PBS-T and incubated with goat anti-mouse peroxidase (1:5000, Vector Labs,

Burlingame, CA). After a final wash with PBS-T, immune complexes were visualized by

enhanced chemilumenescence (ECL) and X-ray exposure. For the detection of ubiquitin,

mouse anti-ubiquitin (1:1000, Santa Cruz Biotechnology, Sacramento, CA) was used.

Dystrophin Immunochemistry and Fiber Morphometrics

The integrity and size of the gluteus medius muscle fibers were evaluated by a-

dystrophin immunostaining. Biopsies were obtained before and immediately after exercise

in controls and horses supplemented with vitamin E. Muscle biopsies were frozen in OCT

frozen tissue embedding media and cryosections were collected (8-12 kim) on SuperFrost

glass slides. Tissue was oriented to give cross-sections of muscle. OCT was removed and

tissue sections were incubated with 5% horse serum in PBS for 20 minutes at room

temperature. Subsequently, tissues were incubated in anti-a-dystrophin (1:400) for one hour

at room temperature. After exhaustive washes with PBS, sections were incubated with goat

anti-mouse Alexafluor 488 (1:250, Invitrogen, Carlsbad, CA). Nuclei were detected by

propidium iodide (1 lg/mL) counterstain. Immunostaining was visualized by epifluorescent

microscopy (Figure 3.2). Representative photomicrographs were captured and analyzed

with NIS Elements software for fiber area. Three images were taken from each of three

cross-sections taken from each horse for a total of nine images per horse. Representative

images were captured at 200X using a Nikon TE2000U inverted microscope equipped with

epifluorescence and a DMI200F digital camera.









Statistical Analysis

Horse weights and grain intake were taken prior to the start of each adaptation and

training period. Statistics were analyzed by analysis of variance (ANOVA) with repeated

measures over time according to treatment. Analyses were performed with Statistical

Analysis System (SAS) version 6.12 using proc GLM for the ANOVAs and proc mixed

for the ANOVAs for measures repeated over time. All analyses with P<0.05 were

considered statistically significant. The blood data for vitamin E, TBARS, oxidized,

reduced, and total glutathione, as well as %reduced glutathione, and TEAC was analyzed

by analysis of variance (ANOVA) with repeated measures over time according to

treatments. The regression relationship between each of the blood parameters and time

was generated for each treatment group over the total sampling times. Analyses were

performed with SAS version 6.12 using proc GLM for the ANOVAs and proc mixed for

the ANOVAs for measures repeated over time. All analyses with P<0.05 were

considered statistically significant. The muscle data for carbonyl content and ubiquitin

content was analyzed using SAS by Fischer's exact test using proc freq with a chi-square

value P<0.05 being considered statistically significant.









Table 3.1. Nutrient composition of hay sampled at selected times during the study


Sample
#1


Sample
#2


Sample
#3


Sample
#4


Sample
#5


Sample
#6


Average


Dry Matter %
Ash %
Crude Protein 0
Phosphorous %
Fat %
Acid Free
Detergent Fiber
(ash free) %
Neutral
Detergent Fiber
(ash free) %
Calcium %
Copper (ppm)
Manganese
(ppm)
Zinc (ppm)
Iron (ppm)


90.90
4.04
6.06
0.14
1.26
41.82


91.09
3.30
5.64
0.14
0.78
43.63


89.50
3.73
5.15
0.15
1.64
39.21


90.75
3.79
9.47
0.20
0.89
36.98


91.05
3.53
8.91
0.14
0.89
34.02


89.54
3.28
9.00
0.19
0.82
34.94


90.5
3.6
5.6
0.2
1.1
38.4


81.79 81.34 80.35 76.19 72.75 76.09 78.1


0.20
5.32
146.00

31.00
180.00


0.18
4.07
144.00

26.00
77.00


0.46
2.29
52.00

23.00
102.00


0.40
2.30
42.00

11.00
103.00


0.40
2.30
42.00

11.00
103.00


0.61
7.75
49.00

30.00
475.00


Table 3.2. Nutrient composition of grain sampled at selected times during study
Sample Sample Sample Sample Sample Sample
#1 #2 #3 #4 #5 #6


0.4
4.3
83.7

23.5
214.5


Average


Dry Matter %
Ash %
Crude Protein 0
Phosphorous %
Fat %
Acid Free
Detergent Fiber
(ash free) %
Neutral
Detergent Fiber
(ash free) %
Calcium %
Copper (ppm)
Manganese
(ppm)
Zinc (ppm)
Iron (ppm)


95.22
6.62
15.15
0.66
3.34
10.02


94.92
6.38
15.18
0.62
3.28
9.15


94.78
6.45
15.70
0.62
2.62
9.46


95.57
7.64
14.53
0.67
2.95
8.34


96.03
7.08
15.11
0.62
1.37
7.36


96.62
7.05
15.27
0.59
1.35
7.60


95.5
6.9
15.3
0.6
2.5
8.7


26.55 23.84 25.01 22.33 21.91 23.26 23.8


0.80
37.00
83.00


1.18
52.00
122.00


1.16
48.00
125.00


1.28
58.00
115.00


1.93
44.00
118.00


2.14
62.00
109.00


1.4
50.2
112.0


90.00 144.00 138.00 111.00 131.00 187.00 133.5
225.00 269.00 268.00 327.00 386.00 508.00 330.5






51


7 weeks supplementation

8 weeks training/supplementation

SET

15 weeks washout/detraining

Cross-over treatments

10 weeks supplementation

8 weeks training/supplementation

SET

Figure 3.1. Summary of experimental timetable.


Figure 3.2. Representative image of a-dystrophin immunostaining. Representative
photomicrographs were captured and analyzed with NIS Elements software
for fiber area. Three images were taken from each of three cross-sections
taken from each horse for a total of nine images per horse. Representative
images were captured at 200X using a Nikon TE2000U inverted microscope
equipped with epifluorescence and a DMI200F digital camera.














CHAPTER 4
RESULTS AND DISCUSSION

Vitamin E

Vitamin E supplementation did not increase circulating levels of the nutrient.

Vitamin E was fed to horses at a concentration approximately five-fold higher than the

NRC recommended level for horses in moderate to intense training (80 IU/kg DM).

Blood samples were monitored for circulating vitamin E content at the beginning and end

of the eight week training period (Table 4.1). No differences in plasma vitamin E

between control and vitamin E supplemented horses were observed.

Due to mechanical problems with the treadmill, the adaptation period for the

second half of the experiment was actually ten weeks rather than seven. During the extra

three weeks, the horses were fed their respective diets and left untrained. Therefore,

there was a total of 25 weeks (6 months) between the end of one training phase and the

beginning of the next. This should have provided adequate time for the horses to detrain

(return to baseline fitness levels) and for blood and muscle vitamin E levels to

equilibrate. Tissue depletion and repletion in adult standardbred horses showed that

plasma vitamin E values in most supplemented groups were stabilized within a few days

of beginning supplementation and upon being taken off supplementation, most horses

reached their baseline value within three weeks, although a few remained slightly higher

even after seven weeks (Roneus et al., 1986). Liver samples showed that this organ

rapidly accumulated vitamin E and the concentration declined following placement on a

low vitamin E diet, indicating that there is no permanent storage of vitamin E in the liver.









By contrast, skeletal muscle vitamin E concentration was most consistent regardless of

supplementation levels. Muscle tissue vitamin E concentrations increased much slower

than plasma and liver and decreased slower upon being placed on a low vitamin E diet.

Skeletal muscle in groups that were supplemented up to 1800 mg dl-a-tocopheryl acetate

(1800 IU) per day returned to baseline values after being taken off the supplement for 7

weeks (Roneus et al., 1986). The group that was supplemented 5400 mg dl-a-tocopheryl

acetate (5400 IU) did not. However, maximum vitamin E levels in the skeletal muscle

tissue were achieved with 1800 mg dl-a-tocopheryl acetate per day. Therefore, the

horses in our study were fed an adequate level of vitamin E to reach maximum tissue

levels within 7 weeks. The blood samples taken before and after each training phase

were not different, as described by others (Roneus et al., 1986).

Throughout the duration of the study, a significant period effect was detected in

several measures of redox potential. This may be explained partially by differences in

tissue vitamin E concentration and retention. After several years of deficient diets,

vitamin E stores can still remain in the body (Blatt et al., 2001). Our plasma vitamin E

levels showed no period effect. However, circulating vitamin E reaches a saturation

point and can only be increased at most three to four fold by supplementation (Blatt et al.,

2001). It is possible that our control horses were at a sufficient level of supplementation

such that additional vitamin E could not further elevate plasma levels. This does not,

however, mean that tissue levels of vitamin E could not have been significantly higher in

period two as compared to period one.









Lipid Peroxidation

Vitamin E supplementation did not alter lipid oxidation levels. The total amount of

oxidized lipids in the plasma was measured before and after a bout of mild exercise over

the duration of the 9 week training period. Plasma thiobarbituric acid reactive

substances (TBARS) is a measure of lipid peroxidation in the body. The TBARS

concentrations remained relatively constant throughout the feeding trial in both control

and vitamin E supplemented horses (Figure 4.1). A decline in plasma TBARS

concentration at week 8 was observed for both groups of horses. The cause of this

reduction is unknown but may reflect a sampling error. Following a training episode,

plasma TBARS concentrations remain unaffected by treatment (Figure 4.2). The

concentration of TBARS when adjusted for plasma volume using a plasma protein ratio

was, however, lower post exercise as compared to pre exercise for both control and

vitamin E supplemented horses (P<0.0001). From these results, it is concluded that

circulating oxidized lipid content in plasma was altered by exercise but was not

influenced by vitamin E supplementation. Feeding an excess of vitamin E does not

reduce plasma TBARS suggesting no beneficial effect at this level of athletic

performance. The effect of vitamin E supplementation on lipid peroxidation was

examined following a single bout of strenuous exercise (Table 4.2). Horses were

exercised to exhaustion and blood TBARS content was measured during the recovery

period. Immediately after exercise, TBARS content declined in control and vitamin E

supplemented horses. These values returned to baseline within 24 hours and remained

unchanged at 48 hours. No differences between control and vitamin E supplemented

horses were observed at any time point. Therefore, vitamin E supplementation does not

improve the antioxidant capacity of exercising horses as measured by TBARS.









The TBARS assay primarily measures malondialdehyde (MDA), a short chain fat

group which is formed from damaged cell membranes. It has been criticized in previous

studies for assay variability and its reflection of other parameters in the body independent

of oxidative stress. In this study, there was no significant difference between treatment

and control groups at any sample point over the training period. There was a significant

decrease in week eight pre TBARS values (Figure 4.1). This was an expected result

since as the horses fitness level increased there should have been less peroxidation in the

body. The exercise performed at week eight was a simple run on a flat surface over the

ascribed training distance. Therefore, minimal stress was placed on these horses.

However, the pre SET blood samples for horses were the highest of all samples drawn.

This is possibly due to stress associated with preparing the horses to run the exercise test

(number of people, equipment used, etc.). An unexpected finding was that TBARS

values actually decreased after exercise when compared to before exercise values if post

exercise concentrations were adjusted for plasma volume (P<0.05). It is possible that this

is due to increased clearance in the body directly after exercise. MDA was observed to

have a short half-life and is therefore cleared quickly from the body (Siu and Draper,

1982). It is also possible that longer duration of exercise yields greater TBARS

concentrations. It has been shown that horses undergoing endurance exercise (140 km

race) have elevated levels of TBARS post exercise, and these concentrations remain

elevated 16 hours post exercise (Marlin et al., 2002). Possibly an increase in TBARS

concentrations is more common in low intensity but long duration exercise which relies

more on free fatty acids as an energy source as opposed to the sprint-type training that

our animals performed. It is also possible that the adjustment for plasma volume using









plasma proteins was not appropriate. Van Beaumont and associates in 1973 concluded

that there may be a net loss of plasma proteins as a result of exercise, and thus plasma

protein concentration may not be a reliable way to adjust for plasma volume. Contrary to

this, a more recent study by Lindinger and colleagues showed that total plasma proteins

in exercising horses are directly correlated to plasma volume (Lindinger et al., 2000).

Plasma concentration of TBARS following a SET was significantly lower than pre

exercise concentrations when adjusted for plasma volume but not when left unadjusted.

However, during the training phase, both unadjusted concentrations and adjusted

concentrations were significantly lower post exercise when compared to pre exercise

concentrations.

Antioxidant Capacity

Plasma antioxidant capacity was unaffected by vitamin E supplementation. Plasma

was analyzed for its ability to scavenge peroxide free radicals (Trolox equivalent

antioxidant capacity, TEAC). The TEAC values increased in both control and vitamin E

supplemented horses after five weeks of moderate exercise (P<0.05) (Figure 4.3). These

results posit that antioxidant defenses are improved as the horses transition through the

fitness period. However, supplementation of excess vitamin E did not improve global

antioxidant capacity (Table 4.4). Exercise did, however, cause antioxidant status of the

blood to decrease. Post exercise TEAC values were lower than pre exercise TEAC

values for both treatment groups when values were adjusted for plasma volume

(P<0.001). To examine the effects of vitamin E on recovery from strenuous exercise,

horses were exercised to exhaustion and plasma TEAC concentrations were measured

(Table 4.5). Immediately upon completion of the exhaustive bout of work, TEAC values

were low in both control and supplemented animals (P<0.05). These values returned to









baseline within 24 hours and remained constant at 48 hours. Thus, control diets sufficient

in vitamin E likely meet the antioxidant demands of horses undergoing mild exercise as

well as a stressful performance regiment.

An interesting finding is that the moderate exercise performed during the training

phase resulted in unadjusted post exercise TEAC values being slightly elevated as

compared to pre exercise samples. This is potentially due to mild exercise causing an

activation of the body's antioxidant system. However, following a strenuous exercise

bout (SET exercise) the TEAC values were significantly lower than pre exercise values

but returned to resting levels by 24 hours post. This possibly indicates that strenuous

exercise taxes the antioxidant system enough to decrease its radical scavenging abilities.

A study on similarly exercised horses showed that horses exercising at 8 m/s on a 6

incline (similar to our highest intensity training exercise) had oxygen consumption of 63

mL/kg/min whereas horses running at 12 m/s on a 6 incline (maximum effort in our SET

prior to fatigue) had oxygen consumption of 150 mL/kg/min (Kavazis et al., 2004).

Therefore, oxygen consumption increased dramatically between our training exercise and

SET exercise, and this possibly explains the horses having a different response post

training exercise versus post SET exercise. Therefore, intensity of exercise must be taken

into account when examining antioxidant capacity of exercising subjects.

Furthermore, training was shown to increase the antioxidant capacity of the

animals regardless of treatment group (P<0.05). Following five weeks of training, horses

had significantly elevated TEAC values pre exercise as compared to week 0 (initial

value). This indicates that fitness level must be taken into consideration when comparing

antioxidant capacities of exercising subjects. Our results are supported by another study









using exercising horses which showed significant increases in citrate synthase activity in

middle gluteus muscle following and intense exercise regime (27% higher activity after

week 1 and 42% higher after week 2). This was accompanied by an increase in capillary

density following five weeks of training (Essen-Gustavsson et al. 1989). Therefore,

training is shown to cause adaptations which may improve the antioxidant status of the

exercising subject.

Glutathione Data

Glutathione participates in the body's antioxidant defense by serving as a sink for

electrons during the reduction of H202 to water. More glutathione in the reduced form

may allow for efficient quenching of newly formed free radicals. Therefore, high levels

of reduced and % reduced glutathione are indicative of improved antioxidant potential.

Total, reduced, and oxidized glutathione were measured in the blood in horses fed control

and excess vitamin E and maintained on a moderate exercise regime. Consistent with

our prior results, vitamin E supplementation did not appear to affect oxidized, reduced, or

total glutathione status in the blood of moderately exercising horses, nor did it affect the

percent of total glutathione which was in the reduced form (Table 4.5). Vitamin E did,

however, cause a significant elevation of % reduced glutathione in response to strenuous

exercise (SET) as compared to the control diet (P<0.006). This effect is attributed to

lower plasma oxidized glutathione levels in vitamin E treated horses (P<0.03). These

results suggest that vitamin E may enhance the glutathione redox system, particularly

during intense exercise.

Exercise training also influenced glutathione parameters. Reduced and total

glutathione decreased in pre exercise blood samples after two weeks of exercise training









(P<0.05). This decrease persisted throughout the remainder of the study. Exercise also

caused an immediate drop in reduced, total, and % reduced glutathione with post exercise

values being lower than pre exercise values (P<0.01). Exercise did not affect oxidized

glutathione concentrations. This pattern persisted following the SET. Post exercise

values for reduced, total, and % reduced glutathione were lower than pre exercise values

(P<0.01). Oxidized glutathione was not affected by exercise. At week 5, when the

horses underwent a more rigorous form of exercise (exercise on a 6 incline as opposed to

flat), post exercise concentrations of reduced and total glutathione were significantly

lower as compared to week 2 values (P<0.01). This was also the case for week 9 (SET)

data. This finding indicates that strenuous exercise caused the horses to utilize their

reduced glutathione stores to a greater extent as compared to milder exercise. Marlin and

colleagues (2002) found a similar trend in endurance horses. When blood was collected

from 40 competitive endurance horses both before and after a 140 km race, all

glutathione measures (reduced, total, oxidized) decreased significantly post exercise

(Marlin et al. 2002). Results of the present study agree with their findings.

Carbonylation and Ubiquitination

Strenuous exercise can lead to formation of carbonylated proteins in muscle fibers.

Carbonyl groups are formed as a consequence of ROS accumulation and may contribute

to loss of contractile function. The presence and extent of carbonylation was examined in

purified myofibrillar proteins isolated from the gluteus medius of control and vitamin E

supplemented horses before and after a single bout of strenuous exercise. Carbonylation

was measured by Western blot (Figure 4.4). Statistical significance was based on either

presence or absence of carbonyl groups in all horses undergoing the standard exercise

test. Three major carbonylated proteins were apparent prior to exercise that corresponded









to the approximate sizes of myosin heavy chain, a-actinin, and a-actin. The presence of

carbonylation was lower in vitamin E supplemented horses (P<0.07). Following the

SET, the relative amount of carbonylated myosin heavy chain and actin remained

substantially lower for the vitamin E supplemented group (P<0.02). The lower amounts

of carbonylated myofibrillar proteins does not equate with improved performance. Times

to exhaustion did not differ between control and vitamin E supplemented horses (Table

4.7).

It is unclear why carbonylation of pre exercise samples was more extensive than

post exercise samples. Carbonylated proteins are present in the gluteus medius four

hours after exercise (Kinnunen et al., 2005a; Kinnunen et al., 2005b). Carbonyls in

plasma were shown to increase post exercise, were the highest at 4 hours post exercise,

and had not returned to baseline values at 24 hours post exercise. In the muscle tissue, 4-

HNE-modified proteins (marker of lipid peroxidation) did not increase between pre

exercise samples and at 4 hours post exercise. This demonstrates that measures of lipid

peroxidation do not always follow the same trend as markers of protein oxidation and

helps to explain the results of our study in which TBARS was not affected in the same

way as protein carbonyls by either exercise or treatment. Protein carbonyl levels

remained high at 24 hours post exercise as compared to pre exercise samples (Kinnunen

et al., 2005a). It is possible, therefore, that the more extensive carbonylated proteins in

our pre exercise samples actually represent carbonylation from chronic exercise training.

These modified proteins may be rapidly eliminated upon commencement of exercise.

The means by which muscle protein carbonyls are degraded is unknown but likely does

not involve the ubiquitin system.









Ubiquitin was not detected in purified myofibrils at any point in time indicating

clearance of damaged muscle fibers by another mechanism (data not shown).

Ubiquitinated proteins were, however, present in the supernatant collected from isolate

myofibrils, demonstrating that ubiquitin pathways are active (data in appendix). It is

possible that by taking biopsies immediately following exercise, the ubiquitin levels

detected were not actually reflective of that particular exercise bout. Radak et al. (2000)

showed that reactive carbonyl derivatives were detectable in the mitochondrial fraction of

rat muscle tissue post exercise but not in the cytosolic fraction. The peptidase-like

activity of the proteosome complex (20S and 26S proteosomes) was increased in the

cytosol following exercise. The 26S proteosome is known to degrade ubiquitinated

proteins (Radak et al., 2000), and therefore, cytosolic expression of ubiquitin may

indicate proteins marked for degradation of the proteosome complex. Since the

myofibrillar fraction of our muscle samples did not express ubiquitination, this may

explain why they are more susceptible to carbonylation.

How damaged myofibrils are degraded and replaced is still unknown. Sangorrin et

al. (2002) states that non-lysosomal pathways are the main regulators of myofibrillar

protein breakdown in the early stages of damage but are replaced by lysosomal

proteolytic enzymes later. Three non-lysosomal pathways suggested in this study are

ubiquitin-proteolytic system, Ca2+-activated proteases (at-calpain and m-calpain), and

ATP-independent proteolysis. Our study indicated that ubiquitin is not present in

myofibers. The calpains are unable to degrade actin and myosin in myofibers (Sangorrin

et al., 2002). Therefore, another system must be present to degrade damaged and

carbonylated myofibers. Sangorrin et al. (2000) reported the presence of a serine-type









protease bound to myofibrils in mouse skeletal muscle. This protease was called protease

M and was able to degrade protein constituents from whole myofibrils in vitro. They

suggest that this protease could be responsible for skeletal muscle myofibrillar ATP-

independent proteolysis (Sangorrin et al., 2002).

Muscle Morphometrics

Exercise training, but not diet, caused a change in muscle fiber size. The integrity

and size of the gluteus medius muscle fibers were evaluated by a-dystrophin

immunostaining (Figure 4.5). No differences in diameter, area, or number of nuclei per

fiber were observed between control and vitamin E supplemented horses (Table 4.8).

Exercise training did, however, decrease fiber diameter size (Table 4.9). The distribution

of fiber cross-sectional area (CSA) was plotted for control and vitamin E supplemented

horses following the SET. As shown in Figure 4.6, very little difference in the

distribution of fiber CSA between the groups was observed. However, the vitamin E

supplemented horses appeared to possess a higher percentage of small muscle fibers.

This suggests a shift toward the smaller diameter, type I oxidative metabolism muscle

fiber. The CSAs of muscle fibers in our study were in a similar range to other studies

documenting fiber characteristics. Serrano et al. (1996) detected three fiber types in the

gluteus medius of horses: (1) Type 1 which had a cross sectional area (CSA) between 20

and 23 am2 X 100, (2) Type IIA which had a CSA between 22 and 25 am2 X 100, and (3)

Type IIB which had a CSA between 26 and 36 jam2 X 100. Essen-Gustavsson et al.

(1989) showed that five weeks of intense exercise training caused an increase in type IIA

fibers and a decrease in type IIB fibers and also a decrease in fiber area of both types IIA

and IIB fibers. Since our study did not fiber type, it is difficult to determine if our horses

had the same fiber type shifting. However, our horses did appear to have a decrease in






63


total fiber diameter following training, and our vitamin E supplemented horses had a

higher percentage of predicted type I fibers post exercise as compared to controls.









Table 4.1. Effect of vitamin E supplementation on resting plasma vitamin E
concentration of horses in a training program
Effect Variable Vitamin E Level
(gg/mL)

Treatment Controla 2.520.17
Vitamin Eb 2.700.15

Time 1 2.480.15
2 2.740.17

Period 1 2.600.12
2 2.61+0.19
a= 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
Time 1 = initial value; before commencement of 8 wk exercise program
Time 2 = pre exercise value at the conclusion of 8 wk exercise program
Period 1 = Adaptation phase, training phase, and SET before diet cross-over
Period 2 = Adaptation phase, training phase, and SET after washout, detraining, and diet
cross-over

Table 4.2. Effect of vitamin E supplementation on plasma TBARS concentration in
horses following strenuous exercise
Time Treatment TBARS Concentration
(mmol/L)

Pre Controla 143.911.3
Vitamin Eb 141.69.2

Post Control 132.1514.9
Vitamin E 140.5813.3

Adj Post Control 110.0+11.8*
Vitamin E 118.6+11.0*

24 Post Control 138.76.4
Vitamin E 138.915.2

48 Post Control 135.1+13.9
Vitamin E 150.38.9
a= 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
* = significantly lower values as compared to pre exercise values (P<0.05)










Table 4.3. Plasma TEAC concentrations in control and vitamin E supplemented
exercised horses


Item


Pre Exercise


Post Exercise


Treatment


Control
Vitamin Eb

Control
Vitamin E


TEAC Values


1.070.02
1.070.02

1.10+0.02
1.100.02


Main Effect
Treatment Time


P= 0.995


P=0.985


P<0.001


P<0.001


Post Ex" Control 1.00+0.03 P=0.867 P<0.001 P=
Vitamin E 1.01+0.04
80 IU vitamin E / kg DM
80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
TEAC values reported in arbitrary TEAC units
Post exercise values adjusted for plasma volume by total plasma protein ratio


Table 4.4. Plasma TEAC concentrations before and after strenuous exercise
Time Treatment TEAC
Value


Pre


Post


Control
Vitamin Eb


1.15+0.01
1.140.02


Control 1.130.01*
Vitamin E 1.130.01*


Post adj" Control
Vitamin E

24 Post Control
Vitamin E


0.940.01*
0.950.04*

1.130.04
1.130.04


48 Post Control 1.160.00
Vitamin E 1.160.01
= 80 IU vitamin E / kg DM
= 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
S= Values lower than pre exercise values (P<0.05)
= Post exercise values adjusted for plasma volume by total plasma protein ratio


Treatment
X Time

P=0.999


P=0.999


Adj

a=
b=
A=
#=


0.972














Table 4.5. Effect of vitamin E supplementation on glutathione levels (mg/dl/%ORBC) in horses undergoing moderate exercise (Means
and SEMs reported)

Pre- Post-
Exercise Exercise

Week Diet Oxid# Reduced# Total# %Red Oxid# Reduced# Total# %Red'
0 CTLa 0.71+0.17 1.550.14 2.25+0.13 69.636.6
Vit Eb 0.620.26 1.71+0.20 2.260.17 75.889.1
2 CTL 0.470.09 1.190.09 1.960.07* 76.264.4 0.470.16 0.890.17 1.330.12 65.179.3
Vit E 0.410.09 1.430.09 1.850.13* 78.834.2 0.340.09 1.030.07 1.370.07 76.025.8
5 CTL 0.460.14 1.220.12* 1.680.10* 73.81+7.3 0.41+0.18 0.920.10 1.270.14 72.01+8.4
VitE 0.680.16 1.080.13* 1.730.21* 62.706.5 0.630.08 0.830.13 1.450.13 54.337.5
52 CTL 0.420.12* 1.220.19* 1.610.11* 72.058.5 0.570.08 0.500.08* 1.070.11* 47.397.3
VitE 0.320.13* 1.360.19* 1.630.18* 81.004.6 0.450.10 0.560.10* 1.000.12* 65.937.8
8 CTL 0.780.11 1.040.11* 1.820.12* 57.91+5.5* 0.750.10* 0.750.10 1.500.18 50.552.8*
VitE 0.800.12 1.100.23* 1.900.21* 54.676.9* 0.680.12* 0.750.12 1.420.21 55.368.7*
9 CTL 0.720.14 1.240.11* 1.950.12* 64.535.7 0.560.11 0.360.11* 0.890.08* 50.277.4*
VitE 0.570.17 1.25+0.09* 1.81+0.18* 72.797.3 0.580.06 0.430.06* 0.960.13* 48.6714.0*
Avg CTL 0.590.13 1.240.13* 1.890.11* 69.036.3 0.550.13 0.680.11 1.210.13 57.08+7.0
Vit E 0.570.16 1.320.16* 1.860.18* 70.986.4 0.540.09 0.720.10 1.240.13 60.068.8
a= 80 IU vitamin E / kg DM
b= 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
Time reported in weeks
# reported in mg.dl-..%rbc-1
Calculated as % of total glutathione that is in the reduced form
2 horses trained on an incline
* significant differences in time points (P<0.05) as compared to time 0 (pre exercise) or time 2 (post exercise)









Table 4.6. Effect of vitamin E supplementation on glutathione measures taken at time


points during a standard exercise test (Means


Time


Treatment Oxidized"*


Control
Vit Eb

Control
Vit E


Post


24 Post Control
Vit E


0.720.14
0.570.17

0.560.12
0.580.20

0.81+0.23
0.640.17


48 Post Control 0.890.32
Vit E 0.650.24
a = 80 IU vitamin E / kg DM


Reduced#


1.240.11
1.25+0.09

0.370.11+
0.430.06+

1.160.09
1.200.10

0.970.51+
0.91+0.52+


and SEMs reported)
Total# %Reduced *


1.950.12
1.81+0.18

0.890.08+
0.960.13+

1.960.16
1.820.17

1.850.11
1.51+0.18


62.905.7
72.807.3


45.767.5+
48.6614.0+

63.158.9
68.047.2

54.5810.5
68.6711.8


b 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
# reported in mg.dl-..%rbc-
Calculated as % of total glutathione that is in the reduced form
* significant differences between treatments (P<0.05) over time points
* significant differences between time points (P<0.05) as compared to pre exercise value




Table 4.7. Effect of vitamin E supplementation on time to fatigue in a SET
Treatment Horse Number Time to Fatigue Average
(minutes) (minutes)


Control 1
2
3
4


Vitamin Eb


5.08
5.25
4.70
5.83

5.02
4.83
5.25


5.220.23




5.090.10


8 5.25
a = 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
n=4
No statistical differences between treatment groups









Table 4.8. The effect of vitamin E supplementation on parameters of muscle fiber sizes
Variable Measured Controla Vitamin Eb

Area ([tm2) 3988820 367612
Diameter ([tm) 625 59+3
#Nuclei/Fiber 4.630.36 4.450.36
a= 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E

Table 4.9. Effects of exercise on muscle cross morphometrics
Variable Measured Initial Pre Post
Area (tm2) 4054169 3736156 3691+132
Diameter (tpm) 693* 57+1 561.07
#Nuclei/fiber 4.510.72 4.300.69 0.780.76
* = significant difference (P<0.05)











O Control
* Vitamin E


200

150

100

50


0 2 5 6 8 9


Week

Figure 4.1. Plasma TBARS concentration is not effected by vitamin E supplementation.
Prior to initiation of treadmill exercise, blood was collected and plasma
harvested. TBARS assays were performed on duplicate samples. Values
were calculated based on known concentrations. Means and SEMs are shown.
Asterics indicate concentrations lower than initial concentration (week 0)
(P<0.05).











O Control
* Vitamin E


200

150

100

50


2 5 6 8 9
Week



Figure 4.2. Plasma TBARS values after exercise are not affected by vitamin E
supplementation. Plasma was isolated after treadmill exercise. TBARS
concentrations were calculated using known amounts and values were
corrected for plasma volume using plasma protein ratio. Means and SEMs of
duplicate samples are shown.












D Control
* Vitamin E


1.20

1.15

1.10

1.05

1.00

0.95

0.90

0.85


Week


Figure 4.3. TEAC values for pre exercise samples show no difference between control
and vitamin E supplemented horses but increase with improved fitness. TEAC
values are reported in arbitrary TEAC units and increase over time in horses
undergoing an exercise training program. indicates significantly different
time points as compared to week 0 (P<0.05).










Pre SET
CTL E












"OEM


Post SET
CTL E

! im


Figure 4.4. Purified gluteus medius myofibrillar proteins are carbonylated. Myofibril
proteins were purified from muscle biopsies obtained from the gluteus medius
prior to SET from control and vitamin E treated horses. Equal amounts of
protein were analyzed by Western blot using mouse anti-carbonyl. Immune
complexes were visualized by ECL. Representative blots are shown.
Predicted myosin heavy chain (MyHC), a-actinin, and a-actin protein sizes
are shown.


- My-H(--


--a-crinir







- aictin -

































Vit E Pre Vit E Post

Figure 4.5. Dystrophin immunostained gluteus medius muscle fiber cryosections
obtained from horses before and after SET exercise. Biopsies were harvested
before and after exercise and stored frozen. Twelve micron cryosections were
collected and incubated with mouse anti-dystrophin. Immune complexes were
detected with goat anti-mouse Alexafluor 488. Representative
photomicrographs at 200X are depicted







74




30.00 -

25.00 Vitamin E Post
** Control Post
20.00 = Initial

15.00- .o

10.00- \ *
*lOOO*.. \ ** *,--._ .
5.00 -

0 .0 0 I I I I I I I I I
1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000
Size (pm)

Figure 4.6. Distribution of muscle fibers sizes from horses before
training/supplementation, and before and after a standard exercise test.
Biopsies were obtained prior to the beginning of the study and from control
and vitamin E supplemented horses upon completion of each 9 week training
phase. Cross sectional area (CSA) was measured and the percentage of fibers
in each 1000m2 quartile was calculated.














CHAPTER 5
IMPLICATIONS

When all of our results are taken together, this study clearly supports the premise

that training enhances the antioxidant status of the animal, and therefore future studies

trying to compare antioxidant supplementation benefits should take into consideration the

fitness level and expected workload of the subjects. It appears from analyzing the data

collected on this study that vitamin E may have a significant effect on the body's ability

to handle oxidative stress. Blood glutathione data and muscle carbonyl data both showed

favorable effects of vitamin E supplementation. It is possible that training somewhat

mutes many of the effects of antioxidant supplementation simply because increasing

fitness will increase the natural antioxidant capacity of the body, as shown by our TEAC

data. However, a single strenuous bout of exercise, such as our SET, is sufficient to show

beneficial effects of vitamin E supplementation on glutathione and carbonyl parameters

measured.

It is however, worth mentioning that vitamin E has not been shown to enhance

performance and in some cases has been shown to decrease performance. Coombes et al.

(2001) showed that extremely high levels of vitamin E supplementation (10,000 IU of

additional vitamin E/kg diet over control diet) combined with a-lipoic acid actually

caused a decrease in maximal twitch tension and titanic force production in the skeletal

muscle of rats when artificially stimulated. In further studies, they used varying levels of

vitamin E and added dihydrolipoic acid at different concentrations to baths containing

diaphragm muscle strips. The results from this study implicate high levels of vitamin E






76


in causing depressed skeletal force production at low stimulation frequencies. Thus, it is

possible that feeding too high a level of vitamin E might be detrimental to some

measurements of performance. Our study did not show any differences in indices of

performance. It is likely that the level of vitamin E, if supplemented, must be adjusted to

reach potential maximum benefits in the prevention of muscle damage and reduction of

peroxidation without causing impaired performance due to decreased muscle

contractility.















APPENDIX A
SUPPLEMENTAL PROTOCOLS

Table A. 1. Weights of horses at the start of each time period
Horse Adaptation Training Adaptation Training
Number Period 1 Period 1 Period 2 Period 2

1 509 501 508 510
2 505 495 516 528
3 503 492 505 514
4 494 500 510 513
5 576 587 610 606
6 525 529 531 559
7 604 581 574 596
8 502 500 517 518
Avg 52714 52314 53413 54314
Weights are reported in kg.
Horses 1-4 were on vitamin E diet for period 1 and control diet for period 2.
Horses 5-8 were on control diet for period 1 and vitamin E diet for period 2.
Period 1 = Adaptation phase, training phase, and SET before diet cross-over
Period 2 = Adaptation phase, training phase, and SET after washout, detraining, and diet
Statistical differences not observed between treatments or periods.









Table A.2. Daily grain intake of horses on each phase of trial
Horse Adaptation Training Period Adaptation Training Period
Number Period 1a Ib Period 2a 2b

1 6.36 7.52 6.35 7.65
2 6.31 7.42 6.45 7.92
3 6.29 7.38 6.31 7.71
4 6.18 7.50 6.37 7.70
5 7.20 8.80 7.63 9.09
6 6.56 7.94 6.64 8.39
7 7.55 8.72 7.18 8.94
8 6.28 7.50 6.46 7.77
Avg 6.590.18 7.850.21 6.670.17 8.150.21
a = Grain fed at 1.25% of body weight
b = Grain fed at 1.5% of body weight
Intakes are reported in kg/day.
Horses 1-4 were on vitamin E diet for period 1 and control diet for period 2.
Horses 5-8 were on control diet for period 1 and vitamin E diet for period 2.
Period 1 = Adaptation phase, training phase, and SET before diet cross-over
Period 2 = Adaptation phase, training phase, and SET after washout, detraining, and diet
Statistical differences not observed between treatments or periods.









Table A.3. Treadmill training schedule for period 1
Week# Monday Tuesday Wednesday Thursday Friday

1 DNT 1.0 DNT 1.0 1.0
2 1.5 1.5 DNT 1.5 2.0*
3 DNT DNT 2.0 2.0 2.5
4 2.5 2.5 DNT 3.0 3.0
5 3.0 3.0 DNT 3.0* DNT
6 3.0i* 3.0 3.0 3.0i DNT
7 DNT 3.0 3.0i 3.0 3.0i
8 3.0 3.0i DNT 3.0* 3.0i
Distance reported in kilometers.
i indicates exercise on 6 incline.
* denotes blood collection day
DNT = "did not train"









Table A.4. Treadmill training schedule for period 2
Week# Monday Tuesday Wednesday Thursday Friday

1 1.0 DNT 1.0 1.0 1.5
2 DNT 1.5 1.5 DNT DNT
3 1.5 2.0* 2.0 2.0 2.5
4 2.5 2.5 DNT 3.0 3.0
5 3.0 3.0 DNT 3.0* 3.0
6 3.0i* 3.0 3.0i DNT DNT
7 3.0i* 3.0 DNT 3.0i DNT
8 3.0 3.0i DNT 3.0* 3.0i
Distance reported in kilometers.
i indicates exercise on 6 incline.
* denotes blood collection day
DNT = "did not train"










Table A.5. Plasma proteins values of training phase and SET used for adjusting for
plasma volume (Values reported SEM)


Week Time Treatment Plasma Proteins
Period 1


0

2



5


Control
Vit Eb
Pre Control
Vit E
Post Control
Vit E
Pre Control
Vit E
Post Control
Vit E
Pre Control
Vit E
Post Control
Vit E
Pre Control
Vit E
Post Control
Vit E
Pre Control
Vit E
Post Control
Vit E
24 Post Control
Vit E
48 Post Control
Vit E


Plasma Proteins
Period 2


6.530.07
6.500.06
6.25+0.09
6.450.01
6.850.07
6.980.05
6.450.10
6.630.02
6.830.14
6.880.02
6.630.19
6.330.04
6.980.12
7.200.03
6.35+0.10
6.480.02
6.850.11
6.650.04
6.670.20
6.580.16
7.870.23
7.70+0.16
6.430.17
6.500.11
6.330.12
6.480.03


a = 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
Period 1 = Adaptation phase, training phase, and SET before diet cross-over
Period 2 = Adaptation phase, training phase, and SET after washout, detraining, and diet
cross-over
2 horses trained on an incline


6.430.03
6.350.06
6.430.02
6.630.09
7.050.06
6.850.11
6.600.02
6.500.11
6.950.04
6.930.07
6.38+0.04
6.480.11
7.180.13
7.230.12
6.330.02
6.630.07
6.780.05
6.880.10
6.450.06
6.600.10
7.830.06
7.930.11
6.430.04
6.480.10
6.300.04


8


9















APPENDIX B
ADDITIONAL TBARS DATA









Table B. 1. Effect of vitamin E supplementation on plasma thiobarbituric acid reactive
substances (TBARS) concentration in exercising horses
Main Effect
Item Treatment TBARS Treatment Time Treatment X
concentration Time
(Mmol/L)


Pre Exercise


Post
Exercise*


Difference


Control

Vitamin Eb


Control


128.589.71

132.137.51

104.2711.51


p=0.494


p=0.972


p<0.001


p=0.072


p=0.343


p=0.20


Vitamin E 104.487.79


Control


24.31+10.56


p=0.613


p<0.006


p<0.05


Vitamin E 27.666.02
a = 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
Difference = pre post
Exercise = average of pre or post exercise samples at 5 time points during trial
* Post exercise values are significantly lower than pre exercise values (P<0.01)









Table B.2. TBARS values for control and vitamin E supplemented horses over time


Pre TBARS ConcentrationA


126.93
130.64


Post TBARS ConcentrationA


N/A
N/A


Week 2


Week 5


Week 5


Week 8


Week 9 (SET)


Control 138.84
Vitamin E 126.39


Control
Vitamin E


141.09
137.69


Control 131.00
Vitamin E 139.51


Control
Vitamin E


89.73
116.93


Control 143.91
Vitamin E 141.60


a = 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
A indicates concentrations reported in Mmol/L and adjusted for plasma volume
@ indicates run on incline


Time

Initial


Treatment

Control
Vitamin Eb


111.13
111.50

88.40
104.54

110.30
91.09

101.91
98.11

111.26
118.59






85


Table B.3. Thiobarbituric acid reactive substance (TBARS) concentration for pre
exercise time points in control and vitamin E supplemented horses by period
Treatment Period TBARS concentration

Control 1 119.01+6.53
Control 2 138.157.10

Vitamin Eb 1 123.534.43
Vitamin Eb 2 140.754.73
a = 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
indicates concentrations reported in Mmol/L SEM and adjusted for plasma volume






86


Table B.4. Thiobarbituric acid reactive substance (TBARS) concentration for post
exercise time points in control and vitamin E supplemented horses by period
Treatment Period TBARS concentration

Control 1 100.778.83
Control 2 108.435.78

Vitamin Eb 1 99.654.50
Vitamin Eb 2 109.884.91
a = 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
indicates concentrations reported in Mmol/L and adjusted for plasma volume















APPENDIX C
ADDITIONAL TEAC DATA









Table C.1. TEAC values pre and post exercise for control and vitamin E supplemented
horses over time
Pre Post
TEAC
Time Treatment TEAC values^ values^

Initial Controla 1.02 N/A
Vitamin Eb 1.02 N/A

Week 2 Control 1.02 0.93
Vitamin E 1.02 0.97

Week 5 Control 1 1.11
Vitamin E 0.99 1.11

Week 5
incline Control 1.12 0.99
Vitamin E 1.12 0.95

Week 8 Control 1.14 1.09
Vitamin E 1.14 1.1

Week 9
(SET) Control 1.15 0.94
Vitamin E 1.14 0.95
a = 80 IU vitamin E / kg DM
b = 80 IU vitamin E / kg DM + 3000 IU supplemental vitamin E
indicates TEAC data reported in arbitrary TEAC units and adjusted for plasma volume