Assessment of Oxidative Stress and Muscle Damage In

ASSESSMENT OF OXIDATIVE STRESS AND MUSCLE DAMAGE IN

EXERCISING HORSES IN RESPONSE TO LEVEL AND FORM OF VITAMIN E

MADISON FAGAN

(Under the Direction of KYLEE JO DUBERSTEIN)

ABSTRACT

Vitamin E is an essential antioxidant noted for reducing oxidative stress. The goal was to determine (1) if supplemental vitamin E is beneficial to exercising horses and (2) if there is a benefit of natural vs synthetic supplements. After a 2wk washout 18 horses were divided into groups and fed a control diet plus: (1) 1000 IU synthetic α-tocopherol

(SYN-L), or (2) 4000 IU/d synthetic α-tocopherol (SYN-H), or (3) 4000 IU/d RRR-α- tocopherol (NAT). Horses began a 6wk exercise protocol, with standard exercise tests

(SET) performed pre and post the protocol. Venous blood samples were collected. NAT horses had higher α-tocopherol (P<0.05). Plasma MDA levels were lower in NAT vs

SYN-L horses post SET2 (P=0.02). Serum AST was significantly lower post SET2 in

NAT horses vs SYN-L or SYN-H (P<0.05). In conclusion, feeding higher levels of the more bioavailable natural vitamin E source had a beneficial effect of reducing oxidative stress.

INDEX WORDS: Vitamin E, Equine, Exercise, Alpha-tocopherol, Oxidative stress,

Nutrition ASSESSMENT OF OXIDATIVE STRESS AND MUSCLE DAMAGE IN

EXERCISING HORSES IN RESPONSE TO LEVEL AND FORM OF VITAMIN E

MADISON FAGAN

B.S., North Carolina State University, 2015

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

Madison Fagan

EXERCISING HORSES IN RESPONSE TO LEVEL AND FORM OF VITAMIN E

MADISON FAGAN

Major Professor: Kylee Jo Duberstein Committee: Robert Pazdro Jarrod Call

Electronic Version Approved:

Suzanne Barbour Dean of the Graduate School The University of Georgia August 2018 This paper is dedicated to my family for their constant encouragement.

iv ACKNOWLEDGEMENTS

I would like to first acknowledge my major professor, Dr. Kylee Duberstien.

Thank you for allowing me to be involved in this project. I would not have been nearly as successful in this program without your constant support and advice. I am thankful for your continued mentorship and guidance throughout this process. Thank you for always being available to help with anything, project related or not. I look forward to continuing to work with you!

I would also like to acknowledge all of the undergraduates that worked long, hard hours during this project. Thank you for committing your weekends and free time to cleaning stalls, exercising horses and being so dedicated to the success of this project.

Each of you played an important role in carrying out a very labor intensive project and I appreciate each of your contributions.

Last but not least, thank you to my husband, Zac, for your support during this project. Thank you for your willingness to help clean stalls early in the morning every weekend and your openness to learning everything about equine care. Thank you for taking the time to help me study and for always suggesting ice cream after the most stressful days!

v TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

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

CHAPTER

1 Introduction ...... 1

2 Literature Review...... 4

Vitamin E ...... 4

Free Radicals ...... 14

Antioxidants ...... 17

Oxidative Stress ...... 22

Muscle Damage ...... 28

Current Recommendations...... 33

3 Assessment of Oxidative Stress and Muscle Damage in Exercising Horses in Response to Level and form of Vitamin E...... 35 Abstract ...... 36

Introduction ...... 37

Materials and Methods ...... 39

Results ...... 44

vi Discussion ...... 46

Tables and Figures ...... 50

Literature Cited ...... 56

4 Conclusions ...... 61

REFERENCES ...... 63

APPENDICES

A TBARS Procedure ...... 73

B Protein Carbonylation Procedure ...... 75

C Diet and Treatment Groups ...... 77

vii LIST OF TABLES

Page

Table 1: Weight, age and sex distribution for treatment groups...... 50

Table 2: Six-week exercise protocol ...... 51

Table 3: Standard Exercise Test preformed before and after six weeks of exercise condition by each treatment group ...... 51

viii LIST OF FIGURES

Page

Figure 2.1: Tocopherol and tocotrienol isoforms differ based on the methylation around the chromonal ring ...... 5

Figure 2.2: Stereoisomers of -tocopherol based on the R or S orientation of methyl groups around 3 chiral carbons ...... 6

Figure 2.3: : -tocopherol inhibits chain propagation of lipid peroxidation ...... 20

Figure 2.4: The p-orbital orientation of electrons on the aromatic ring and small  of - tocopherol enhances stabilization of the molecule as it neutralizes free radicals ..22

Figure 2.5: Various pathways that contribute to protein carbonylation, leading to oxidative stress ...... 26

Figure 3.1: Timeline of project ...... 51

Figure 3.2: Average serum -tocopherol concentrations ...... 52

Figure 3.3: Average change in aspartate aminotransferase concentrations ...... 52

Figure 3.4: Average change in creatine kinase concentrations ...... 53

Figure 3.5: Average plasma protein carbonylations ...... 53

Figure 3.6: Average total glutathione concentrations ...... 54

Figure 3.7: Average plasma malondialdehyde concentrations as measured by TBARS ...54

Figure 3.8a: Percent change of total stride duration for front legs ...... 55

Figure 3.8b: Percent change of total stride duration for hind legs ...... 55

ix LIST OF COMMONLY USED ABBRVEATIONS

SET Standard Exercise Test

-TPP Alpha-tocopherol transport protein

IU International Unites

•- O2 Superoxide Radical

OH• Hydroxyl Radical

H2O2 Hydrogen Peroxide

ROS Reactive Oxygen Species

ATP Adenosine Triphosphate

ETC Electron Transport Chain

TBARS Thiobarbituric Acid Reactive Substances

MDA Malondialdehyde

DI Deionized

TEP 1,1,3,3-Tetraethoxypropane

CK Creatine Kinase

AST Aspartate aminotransferase

SOD Superoxide dismutase

GPx Glutathione Peroxidase

GSH Reduced Glutathione

GSSG Oxidized glutathione

x PUFA Polyunsaturated Fatty Acid

DOMS Delayed Onset Muscle Soreness

xi CHAPTER 1

INTRODUCTION

Reactive oxygen species (ROS) are unstable, highly reactive molecules that contain an unpaired electron in their outer orbital and are capable of damaging proteins, lipids and DNA. Oxidative stress occurs if the production of free radicals outweighs the level of antioxidants needed to stabilize them (Devasagayam et al., 2004). The occurrence of oxidative stress is much more likely as exercise intensity increases and the electron transport chain becomes overwhelmed. Oxidative stress has been implicated as a cause of muscle damage and decreased athletic performance. This has lead researchers to investigate supplemental antioxidants for their potential to inhibit or reduce oxidative stress.

Vitamin E is a major nutrient considered when formulating diets due to its ability to break free radical chain reactions and protect the body from cellular damage resulting from oxidative stress. Vitamin E exists as a group of fat soluble molecules, each comprised of a chromonal ring accompanied by a saturated (tocopherol) or unsaturated

(tocotrienol) side chain. Tocopherol and tocotrienol forms each have four isoforms (, ,

, ) that differ based on the location and amount of methylation of the chromonal ring.

Each isomer has stereoisomers classified (R) or (S) based on the orientation of chiral carbons on the side chain.

The ability of the body to absorb different forms of vitamin E depends on the chemical structure of each molecule. Of the eight -tocopherol stereoisomers, RRR--

1 tocopherol preferentially binds to the tocopherol transport protein and is therefore found in the highest concentrations in the body (Rey et al., 2013). This is the only form of - tocopherol produced in nature, and it is abundant in fresh forages. However, due to an extended shelf life, supplements and commercially manufactured feeds typically include vitamin E as synthetic-all-rac--tocopherol acetate. This synthetic form is an equal mixture of the eight -tocopherol stereoisomers resulting in it containing only 12.5% of the body’s preferred RRR--tocopherol isomer.

Performance horses are thought to require higher doses of vitamin E as they are often housed in stalls, lacking access to fresh pasture. Additionally, they have a higher intensity workload, predisposing them to more oxidative stress. Currently, the National

Research Council dietary guidelines for equines suggests supplementation may improve the vitamin E status of intensely exercising horses (NRC, 2007). Recent research has found supplementary evidence to these recommendations reporting that higher levels of vitamin E help to maintain plasma -tocopherol concentrations in both Quarter Horse and Thoroughbred race horses following an intense bout of exercise (Siciliano et al.,

1997; Rey et al., 2013).

While researchers have examined the bioavailability of different vitamin E sources as well as different levels of synthetic vitamin E supplementation (Tiidus et al.,

1993; Lawrence et al., 1975), to date very few studies have examined these with regards to exercise, specifically in terms of exercising horses. To address this, the objectives of this study were (1) to determine if supplemental vitamin E above NRC recommendations is beneficial to exercising horses and to (2) determine if there is benefit to natural as compared to synthetic vitamin E by increasing physical activity to a level sufficient to

2 elicit oxidative stress. If successful, the antioxidant properties of vitamin E would counteract excessive free radicals formed during exercise resulting in less oxidative stress and muscle damage as determined by biomarkers that can be measured in blood.

3 CHAPTER 2

LITATURE REVIEW

Vitamin E

History

Vitamin E was first discovered in 1923 by Evans and Bishop at UC Berkley when they found that a diet consisting of lard alone caused fetal reabsorption in rats, but supplementing the same diet with alfalfa, lettuce or wheat germ allowed the rats to resume normal reproduction (Evans, 1925). Originally called Factor X and then later vitamin E, once isolated the nutrient was finally dubbed tocopherol from the Greek tokos meaning childbirth, pherein meaning to bring forth and –ol added to designate the alcohol group on the structure (Schneider, 2005). More research on the vitamin led to the finding of multiple structures of tocopherol as well as its beneficial role as an antioxidant (Niki and Traber, 2012). In 1968, the Food and Nutrition Board of the National Academy of

Sciences officially acknowledged vitamin E as an essential nutrient (Bendich, 2001).

Chemistry

Vitamin E does not exist as one structure, but as a group of fat soluble molecules.

Each vitamin E molecule consists of a chromanol ring, which possess a phenolic group, accompanied by a side chain that stems from carbon 2 on the ring. Tocopherols and tocotrientols are differentiated by the saturation of their side chain; tocopherols possess a saturated isoprenoid side chain while tocotrientols have an unsaturated farnesyl side

4 chain with three double bonds at carbons 3, 7 and 11 (Jiang, 2017). Tocopherols and tocotrienols each have four isoforms (, , , ) that differ based on the location and amount of methylation on the chromonal ring. For both tocopherols and tocotrienols,  isomers are trimethylated,  and  isomers are both dimethylated although the location of methyl groups differ, and  isoforms are monomethylated (Bendich, 2001).

Figure 2.1: Tocopherol and tocotrienol isoforms differ based on the methylation around the chromonal ring (Cook-Mills, 2013).

Further, each isomer has stereoisomers classified as R or S by the orientation of chiral carbons on the side chain. Tocopherol side chains possess three chiral carbons allowing for eight possible R or S combinations (RRR, RRS, RSR, SRR, SSS, RSS, SRS or SSR). The chiral centers are at carbon 2 on the chromonal head as well as carbons 4 and 8 on the side chain. Alternately, due to the double bonds of the tocotrienol side chain, the only chiral carbon is at the initiation of the side chain, at carbon 2, allowing for only two stereoisomers of each isoform (R--tocotrienol or S--tocotrienol) (Jensen and

Lauridsen, 2007).

5 Figure 2.2: Stereoisomers of -tocopherol based on the R or S orientation of methyl groups around 3 chiral carbons (https://forageplus.co.uk/natural-versus-synthetic- vitamin-e-horses/).

Although the different forms of vitamin E have strikingly similar antioxidant properties due to the same chromonal ring found on each molecule (Blatt et al., 2001), the bioavailability (quantification of concentration found in plasma or tissue) and bioactivity

(biological effect) greatly differs between stereoisomers. -tocopherol is the most biologically active form of vitamin E. Specifically, RRR--tocopherol is the standard to which other isomers are compared.

In order to be removed from the liver and enter circulation, vitamin E molecules must bind to hepatic -tocopherol transport protein (-TTP). Given this name due to its affinity for -tocopherol, -TTP preferentially recognizes and binds to this isoform resulting in -tocopherol being found at 10x higher concentrations than -tocopherol in plasma (Wolf, 2006). RRR--tocopherol has also been described as having a half-life three times greater than SRR--tocopherol or -tocopherol most likely due to its primary

6 binding and subsequent easier absorption (Traber et al, 1994; Leonar et al, 2005). It has been reported that the requisites for -TTP recognition and binding are (1) a trimethylated chomonal ring, as is only characteristic of - molecules (DellaPenna and

Pogson, 2006), (2) a phytyl tail, noted only in tocopherols (IUPAC, 1982) and (3) the tail attaching to the chromonal head in the R configuration (Panagabko, 2003). According to the third requirement of -TTP, other 2R--tocopherols (RRS, RSR, RSS) can also bind, however they are still not preferentially bound. Further, mammals do not have the ability to convert between vitamin E isomers. For these reasons, -tocopherol has been shown to be the only isomer that is maintained in human plasma (Traber, 1999). Similarly, RRR-- tocopherol has been demonstrated to be more bioavailable than other vitamin E forms in horses (Fiorellino et al., 2009; Pagan et at., 2010).

Sources and Supplements

Vitamin E is abundant in leafy greens, with the part of the plant responsible for photosynthesis being a major source of vitamin E for horses with access to fresh pasture

(DellaPenna, 2005). Additionally, vitamin E is available as -tocopherol in nuts, seeds and vegetable oils. However, when pasture is cut, processed and stored as hay, 30-80% of the vitamin E concentration is lost, making hay an inadequate source of the vitamin

(Schingoethe, 1978). For this reason, many commercially manufactured feeds include vitamin E, making it the most commonly supplemented antioxidant for horses. Vitamin E can be supplemented as natural vitamin E or synthetic vitamin E.

Natural vitamin E supplements are comprised of 100% of the body’s preferred

RRR--tocopherol. To make these supplements, RRR--tocopherol is isolated from oils

that naturally produce this isomer such as vegetable oil. Due to this form of vitamin E being preferentially absorbed by the body, many researchers have come to recommend natural vitamin E as a superior supplement, reasoning that it accumulates in tissue more proficiently (Hoppe and Krennrich, 2000). Pagan et al. (2005) compared three forms of vitamin E supplementation (two natural forms and one synthetic) using Thoroughbred geldings over six weeks. Results from this study indicated natural supplementations were superior to synthetic supplements in terms of bioavailability. Enhanced bioavailability of natural supplementation has also been demonstrated in piglets, turkeys and dairy cows

(Amazan, 2012; Soto-Solanoa, 1995; Pumfrey, 1993).

RRR--tocopherol can undergo a micellization process to enhance absorption of the supplement. Micelles are small transporters that have a hydrophobic core surrounded by a hydrophilic shell (Jones and Lerous, 1999). Micellizing vitamin E allows the fat soluble molecule to become water soluble resulting in a more efficiently absorbed supplement.

Chemically combining tetramethylhydroquinone (a benzene ring with four methyl groups and two OH groups that serves as the aromatic ring of synthetic tocopherol,

C10H14O2) and racemic isophytol (carbon chain altered to attach as the side chain of synthetic tocopherol, C20H40O) results in synthetic vitamin E. Since the racemic isophytol has three chiral centers, the reaction creates a mixture of the eight stereoisomers of - tocopherol leading synthetic supplements to contain only 12.5% RRR--tocopherol

(VERIS Research summary, 1999). Also known as all-racemic--tocopherol (or all-rac), this is the most popular form of supplementation due to its lower cost and longer shelf life (from the addition of the acetate molecule), however it has been demonstrated to be

8 less bioavailable than the natural supplements. Nevertheless, it has also been shown that all-rac--tocopherol can increase plasma vitamin E levels when compared to a non- supplemented control group. One study on cows compared vitamin E status between a control group (1000 IU/d all-rac--tocopheryl acetate), synthetic supplemented group

(2500 IU/d all-rac--tocopheryl acetate) and natural supplemented group (2500 IU/d

RRR--tocopheryl acetate). Each supplement was formulated to provide the same amount of vitamin E, hypothesizing that both supplemented groups would be similar and higher than the control in terms of vitamin E status of the cow. This study found the highest vitamin E status in the naturally supplemented cows as measured by plasma, colostrum and milk. However, the synthetic group demonstrated higher vitamin E concentrations than the control group, providing evidence that all-rac supplementation does improve vitamin E status (Weiss et al., 2009).

Due to propensity for oxidative degradation, forms of supplementation must undergo chemical processing in order to increase their stability and shelf life. If left unmodified, contact with air causes degradation leading the vitamin to lose its antioxidant properties and thus become ineffective. Often an acetate acid is added to the vitamin E molecule in a process called esterification. Through chemical manipulation an ester group replaces the –OH group on the chromonal ring, acting as a cap to protect the antioxidant properties from oxidation. This ester group cap is easily removed during digestion by esterase enzymes, allowing the molecule to be absorbed as -tocopherol. Pagan et al.

(2013) demonstrated that esterification does not affect the bioavailability of -tocopherol.

Since the esterification process increases shelf life, all-rac supplements can be mixed into commercially manufactured feed without fear of decreased antioxidant status.

9 Alternately, micellization does not protect the molecule from degradation, thus these supplements tend to have a shorter shelf life and are often provided as a top-dressing that must be added to grain daily. All-rac supplements are also available as top-dressing.

In order to compare bioavailability of natural and synthetic sources, a conversion factor was developed by Weiser and Vecchi (1982) using a fetal rat reabsorption model.

They determined a ratio of 1.36:1 for biopotency of natural: synthetic supplementation.

However, some controversy surrounds this ratio as recent research has suggested horses may absorb supplements differently than laboratory animals originally used for this research. Pagan et al. (2010) proposed that natural source, RRR--tocopherol is 2.52 times more potent than all-rac--tocopherol in horses. The vitamin E absorption pathway is similar across species and includes intestinal absorption, prehepatic transport via lipoproteins, hepatic uptake via -TTP, posthepatic transport by additional lipoproteins, and finally uptake by various cellular receptors. However, differences in the type of lipoproteins that transport vitamin E may impact the absorption and thus conversion factor between species. Leat et al. (1978) noted a difference in the distribution of types of lipoproteins between members of the perissodactyla family (mammals with odd numbered hooves that are hindgut fermenters). High density lipoproteins accounted for over 80% of the lipoproteins in domestic horses while no high density lipoproteins were detected in the plasma of rhinoceros, contributing to the low tocopherol concentrations noted in rhinoceros. There may also be differences in concentrations of lipoproteins between horses and rats which can account for the discrepancy in conversion factor.

The International Unit (IU) takes this conversion factor into account and is often used a measurement of alpha-tocopherol in food and supplements as it attempts to

provide the most accurate measure of the amount of vitamin E absorbed by the body. The

National Research Council (NRC) recommends that horses at maintenance require 500

IU of vitamin E per day. Knowing the IU of various supplement forms allows for easier calculation of the amount of supplement that should be given to horses regardless of the form.

Deficiency

Many horses are able to take in adequate amounts of vitamin E via fresh pasture or manufactured grain; however, some horses do not have access to fresh pasture and do not ingest enough vitamin E. Apart from not consuming enough vitamin E, intense exercise has also been implicated as a means for reduced vitamin E levels. Reduced plasma vitamin E levels have been reported in horses exercised over a 4-month period compared to a non-exercised control group (Petersson et al., 1991). Additionally, exercise has been shown to reduce vitamin E levels in other species including rats and humans

(Bowles et al., 1991; Meydani et al., 1993).

For some horses, especially performance horses that regularly exercise at high intensity, vitamin E deficiency may contribute to exercise induced muscle damage

(EIMD). Siciliano et al. (1997) reported the effects of supplemental vitamin E on muscle integrity of exercising horses. Nineteen horses were split into three groups: no supplemental vitamin E, 80 IU/kg DM vitamin E and 300 IU kg/DM vitamin E. Horses were exercised 5 days per week for 90 days. They found serum -tocopherol to decrease in both the control and 80 IU groups. Additionally, they reported a horse in the control group (with no supplemental vitamin E) displayed symptoms of exertional

11 rhabdomylosis, an extreme form of EIMD in which muscles severely cramp inhibiting normal movement.

Vitamin E deficiency has also been associated with a number of neuromuscular diseases in horses. Neuroaxonal Dystrophy and Equine

Degenerative Myeloencephalopathy (NAD/EDM) are closely related diseases, differing by location of axon degeneration, with genetic origins in which gene expression is influenced by -tocopherol concentrations within the first year of a foal’s life

(Baumgartner et al., 1990). Additionally, Equine Motor Neuron Disease (EMND) is an acquired neurodegenerative disorder seen in adult horses with low vitamin E levels that effects the lower motor neurons in the spinal cord and brain stem (Divers et al, 2006).

Toxicity

Supplementing over the recommended amount of vitamin E has shown little to no evidence of toxicity in horses. The body has mechanisms in place to halt accumulations of dangerous levels of the vitamin in the body; rather than excess vitamin E being stored in tissues, it is readily excreted in bile (Kayden and Traber, 1993). There has been evidence that supplementing well above the NRC’s upper safety levels of 20 IU/kg BW per day may cause coagulopathy and poor bone mineralization (NRC, 2007). In addition, supplementing at 10x the NRC requirements may impair beta-carotene absorption

(Williams and Carlucci, 2006).

12 Storage

Once absorbed in the small intestine and processed in the liver, vitamin E is bound to lipoproteins to be distributed to various tissues throughout the body. Due to its fat soluble nature, Blatt et al. (2001) noted that vitamin E is circulated to tissues in a manner similar to the perfusion of fat soluble drugs. They categorized tissues as rapidly perfused central compartments (heart, lungs, brain, kidney, liver), slowly perfused peripheral compartments (other organs, muscle, skin) and a very slowly perfused compartment (adipose tissue). Although the rapidly perfused compartments are able to reach equilibrium faster, due to redistribution 90% of vitamin E is stored in adipose tissue

(Blatt et al., 2001). However, it was found that -tocopherol stored in adipose tissue has poor mobilization as it has been reported to release very slowly during experimental vitamin E deficiency (Machlin and Gabriel, 1982; Machlin et al., 1979). Similarly, neural tissue has also demonstrated a high retention rate of vitamin E indicating poor mobilization. Rats fed a vitamin E deficient diet were able to maintain normal neural function for up to 40 weeks (Pillai et al., 1994). Comparable neural retention rates were also reported in dogs (Pillai et al., 1993). Due to inefficient release of vitamin E from adipose tissue, it has also been reported that supplements must be ingested daily to maintain plasma concentrations as available vitamin E is quickly depleted (Jensen et al.,

1990; Traber and Sies, 1996).

In consideration of the quantification of tissue vitamin E stores, studies have reported consistency between serum vitamin E and tissue vitamin E levels. After pigs were supplemented with vitamin E for 7 weeks, an increase in serum, liver, adipose and skeletal muscle tissue was detected, but after two days of a non-supplemented diet the

13 liver concentration dropped by 80% along with a drop in serum concentration whereas adipose and muscle tissue remained relatively unchanged. This allowed for the conclusion that serum and liver vitamin E values relate to the current vitamin E status while adipose and muscle tissue correlate to nutritional history. This study also pointed to the conclusion that serum vitamin E values are indicative of tissue values (Jensen et al.,

1990). Additionally Fry et al. (1993) considered the association between tissue and plasma -tocopherol in sheep during various weeks of a vitamin E deficient diet, reporting that the plasma -tocopherol provided a reliable indication of vitamin E status.

Free Radicals

A free radical is a molecule with an unpaired electron in its outermost orbital,

•- often due to incomplete reduction of oxygen. Superoxide (O2 ) and the hydroxyl radical

(OH•) are free radicals. Additionally, while not a free radical itself, hydrogen peroxide

• (H2O2) has the potential to produce OH via the Fenton Reaction. Collectively, these free radical species are referred to as reactive oxygen species (ROS). Free radicals are highly reactive species; they have the ability to interact with other molecules in an attempt to gain the missing electron needed for stabilization. When a radical is successful in acquiring an electron from a nonradical, a free radical chain reaction can occur as new radicals are formed which interact with more nonradicals, propagating the formation of more free radicals.

While the build-up of free radicals can be dangerous to the body, low levels of

ROS are important and necessary to maintain optimal health. As a part of the immune response, phagocytes use free radicals to abolish unwanted pathogens (Droge, 2002).

Additionally, ROS has been found to play a role in cellular signaling, gene activation, biosynthesis of prostaglandins and cellular growth (Kohen and Nyska, 2002).

Electron Transport Chain

The body’s main producer of adenosine triphosphate (ATP) is the electron transport chain (ETC). Located in mitochondria, the ETC is comprised of four complexes which work to transfer electrons, reduce O2 to H2O and ultimately break down glucose for fuel. NADH and FADH produced by the TCA cycle donate electrons to carriers at complexes I and II, respectively. As the electrons are passed along to complex III and finally to oxygen at complex IV, a shuffling of hydrogens into the inner mitochondrial membrane space occurs resulting in an electrochemical membrane potential that powers

ATP synthesis at a separate complex V (Fermie, 2004). Normally, 98% of oxygen is successfully reduced to water during this process; however, 2-5% of oxygen consumed ends in the formation of free radicals as electron leakage occurs in the ETC. Complex I and complex III have been implicated as major sites of ROS production in the ETC (Raha and Robinson, 2000).

Complex I, also known as NADH-ubiquinone oxioreductase, is a multiprotein complex that spans the mitochondrial membrane, exposing it to both the matrix and inner membrane space. Here NADH is oxidized to NAD as it donates electrons to coenzyme Q

(Ubiquinone, CoQ). CoQ carries electrons through various iron-sulfur clusters resulting in the movement of hydrogen atoms into the intermembrane space and generating a membrane potential. When electrons leak from this complex, oxygen can receive them resulting in O2•- formation. While some is known of the route of electrons through this

15 complex, the exact mechanism of ROS production has yet to be elucidated. Rotenone is a naturally occurring compound that inhibits electron transfer between the iron-sulfur clusters and CoQ, thus stopping overall oxygen consumption (Suzuki and King, 1983)

•- allowing for a method to study O2 production. Studies using rotenone have hypothesized that the iron-sulfur clusters are the cause of free radical production at complex I

(Kussmaul and Hirst, 2006; Lenaz et al., 2006).

The second common site of ROS production, complex III (bc1 complex, ubiquinone: cytochrome C reductase) is comprised of three main structures that contribute to the movement of protons and electrons along the ETC. This movement is described by the Q cycle in which electrons are accepted from ubiquinol of complexes I and II and transferred to cytochrome C, the next electron carrier. The full Q cycle is made up of two half cycles and is initiated when ubiquinol connects to the CIII complex and transfers two electrons. One of these electrons is passed to the Rieske center, then to cytochrome C1, then to cytochrome C. The other electron from ubiquinol travels along the heme groups of cytochrome b and is then passed to ubiquinone resulting in the formation of a semiquinone radical. The original two electrons are finally transported to the innermembrane space of the mitochondria. The second half-cycle is initiated when a second ubiquinol molecule binds to CIII causing two more electrons to follow the same path as the previous two electrons. Overall, the full Q cycle results in the reduction of two cytochrome C molecules, the formation of one ubiquinol, four protons moved to the innermembrane space and two protons taken from the matrix. It has been theorized that the semiquinone radicals produced during the Q cycle are the molecules responsible for

ROS formation in this complex (Rich and Bonner, 1978; Grigolava et al., 1980)

The state of respiration in the ETC can affect the amount of ROS produced

(Chance and Williams, 1955). In vitro, respiratory states 1-5 exist depending on various factors including available oxygen, ADP and respiration rate. These factors can be manipulated in order to study ROS production. In vivo, mitochondria typically exist in state 3. State 3 respiration is considered normal as CoQ and CytoC are fluctuating between reduced and oxidized states to deliver electrons to their final destination; it is characterized by high ADP, a fast respiration rate and more NADH than NAD. When conducting experiments in a closed system, state 4 respiration can be achieved when

ADP levels are altered to drop resulting in no substrate for ATP synthase. This causes a temporary back-up in the ETC in which coenzyme Q and cytochrome C are locked in reduced states as there is no acceptor for their electrons. This allows for electrons to leak out of the ETC as they are still being donated by NADH, but have no carrier to accept them; the leaks in the ETC result in more ROS. Although free radical production is normal in the body, and even needed to maintain homeostasis (Kohen and Nyska, 2002), increased oxygen consumption leads to a higher rate of free radical production.

Antioxidants

Enzymatic and Non-enzymatic

Antioxidants exist to neutralize free radicals and inhibit cellular damage that may occur from ROS. The antioxidant system includes both enzymatic and non-enzymatic components which can work directly or indirectly to prevent the build-up of damaging

ROS.

17 Enzymatic antioxidants directly interact with free radicals and include the enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx).

•- Often considered the first line of antioxidant defense, SOD breaks down O2 to form

H2O2 and O2 by catalyzing the addition of hydrogen ions resulting in the conversion of two superoxide ions into hydrogen peroxide as explained by the Haber-Weiss reaction:

•- + 2O2 + 2H → H2O2 + O2 (Kohen and Nyska, 2002). SOD exists in three isoforms found in cytosol, mitochondria or extracellular space. Further decomposition of H2O2 into water and O2 (2H2O2 → 2H2O + O2) is accomplished by the enzyme catalase. This antioxidant enzyme is distributed throughout the cell and thought to have higher levels of activity in type I, oxidative muscles. Similarly, glutathione peroxidase (GPx) is another enzymatic antioxidant that works to detoxify H2O2 by converting it to water. GPx, however, has a higher affinity for H2O2 than catalase, making it an important component of the antioxidant system. GPx uses reduced glutathione (GSH) as an electron donor, resulting in the formation of oxidized glutathione (GSSG). GSSG is converted back to GSH by glutathione reductase to propagate further H2O2 removal (Kohen and Kyska, 2002). The general removal of H2O2 by enzymatic antioxidants can be described with the following equation:

• O2 → (SOD) → H2O2 → (CAT or GPx)→ H2O + O2

Non-enzymatic antioxidants include a variety of water and fat soluble dietary components that are radical scavenging, functioning to interrupt free radical chain reactions by donating an electron resulting in stabilization of radicals (Irshad and

Chaudhuri, 2002). Vitamin C and uric acid are among the water soluble antioxidants, while those considered fat soluble include, carotenoids, ubiquinol and vitamin E.

18 Vitamin C, or ascorbate, is generated by the horse and therefore is not

•- • supplemented. It is a key antioxidant for the scavenging of O2 , OH , H2O2 as well as singlet oxygen as it undergoes a two electron reduction to become a semidehydroascorbyl radical, then dehydroascorbate radical. Once fully oxidized, NADH or glutathione can reduce dehydroascorbate back to vitamin C for further radical scavenging (Levine et al.,

1999). Uric acid’s antioxidant properties have been shown to protect cells from singlet oxygen, lipid peroxidation, and protect DNA from ROS destruction (Grootveld and

Halliwell, 1987). Carotenoids, specifically -carotene, act as antioxidants by scavenging singlet oxygen and stopping peroxyl radicals. Additionally, this group is a precursor to vitamin A, which itself has antioxidant properties (Fukuzawa et al., 1998). Ubiquinol is a highly successful lipid peroxyl radical scavenger that also has the ability to recycle vitamin E (Lass and Sohal, 1998).

Vitamin E

Vitamin E is often considered to be the most potent free radical scavenging antioxidant for its role in preventing the propagation of lipid peroxidation chain reactions

(Liebler, 2008). This lipid peroxidation chain reaction occurs when a hydrogen atom is taken from a lipid (LH) by the peroxyl radical (LO•2) to form a lipid hydroperoxide

(LOOH) and a lipid radical (L•). The lipid radical then reacts with oxygen to create an additional lipid radical, thus resulting in a continual chain reaction of lipid peroxidation.

Vitamin E (TO) acts as a lipid radical scavenger, functioning to neutralize the radical before it can attack the lipid, resulting in the termination of the chain reaction. An intermediate and less reactive -tocopheroxyloxyl radical is formed in the process and is

19 recycled by other antioxidants. This reaction can be described by the following equations and animation (Wang and Quinn, 1999):

LO•2 + LH → LOOH + L•

L• + O2 → LO•2

LO•2 + TOH → LOOH + TO•

Figure 2.3: -tocopherol inhibits chain propagation of lipid peroxidation (Wang and

Quinn, 1999).

The chromonal head of the vitamin E structure is responsible for the antioxidant activity, while the tail structure is a determinant of absorption during digestion and needed for preservation of the structure in cellular membranes (Burton and Traber, 1990).

Niki et al. (1985) investigated the phytyl side chain’s influence on antioxidant activity in

-tocopherol and three other structurally related antioxidants by studying the oxidation of methyl linoleate and soybean phosphatidylcholine in conjunction with the four antioxidants. From these experiments they were able to conclude that vitamin E and its most similar model (lacking the side chain) have comparable antioxidant properties;

20 however, the molecule with the side chain showed superior retention within membranes.

These results further evidence that the antioxidant properties of vitamin E are maintained in the chromonal head of the structure.

A pair of electrons maintained on the oxygen of the chromonal head allow for stabilization of the -tocopheroxyloxyl radical which results from the neutralization of free radicals. These electrons occupy a p-orbital around the nucleus of the oxygen; since it is impossible to predict the exact path of electrons the orbital describes the region in which there is a 95% chance of finding an electron (the p-orbital is depicted by the balloon shapes in figure 1.4). The p-orbital orientation allows the electrons to sit perpendicular to the ring structure, this accounts for the enhanced stabilization of the vitamin E radical, contributing to the superior chain breaking properties. Specifically, when neutralizing free radicals, the p-type lone pair of electrons overlap with the singly occupied molecular orbital of the free radical. The resulting molecule is stabilized via resonance, or when electrons continually redistribute themselves into two forms of the same molecule. The degree of overlap between the free radical and the - tocopheroxyloxyl radical is determined by , the angle between the direction of the p- orbital and the line perpendicular to the aromatic plane. -tocopherol has a relatively small . Other structurally similar molecules with a larger  have more steric hindrance, resulting in less overlap between the molecule and radical thus reduced antioxidant potential (van Acker, et al., 1993).

21 Figure 2.4: The p-orbital orientation of electrons on the aromatic ring and small  of

-tocopherol enhances stabilization of the molecule as it neutralizes free radicals

(van Acker, et al., 1993).

The efficiency of antioxidants can be measured by the rate at which they react with radicals. Vitamin E is an extremely capable antioxidant and has been deemed one of the most efficient chain breaking antioxidants for its ability to quickly react with radicals

(Burton and Traber, 1990). In an experiment conducted by Burton et al (1985), it was found that -tocopherol reacts with peroxyl radicals 200x faster than butylated hydroxytoluene, a commercial antioxidant. Vitamin E also has the ability to scavenge superoxide, hydroxyl radicals and most other free radical species. The stereoelectronic features (lone pair of electrons in the p-orbital) allowing for a more stable -tocopherol radical intermediate also contribute to the fast reaction rate (Burton and Ingold, 1981).

Oxidative Stress

During intense exercise, more oxygen is required to meet the demands of the ETC during oxidative phosphorylation for the production of ATP. Augmented oxygen flux to skeletal muscle results in a greater production of ROS. Free radicals detected via spin

resonance spectroscopy showed a two to three-fold increase in the muscle and liver of rats after intense exercise (Daries et al., 1982). Once ROS rise to levels superior to the antioxidant capacity, oxidative stress can occur. Oxidative stress has been defined as an imbalance of ROS and antioxidants and has been shown to damage cellular components such as proteins, carbohydrates, lipids and nucleic acids (Yilmaz, 2012).

Oxidative stress is of special interest to the equine industry. Over millions of years, horses have evolved certain biomechanical features to allow them to excel as elite athletes. Features include the capacity to increase heart rate ten-fold from resting, an advanced muscular system and the ability to release a large number of red blood cells with splenic contractions. Equine sports such as racing, jumping and dressage require the horses to work at maximal levels, leading to a possible increase in exposure to oxidative stress which could result in fatigue, muscle damage and a general decrease in performance. A myriad of studies exist examining oxidative stress in equine athletes, however there are few conclusive results between studies. This may be due to a variation in exercise protocol, level of fitness, nutrition and variables across studies. There are various biomarkers in the blood that can be measured which correlate to incurred oxidative stress.

Lipid Peroxidation

Lipid membranes are particularly susceptible to free radical damage. Lipid peroxidation is the result of a free radical chain reaction that is thought to cause decreased cell function due to a damaged cell membrane and the prevention of nutrient passage (Kerksick and Willoughby, 2005). Lipid peroxidation has been summarized in

23 three steps: initiation, chain-propagation and termination. The process is initiated when a hydrogen atom is removed from the backbone of a polyunsaturated fatty acid (PUFA), allowing for oxygen interaction with free radicals which gives way to an exceptionally reactive peroxyl radical. The peroxyl radical attacks other compounds, forming new radicals resulting in a chain reaction. The chain can be terminated when another molecule is available to donate an electron, resulting in a more stable free radical that can be disposed of by the body (Clarkson and Thompson, 2000).

Malondialdehyde is a major product of cellular membrane breakdown associated with lipid peroxidation. Thiobarbituric acid reactive substances (TBARS) assays are commonly used in equine medicine to demonstrate oxidative stress by measuring MDA levels. However, the TBARS method has been criticized for lack of specificity, sometimes leading to inconsistent results. Brady et al. (1978) reported an increase in plasma MDA after horses ran for 10 minutes. Balogh et al. (2008) demonstrated a decrease in MDA concentrations in plasma when horses were subjected to two 1-minute bouts of intense exercise over jumps. Additionally, a third study reported no change in

MDA concentrations obtained via muscle biopsy after horses participated in a submaximal exercise test (Siciliano et al., 1998). While the inconsistency in these results may be due to the lack of specificity of the assay, it may also be caused by different exercise protocols as well as different fitness levels of the horses used. Regardless of these inconsistencies, it is generally agreed upon that TBARS levels increase post exercise, as associated with increased ROS that tend to develop with more oxygen intake.

24 Protein Carbonyls

Similar to lipid peroxidation, oxidation of amino acids (protein carbonylation) can be caused by exposure to ROS. Protein carbonyl groups, consisting of aldehydes and ketones, form on the side chains of amino acids (especially arginine, lysine, threonine and proline) as a result of oxidation. This process is irreversible and gives rise to proteins that are nonfunctional and susceptible to proteolytic degradation (Stadtman and Levine,

2000). Carbonyl groups can be bound to amino acids by various oxidative pathways.

Proteins can be directly oxidized by ROS, often via a metal-catalyzed oxidation effecting amino acid side chains. Additionally, carbonyls can be formed by direct oxidation of proteins via the -amidation pathway which causes cleavage of the protein backbone.

Lipid peroxidation products such as  or  unsaturated aldehydes from the breakdown of

PUFAs can also contribute to the formation of protein carbonyls when they chemically react with amino acid side chains of proteins via glycation. Furthermore, in a secondary reaction, the oxidation products of reducing sugars produce reactive carbonyl derivatives that react with lysine amino groups to result in protein carbonylation (Dalle-Donne et al.,

2006).

25 Protein backbone Lipid peroxidation cleaved by ROS residues react with oxidation amino acids

Reactive carbonyl Protein oxidized by derivatives from ROS directly reducing sugars Protein react with lysine Carbonylatoin amino groups

Oxidative Stress

Figure 2.5: Various pathways that contribute to protein carbonylation, leading to oxidative stress. (Adapted from Dalle-Donne et al., 2006).

Proteins tend to oxidize at a higher rate than DNA or lipids. Raddak et al. (2002) reported that 5-10% of cellular proteins become oxidized vs less than 0.1% of lipids or

DNA that undergo oxidation. Additionally, protein carbonyls are relatively stable. These characteristics of carbonyls allow them to be easily detected, thus making them reliable indicators of oxidative stress. As such protein carbonylation assays are among that most widely used as a way to quantify protein oxidation.

There is an abundance of studies covering multiple species that have used protein carbonylation as a measure of oxidative stress, many showing an increase in carbonylation with intense exercise. Packer (1997) analyzed the carbonyl content in the hind leg muscle and liver of 12 rats that were either endurance trained for 12 weeks, trained then sedentary or untrained. While there was no difference reported in the liver between groups, it was found that the carbonyl content in the muscle of the trained group was twice as high as the sedentary group. In one of the first studies to examine equine

carbonylation, Kinnunen et al. (2005) demonstrated with eight Standardbred trotters that a moderate bout of exercise was sufficient to cause elevated protein carbonyls measured in plasma. Further, Duberstein et al. (2009) reported differences in protein carbonylation in muscle between samples of pre- and post-exercise horses during a standard exercise test.

Glutathione

Exercise reliably prompts a change in the oxidative status of glutathione, making this antioxidant an additional marker often used to examine oxidative stress. Glutathione is a multifunctional antioxidant. Reduced glutathione (GSH) plays a role in directly scavenging ROS to prevent lipid peroxidation. As a thiol, the active group of glutathione is a sulfhydryl (-SH) group which allows for neutralization of radicals by glutathione. In this interaction, GSH is oxidized to a disulfide (GSSG). With the addition of NADPH,

GSSG is reduced back to GSH by glutathione reductase. Under oxidative stress conditions produced by intense exercise, GSSG is seen at augmented levels. Therefore, the ratio of GSSG: GSH has been employed as an indicator of oxidative stress (Mills et al., 1996). In eight moderately fit humans, Gohil et al. (1988) reported that during extended submaximal exercise GSSG levels increased 100% as well as in increase in

GSH and total glutathione during exercise recovery. Similarly, using six fit horses, increased levels of GSSG have been reported with a prolonged exercise protocol simulating the endurance phase of a three-day event (Mills et al., 1996).

Total glutathione (GSH + GSSG) levels have also been reported as a measurement of oxidative stress. After exhaustive exercise in rats, total glutathione levels

27 were reported to significantly increase in the plasma, skeletal muscle and liver (Lew et al., 1985). Similarly, when eight trained male athletes were subjected to two hours of cycling exercise at 70% VO2max, their total glutathione levels were significantly elevated as compared to pre-exercise levels (Ji et al., 1993). Alternately, Hargreaves et al. (2002) reported an overall decrease in glutathione levels in 35 horses competing in an 80 or 160 km endurance race; the difference is findings may be due to the nature of exercise used in each study (aerobic vs anaerobic).

Another important role of GSH is indirectly scavenging ROS through the recycling of the reduced -tocopherol radical back to active -tocopherol to continue in the intervention of lipid peroxidation. GSH interacts with the free radical reductase enzyme and is again oxidized to GSSG in the process of recycling -tocopherol (van

Haaften et al., 2003). Correlations between levels of vitamin E and GSH have been reported, with increased GSH found in the liver of vitamin E deficient rats (Chow et al.,

1973). It is thought that the augmented GSH may compensate for reduced vitamin E in attempts to prevent oxidative stress.

Muscle Damage

Delayed Onset Muscle Soreness

Unaccustomed physical activity often results in muscle soreness. Delayed onset muscle soreness (DOMS) is a type 1 muscle strain injury and may be the most common and recurring sports injuries for both novice and expert athletes (Cheung, 2003). Muscles contract concentrically (shortening), eccentrically (lengthening) or exist in an isotonic state (no muscle movement); various combinations of these states result in physical

28 movement. Using 60 human subjects assigned to concentric, eccentric or isotonic exercise groups, Talag (2013) reported that eccentric movements caused the most delayed muscle soreness (peaking at 48 hours post-exercise). These types of movements are characterized by active lengthening of the muscle, or when force is applied in conjunction with muscle elongation. In relation to equine anatomy, eccentric contractions occur in the bicep brachii muscle when the limb is on the ground in a stance phase; as the shoulder flexes and the elbow extends, the bicep brachii extends while the force of the ground acts on the muscle. Additional examples of eccentric movement performed by the horse include when a horse lands a jump or when they are moving downhill.

The mechanism of DOMS has yet to be elucidated, however several theories have been developed as to why a pain sensation is associated with eccentric exercise. The most prevalent theories include the lactic acid theory, muscle spasm theory, torn tissue theory, connective tissue theory, and enzyme efflux theory (Gulick and Kimura, 1996).

Some researchers have proposed that DOMS transpires due to a series of events that combine each of the individual theories. The combination theory proposes that the forces produced from eccentric contractions disrupt structural proteins of muscle fibers and cause strain of connective tissue in surrounding muscle fibers. Resulting damage of the sarcolemma causes an accumulation of calcium inhibiting ATP production. The elevated calcium concentrations also stimulate enzymes that damage sarcomeres, troponin and tropomyosin. An increase in neutrophils is then noted. Biomarkers of tissue damage such as creatine kinase leak into plasma, attracting monocytes which in turn convert to macrophages. This stimulates mast cells and histamine production, causing another significant increase in neutrophils. The monocytes and macrophages continue to increase

29 until 48 hours post-exercise, they then produce prostaglandin which stimulate nerve endings. The build-up of histamine, and compounds from phagocytosis in conjunction with pressure from edema and increased temperature stimulate nociceptors in the muscle, all leading to the soreness associated with DOMS (Armstrong, 1990; Smith, 1991; Smith and Jackson, 1990).

While the mechanism is still hypothetical, the muscle damage and corresponding soreness associated with DOMS are known to peak at 24-48 hours post-exercise thus altering athletic performance (Byrne and Eston, 1998). Although equine studies are limited, many researchers have reported a change in biomechanics and gait kinematics in humans following eccentric exercise and used them as a way to quantify muscle damage.

Bruan and Dutto (2003) investigated the effects of eccentric exercise and DOMS in nine highly trained endurance athletes. Running economy (oxygen spent at a fixed, submaximal speed) and stride length were measured at 65%, 75% and 85% VO2max before and 48 hours after the athletes preformed a 30 min run on a treadmill set at 10% decline. The authors reported a decrease in stride length and an increase in running economy, resulting in an overall change in gait attributed to DOMS. An additional study considered the effects of DOMS on leg mechanics by analyzing kinematics of the ankle and knee joint in nine highly trained runners. Runners were recorded with a high speed video camera at 75% VO2max before and 48hours post a 30-minute declined treadmill run.

Results from this study showed a decrease in knee and ankle range of motion coinciding with DOMS at the 48-hour post run time point (Dutto and Braun, 2004).

Vitamin E has been administered as a way to reduce muscle soreness. Although there is documentation of natural RRR--tocopherol reducing muscle damage, there is

little literature published on their effects on DOMS in horses. In contrast, much research has been published on their effects in humans presenting symptoms of DOMS. A literature review citing 36 scientific studies reported mostly positive findings when antioxidants were used as a treatment for DOMS. It was found that antioxidants are most successful at reducing muscle soreness versus a placebo immediately after exercise and at

48 hours post exercise. Furthermore, they reported antioxidant rich foods such as blueberries were more successful at reducing muscle soreness when compared to supplements (Ranchordas et al., 2017). This may be due to RRR--tocopherol in natural foods being more readily absorbed by the body than all-rac-alpha-tocopherol mixtures found in supplementation.

Creatine Kinase and Aspartate aminotransferase

Upon the initiation of intense exercise, the phosphocreatine pathway is the first to respond with the production of ATP. This reversible reaction occurs as a phosphate group is transferred from phosphocreatine to ADP to generate ATP which is catalyzed by the enzyme creatine kinase (CK). However, since phosphocreatine stores are limited and thus rapidly depleted, this form of energy production usually lasts less than five seconds until subsequent energy pathways such as glycolysis are mobilized.

Lipid peroxidation from oxidative stress increases cell membrane permeability allowing for protein compounds, including CK, to leak into the blood. This phenomenon allows CK to be used as an accurate parameter for exercise induced muscle damage as the concentrations of CK in circulation have been shown to correlate to the intensity and duration of physical activity. Using 10 Thoroughbred horses subject to three different

31 exercise protocols, Anderson (1975) demonstrated that strenuous exercise causes a significant increase in CK proportional to the extent of exercise. Further, upon a review of literature, Buzala et al. (2015) confirmed the finding that the increase in serum CK is indicative of workload in horses as it is reported that horses completing endurance rides show a significantly greater increase in CK than those completing lower-effort exercise such as a short bout of treadmill exercise.

CK is generally known to be a reliable parameter for measuring muscle damage.

It has been shown to peak at 4-6 hours post-exercise with a half-life of 90-120 minutes; this allows for values to return to normal in a short time as the enzyme is relatively quickly removed from the blood (Harris et al., 1998) However, some researchers have reported variations of the enzyme depending on individual horse response (Anderson,

1975; Milne et al., 1976). Similar reports of CK variability between subjects have also been noted in man (Clarkson and Ebbeling, 1988). Additionally, differences in exercise protocol, horse fitness and sampling time between studies can cause variations of CK values.

An additional enzyme regularly measured for the evaluation of muscle damage is aspartate aminotransferase (AST). This enzyme is present in most soft tissues and catalyzes the reaction of aspartate and 2-oxoglutataeate to oxaloacetate and glutamate.

Relative to CK, AST has a longer half-life (around 7 days) and does not reach peak concentrations until 24 hours post exercise (Hoffmann, 2008). Regardless, AST is often measured in conjunction with CK to evaluate physical load (Lindholm, 1987). Similar to

CK, AST has been shown to increase post exercise. Freestone et al. (1989) assessed the change in AST for nine Thoroughbreds during 11 weeks of training. Blood was collected

and assessed weekly with increases in AST activity noted after each exercise bout.

Siciliano et al. (1995) reported submaximal exercise elevates serum AST levels, specifically when horses were subjected to repeated submaximal exercise as opposed to short term high intensity exercise.

The changes in enzyme concentration may be due to increased cell membrane permeability often noted in response to lipid peroxidation. It has been reported that supplemental vitamin E may attenuate the leakage of these enzymes, especially when the muscle is subjected to oxidative stress. Itoh et al. (2000) reported a lower concentration in serum enzyme concentrations for men supplemented with -tocopherol vs. a placebo group subjected to six days of running. A study measuring the antioxidant status and subsequent muscle cell leakage of 35 horses during an 80 or 160 km endurance race reported no changes in the plasma vitamin E status of the horses and increases in circulating CK and AST during and after the races (Hargreaves et al., 2002). The author concluded that the maintenance of vitamin E in the system may have contributed to the reduced enzyme leakage as a positive correlation between CK and lipid peroxidation have been reported (Frankiewicz-Jozko and Szarska 2000).

Current Recommendations

The 2007 version of the NRC increased vitamin E recommendations for intensely exercising horses over their previous 1989 recommendation, currently suggesting that intensely exercising horses receive approximately 2 IU/kg BW per day of vitamin E. This newer suggestion has resulted in commercial equine feeds containing higher vitamin E levels. Mostly due to a longer shelf life, the majority of these feeds contain a synthetic,

33 all racemic mixture of vitamin E. While some studies have shown that synthetic supplements are successful in increasing serum vitamin E levels, there is also some evidence that natural supplements may be even more beneficial to exercising horses.

It has been demonstrated that vitamin E is a potent antioxidant that is able to protect cells from ROS that is produced with exercise. However, current literature lacks evidence as to whether or not there is a defined benefit to supplementing vitamin E above

NRC recommendations to exercising horses. Additionally, there is a need to evaluate potential benefits of supplementing horses with natural as compared to synthetic vitamin

34 CHAPTER 3

ASSESSMENT OF OXIDATIVE STRESS AND MUSCLE DAMAGE IN

EXERCISING HORSES IN REPSONSE TO LEVEL AND FORMA OF VITAMIN E1

1 M. M. Fagan, R. Pazdro, P. Harris, A. D. Krotky, J. A. Call, A. Abrams, K. J. Duberstein. To be submitted to The Journal of Equine Veterinary Science

Abstract

Vitamin E is an essential antioxidant that may benefit athletes by reducing oxidative stress. With multiple vitamin E structures, supplements can be derived from natural sources or manufactured as a synthetic tocopherol mixture. This study aimed to determine (1) if supplemental vitamin E is beneficial to exercising horses and to determine (2) if there is a benefit of natural vs synthetic vitamin E. After a 2wk washout

18 horses were divided into three groups and fed a control diet plus: (1) 1000 IU synthetic α-tocopherol (SYN-L), or (2) 4000 IU/d synthetic α-tocopherol (SYN-H), or (3)

4000 IU/d RRR-α-tocopherol (NAT). Horses began a 6wk exercise protocol, with standard exercise tests (SET) performed pre and post the 6wk protocol. Venous blood samples were collected at day 0, 7, 29 and 49. NAT horses had higher α-tocopherol

(P<0.05) at post SET1 through post SET2. Plasma MDA levels were lower in NAT vs

SYN-L horses post SET2 (P=0.02). Serum AST was significantly lower post SET2 in

NAT horses vs SYN-L or SYN-H (P<0.05). In conclusion, feeding higher levels of the more bioavailable natural vitamin E source did appear to have a beneficial effect of reducing oxidative stress.

36 Introduction

As an essential antioxidant, vitamin E is crucial to consider when formulating equine diets. Vitamin E’s main role as an antioxidant is to break free radical chain reactions in an attempt to protect the body from oxidative stress. It is abundant in fresh forages, making it readily available to horses with access to pasture, however it is unstable in the baling and storage process of hay [1]. In order to account for horses that do not have access to fresh pasture, many commercial equine feeds include vitamin E.

Performance horses should be especially regarded when considering vitamin E supplementation. They often are housed in stalls, lacking access to pasture, and due to their high intensity work load tend to incur more oxidative stress than the average horse.

The latest (2007) version of NRC (National Research Council) dietary guidelines for equines [2] left vitamin E recommendations unchanged from the previous (1989) NRC recommendations [3], but states that in some exercise situations vitamin E supplementation above current recommendations may improve vitamin E status. Recent research has supported this recommendation, finding supplementing higher levels of vitamin E helps to maintain plasma -tocopherol concentrations in both Quarter Horse and Thoroughbred race horses following an intense bout of exercise [4,5].

Vitamin E exists not as one structure, but as an entire group of fat soluble molecules. Each vitamin E molecule is comprised of a chromonal ring accompanied by a saturated (tocopherol) or unsaturated (tocotrienol) side chain. Tocopherol and tocotrienol each have four isoforms (, , , ) that differ based on the location and amount of methylation of the chromonal ring. Further, each isomer has stereoisomers classified (R) or (S) based on the orientation of chiral carbons on the side chain [6]. Of all vitamin E

variations, -tocopherol is the preferred isomer for supplementation as it is the most potent radical scavenging antioxidant and the most biologically available, making it the most abundant form in the body. Specifically, the stereoisomer RRR--tocopherol preferentially binds to the tocopherol transport protein to be transferred out of the liver and into circulation in horses [6].

Due to its extended shelf life, supplements and feed typically include vitamin E as synthetic all rac--tocopherol acetate. This synthetic form is an equal mixture of the eight

-tocopherol stereoisomers resulting in it only containing 12.5% of the preferred RRR-- tocopherol. There are also forms of vitamin E available for supplementation that are derived from natural sources resulting in composition of 100% RRR--tocopherol.

Recent research has demonstrated that natural supplementation may be more successful in raising blood -tocopherol levels not only in the horse, but also in humans, piglets, and dairy cows [7-10].

Free radicals or reactive oxygen species (ROS) are highly reactive, unstable molecular substances that contain an unpaired electron in their outer orbital. These species are capable of damaging molecules such as DNA, proteins and lipids by extracting their electrons so the free radicals can stabilize themselves [11]. Although some ROS production is necessary to maintain cellular homeostasis, oxidative stress can occur when the levels of ROS overtake the amount of antioxidants needed to neutralize them [12]. Research abounds examining different levels of vitamin E supplementation on oxidative stress caused by exercise, with some studies finding that supplemental vitamin

E may be able to reduce some oxidative stress biomarkers in horses and humans [13-15], but other research showing a lack of results in the ability of synthetic supplemental

vitamin E to reduce oxidative stress [16, 17]. While researchers have examined the bioavailability of different vitamin E sources, to date very few studies have examined these with regards to exercise. Rats and swine have exhibited preferential response to natural source -tocopherol compared to synthetic supplements [19, 20]. Additionally,

Pagan et al [18] demonstrated a higher bioavailability of natural supplements given to

Thoroughbred horses.

The objectives of this study were to (1) determine if there is any additional benefit of supplementing vitamin E above NRC recommendations to moderately exercising horses and to (2) evaluate potential benefits of supplementing with natural as compared to synthetic vitamin E.

Materials and Methods

Diet and Treatment Groups

Eighteen unconditioned stock-type horses were placed into three treatment groups with six animals per treatment. Horses were blocked by age, sex, and weight, and each block was randomly assigned a treatment group (Table 1). Treatment groups consisted of synthetic low (SYN-L, n=6, 559±60 kg, 11.8±2.6 yrs; 1000 IU/d synthetic all rac-- tocopherol acetate), synthetic high (SYN-H, n=6, 550±30 kg, 12.0±3.9 yrs; 4000 IU/d synthetic all rac--tocopherol acetate) and natural (NAT, n=6, 547±62 kg, 11.2±3.9 yrs;

4000 IU/d micellized RRR--tocopherol) supplementation. All horses were initially placed on a control diet of a formulated grain mixture with no added α-tocopherol for a two-week washout period. The grain was determined via feed analysis to contain 7.8599 mg/l of vitamin E. For the duration of the project, horses were housed in stalls and given

39 access to dry-lot turn out for approximately 6 hours per day. During the washout period, each horse was offered 1.5% body weight (BW) in Russell bermudagrass hay and 0.5%

BW in grain. Following the two-week washout period, horses were placed on treatment diets for one week prior to beginning a 6-week conditioning program (Figure 1). Grain was top dressed with supplemental vitamin E respective to treatment group with NAT receiving 8mL of 500 IU/ml micellized RRR--tocopherol, SYN-H receiving 90.8g of

44.05 IU/g synthetic all-rac α-tocopherol, and SYN-L receiving 22.7g of 44.05 IU/g synthetic all-rac α-tocopherol. All horses also received 56.69g of grain top dressing once daily which provided 1.26mg biotin, 3005.1 IU vitamin D and 36099 IU vitamin A to account for vitamins lacking in the grain. Following this one-week adaptation period, horses began a 6-week conditioning program, and grain was increased to 0.75% BW in order to account for higher energy demands.

Weekly Exercise Protocol

Beginning on week three of the trial (post washout and after one week on treatment diets), horses performed a standardized exercise test (SET) to establish baseline fitness (see section 2.3) and then began a six-week conditioning program of increasing intensity with a second SET performed at the end of this program. Exercise was performed by free lunging in an enclosed, climate controlled arena (85-meter circumference) four times per week for six weeks. Handheld timers were used to monitor speed during the conditioning program, and electronic timers (Farmtec, Wylie, TX) were used to monitor speed during the SETs. Additionally, barriers were placed approximately

3.5m inside the outer wall of the arena in order to ensure that horses were traveling the same distance. Table 2 outlines the weekly exercise protocol.

Standard Exercise Tests

All horses completed a standard exercise test (SET) at the beginning and end of the 6-week conditioning program. This test was performed in the same enclosed arena as the weekly exercise protocol and was adapted from Taylor et al. [21] and Galloux et al.

[22]. Electronic timers were monitored during the test to ensure speed consistency.

Additionally, saddle bags with 45kg of weight were fit to each horse prior to the start of their SET in order to increase the workload by simulating the effects of a rider. Each horse was individually fed their evening grain and respective supplement 12 hours prior to the start of their SET. The protocol for each SET is shown in Table 3.

Blood Collection

Blood was collected via jugular puncture at days 0, 7, 29 and 49. At day 0 and 29 samples were taken at rest and pre-feeding. At days 7 and 49 (the days the SETs were performed) blood was taken immediately pre and 2 hours post SETs as well as 24 hours post SET 2 (Figure 1). At each SET, pre-exercise blood samples were collected in the barn immediately prior to each horse being led to the arena to begin their exercise test.

Post exercise blood samples were collected on each horse at 2 hours following completion of the exercise test. Serum, plasma and whole blood were collected at each time point.

Serum was collected in glass vacutainers with no additive and was allowed to clot at room temperature for approximately 20 min. It was then centrifuged until completely separated and stored in a -80˚C freezer until analysis. Whole blood was collected into two K2EDTA coated vacutainers (one for whole blood analysis and one for plasma analysis) and immediately placed on ice until transported to the lab (approximately 10 min). Whole blood was then aliquoted into storage vials and immediately snap frozen on liquid nitrogen. For plasma analysis, blood was centrifuged and plasma pipetted into storage vials and immediately snap frozen on liquid nitrogen. All blood was then stored in a - 80˚C freezer until analysis.

Blood Analyses

Vitamin E Analysis: Serum was analyzed for -tocopherol levels using high performance liquid chromatography at Iowa State Diagnostics Lab (Ames, Iowa).

Oxidative Stress Analysis: Total glutathione levels were analyzed in whole blood via HPLC-boron doped diamond detection as described by Parks et al [23]. Protein carbonyl content was analyzed in plasma using a protein colorimetric assay kit produced by Cayman Chemical (10005020, Ann Arbor, MI, USA). Results were adjusted for protein content of the sample using a standard BSA (bovine serum albumin) curve.

Thiobarbituric acid reactive substance (TBARS) was measured in plasma according to the centrifugation method described by Ahn et al. [24] and Jo and Ahn [25].

42 Muscle Damage Analysis: Levels of Creatine Kinase (CK) and Aspartate aminotransferase (AST) were analyzed at the UGA Veterinary Diagnostics Lab. CK was analyzed using cobas INTEGRA CKL (Cat # 04524977190) and AST was analyzed using cobas INTEGRA Aspartate Transaminase 500 (Cat # 20764949322). Both parameters were run on Roche Diagnostics cobas c501 chemistry analyzer (Indianapolis,

IN).

Biomechanical Analysis: Horses were filmed pre-SET1 and at 24 and 48 hr post

SET 1 and 2 in order to assess any changes in gait that might be attributed to muscle soreness. Horses were trotted in hand at 4m/s, with speed measured using electronic times (Farmtec, Wylie, TX, USA) to ensure horses were traveling at the correct pace.

Horses were trotted until 6 usable repetitions at 4 m/s (±10% max) were achieved (no more than 10 total repetitions). Video footage was captured using a high speed Ethernet

GigE uEyeTM camera (IDS Imaging Development Systems, Obersulm, Germany) placed perpendicular to the line of travel set to record at 100 frames per seconds. Video clips were downloaded into the Kinovea motion capture analysis program (version 8.15), where they were analyzed for duration of the swing, stance, total stride, and suspension periods. The swing duration was defined as the time from the frame where the hoof first lost contact with the ground to the frame where initial ground contact was made by some portion of the hoof. Stance duration was defined as the time between the frame of initial ground contact by some portion of the hoof to the frame in which the hoof completely lost contact with the ground. Total stride duration was defined as the swing plus the

stance durations of one leg. Suspension duration was defined as the length of time all hooves were off the ground between diagonal pairs.

Statistical Analysis

Blood and biomechanical parameters were statistically analyzed with SAS version

9.4 (Cary, NC, USA) using proc mixed for repeated measures over time. Time, treatment and treatment by time interactions were analyzed for each parameter. Data with P<0.05 were considered statistically significant.

Results

Analysis of serum -tocopherol levels revealed that there was no statistical difference between treatment groups at the initial time point (day 0) nor pre SET1. It was seen, however, that NAT horses had significantly higher serum -tocopherol levels than either SYN-L or SYN-H at all other time points through 2 hours post SET 2 (P<0.05)

(Figure 2). At 24 hours post SET 2, NAT horses had higher levels of serum -tocopherol than SYN-L horses (P< 0.05), but SYN-H horses were not statistically different from either treatment. It was also noted that there were no significant differences in serum α- tocopherol levels between SYN-L and SYN-H groups at any time point.

The change in aspartate aminotransferase (AST) following exercise (2hrs post- pre) at each SET was evaluated as a marker of muscle damage. There were no statistical differences between treatment groups at SET 1. At SET 2, NAT horses had a significantly reduced change in AST as compared to SYN-H (P=0.03) and SYN-L

44 (P=0.02). There were no statistical differences between SYN-L and SYN-H (P=0.83)

(Figure 3).

The change in creatine kinase following exercise (2hrs post-pre) was also evaluated as a marker of muscle damage. No statistical differences were found between treatment groups at either time point potentially because there was a large variation in individual horse response. CK levels did follow the same pattern as AST with the natural group having numerically lower CK levels at SET 2 and the synthetic high and synthetic low groups having higher, comparable CK levels (Figure 4).

Carbonyl content was measured in plasma with few statistical differences between treatments. At post SET 1, it was seen that NAT horses had significantly higher levels of protein carbonylation than the other two groups. However, a slight and non-significant drop in plasma protein carbonyl content was noted for all three groups at the pre SET2 time point. Otherwise, all three treatment groups had comparable carbonylation levels

(Figure 5).

The average total glutathione (GSH + GSSG) measured at each time point revealed no significant differences between treatment groups. However, it was seen that glutathione levels in all three treatment groups numerically (P > 0.05) increased at the three-week time point coinciding with the lowest serum vitamin E levels (Figure 6).

As an additional measure of oxidative stress, TBARS was measured in plasma to assess lipid peroxidation. In accordance with other parameters measured, differences in treatment group response were seen at the second exercise test, but not the first. It was noted that the naturally supplemented horses had lower plasma TBARS levels post- SET

2 as compared to SYN-L or SYN-H (P<0.05) (Figure 7).

The percent change in both front and hind stride duration [(Pre-Post)/Pre *100] was also analyzed as a potential indicator of muscle soreness. As with other parameters measured, the naturally supplemented group had significantly different results than both synthetic groups. There was no statistical difference in the change in front or hind stride duration at the 24-hour post time points between the two synthetic groups. However,

NAT horses showed less reduction in their front (P= 0.0056) and hind (P= 0.012) stride duration as compared to SYN-L and SYN-H horses, potentially due to less muscle soreness (Figures 8a and 8b).

Discussion

The results from this project show that the micellized RRR isomer of vitamin E was able to raise and maintain higher levels of serum -tocopherol throughout the six- week exercise protocol, while increased supplementation of synthetic vitamin E did not significantly raise serum levels. Therefore, in this study the amount of supplementation was not as important as the form of supplementation given to horses. Multiple studies have also demonstrated that the form of vitamin E affects the bioavailability of the supplement. Fiorellino et al. showed that the absorption of vitamin E is highly dependent on the form administered, with micellized supplementation resulting in the highest plasma tocopherol concentrations in horses [6]. In a separate study Pagan et al. found that natural vitamin E sources are more bioavailable than synthetic [18]. Additionally, enhanced effects of natural micellized vitamin E have been demonstrated in piglets [26], turkeys [27] and dairy cows [28].

Research focusing on recovery from intense exercise by vitamin E supplementation has produced confounding results. However, many previous studies used only synthetic supplementation to elicit a response [16, 17, 29, 30]. The present study suggests that natural micellized vitamin E supplementation may play a role in reducing muscle damage, as suggested by the reduced change seen in the NAT treatment group. In this study, many other parameters measured followed the inverse pattern of - tocopherol levels with horses on natural supplementation having less muscle damage, lipid peroxidation, and stride reduction as compared to the two synthetically supplemented groups; this was seen with most significance following the second SET.

Although the synthetic high and natural supplementation were given in equal IU doses, results from this study may indicate the natural supplements are providing a greater dose of active vitamin E. When absorbed during digestion, -tocopherol transport protein (TTP) preferentially binds to RRR--tocopherol. This preferred stereoisomer has a 100% affinity for TTP while its SRR--tocopherol counterpart only has an 11% affinity for binding to the protein [47]. Therefore, having access to significantly more

RRR--tocopherol (100% in natural vs 12.5% in synthetic) may explain the elevated serum vitamin E levels in this study as compared to those that only used all-rac mixtures.

While not noted in the present study, there has been evidence that supplementing well above the NRC’s upper safety levels of 20 IU/kg BW per day may cause coagulopathy and poor bone mineralization [45] and supplementing at 10x the NRC requirements may impair beta-carotene absorption [46].

It has been previously demonstrated that exercise at a high intensity and long duration can result in increased levels of CK and AST, making these enzymes suitable for

evaluating muscle damage incurred by the horse during exercise [31, 32]. Art et al.,

Lekeux et al., and Dias et al. each individually reported elevated CK and AST levels in horses submitted to show jumping competitions [33-35]. While CK and AST followed similar trends in treatment groups in this study, the difference in significance between the parameters may be attributed to more individual variation seen in CK opposed to AST as reported by Anderson [36].

Exercise reliably prompts a change in the oxidative status of glutathione, making this antioxidant an additional marker often used to examine oxidative stress. Usually, the ratio of oxidized to reduced glutathione is reported as a measurement of oxidative stress, however due to the oxidized state of the samples, only total glutathione is able to be reported for this study and resulted in no significant differences between treatments.

Reporting reduced to oxidized glutathione ratios may have prompted more significant results and should be investigated in further studies as past research has reported an increase in oxidized glutathione in correlation to increased oxidative stress in horses and humans [48, 49]. However, at the three-week time point, a numerical increase in glutathione for all treatment groups was noted in conjunction with the lowest measured levels of serum -tocopherol. It is thought that augmented levels of GSH may compensate for reduced vitamin E to prevent oxidative stress as increased glutathione levels have been reported in the liver of vitamin E deficient rats [50].

Evaluation of TBARs has also been historically used as an indicator of oxidative stress in equine studies, specifically as a measure of lipid peroxidation. It is thought that increased TBARs concentrations correlates to increased oxidative stress. In accordance with the increase in TBARs seen post-SET2, multiple studies have reported increases in

48 TBARs in horses following exercise [37-39]. However, when assessed in conjunction with vitamin E supplementation, results are variable. Sumida et al. found that supplementing humans with natural source vitamin E inhibited lipid peroxidation upon analysis of TBARs [40]. When supplementing natural vitamin E to Thoroughbred racehorses, Rey et al. reported finding no effect on TBARs immediately after intense exercise, but did find a decrease in TBARs 8 hours post-exercise as compared to a non- supplemented control group [6]. Alternately, Duberstein et al. noted TBARs levels were unaffected in exercised horses supplemented with synthetic vitamin E versus non- supplemented horses [41]. Results of the present study indicate the naturally supplemented horses had significantly lower TBARs levels at post-SET2. This may further support the idea that natural vitamin E supplementation is superior to synthetic supplements in reducing oxidative stress levels in the body.

Duberstein et al. [41] reported differences in protein carbonylation between samples of pre- and post-exercise horses as a further measure of oxidative stress. In the present study, the only significant difference detected between treatment groups was at 2 hours post SET 1 with the naturally supplemented horses showing significantly higher protein carbonylation levels. This anomaly, in addition to insignificant results, may be explained by the differences in sensitivity of the assays used. Duberstein [41] and Radak

[42] both found favorable results by taking muscle biopsies of horses and rats, respectively instead of analyzing carbonyl content via plasma.

It is also worth noting that on day 42, one horse in the SYN-L treatment group experienced clinical symptoms of rhabdomyolysis immediately at the conclusion of her exercise session. This horse had no known history of rhabdomyolysis, and this event

occurred at the three week mark when the synthetic groups had the lowest serum - tocopherol levels seen during the entire duration of the project (Figure 2). Although there is no evidence linking oxidative stress to rhabdomyolysis, some studies have indicated that a deficiency in vitamin E/ selenium may play a role in the development of rhabdomylosis [44]. Symptoms have been reported in other equine studies in response to low vitamin E diets [5].

Vitamin E is the most commonly supplemented antioxidant for horses [43] with a majority of equine feed companies including synthetic vitamin E additives in their formulated grains. Data from this study may indicate that micellized RRR--tocopherol is superior to synthetic -tocopherol acetate in raising blood levels of -tocopherol and reducing oxidative stress and muscle damage in equine athletes. Further research in this area may investigate the effects of various levels of natural -tocopherol as opposed to the single dosage level investigated in this project to determine the efficacy of lower doses of the natural supplement as compared to the synthetic supplement. Additionally, future research may consider the effects of these treatment diets for an extended treatment time period.

Tables and Figures Table 1: Weight, age and sex distribution for horses receiving NAT (n=6; 4000 IU/d micellized RRR--tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac--tocopherol acetate), or SYN-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E supplementation. Treatment Weight (kg) Age (years) Mares/Geldings Supplement Natural 547  62 11.17  3.869 2/4 8mL Synthetic Low 559  60 11.83  2.563 3/3 22.7g Synthetic High 550  62 12.00  3.899 3/3 90.8g

50 Table 2: Six-week exercise protocol. Each treatment group (Nat, SYN-H, SYN-L) performed the same weekly protocol in an enclosed, climate controlled arena four times per week to increase physical fitness. Week Repetitions Exercise Total Time 1 6 3 min trot (220mpm), 1 min walk (untimed) 18 min trot, 6 min walk 2 4 3 min canter (350mpm), 2 min trot (220mpm) 12 min canter, 8 min trot 3 4 4 min canter (350mpm), 1 min trot (220mpm) 16 min canter, 4 min trot 4 4 3 min gallop (450mpm), 2 min trot (220mpm) 12 min gallop, 8 min trot 5 4 1 min trot (220mpm), 3 min canter (350mpm) 4 min trot, 12 min canter 6 4 1 min gallop (450mpm), 3 min canter (350mpm) 4 min gallop, 12 min canter

Table 3: Standard Exercise Test preformed before and after six weeks of exercise condition by each treatment group (Nat, SYN-H, SYN-L) in order to assess level of physical fitness. The SETs were performed in the same arena as the weekly exercise protocol. Each 500m bout of intense exercise was separated by a shorter trot bout in to allow for partial recovery, as in interval training. Revolutions Distance Speed Direction 6 500m 220mpm (trot) Right (warm-up) 6 500m 220mpm (trot) Left (warm-up) 6 500m 350mpm (canter) Left 4 340m 220mpm (trot) Right 6 500m 350mpm (canter) Right 4 340m 220mpm (trot) Left 6 500m 450mpm (gallop) Left 4 340m 220mpm (trot) Right 6 500m 450mpm (gallop) Right 4 340m 220mpm (trot) Left

Figure 3.1: Timeline of project. * indicates blood draw via jugular puncture. Blood was drawn resting, pre-feeding at day 0 and 21. Blood was drawn immediately pre and 2 hours post each horse completing the SETs on day 7 and day 49. Additionally, blood was drawn 24-hours post each horse completing their SET on day 49.

* * * *

-14 Days Day 0 Day 7 6-week incrementally Day 49 Washout Period Start 1st SET increasing exercise program 2nd SET (Low Vit E Diet) Diets

51 Figure 3.2: Average serum -tocopherol concentrations for NAT (n=6; 4000 IU/d micellized RRR--tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac--tocopherol acetate) and SYN-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E supplemented horses at day 0, 7, 28 and 49 of treatment diets as well as pre and 2-hr post standard exercise tests. * NAT treatment group had significantly higher serum -tocopherol levels than SYN-H or SYN-L group (P<0.05) ^ NAT groups had significantly higher levels of serum -tocopherol than SYN-L but only numerically higher than SYN-H. Serum α-tocopherol levels 12 * 10 * * ^ 8 * 6 Natural tocopherol (ppm)tocopherol -

α 4 Syn High Syn Low 2 Serum 0 Initial Pre SET1 Post SET1 3 week Pre SET2 Post SET2 24hr Post SET2

Figure 3.3: Average change in aspartate aminotransferase concentration for NAT (n=6; 4000 IU/d micellized RRR--tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac-- tocopherol acetate) and SYN-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E supplemented horses calculated from post to pre for standard exercise tests performed before (SET1) and after (SET 2) six weeks of exercise conditioning. a,b: Treatments with differing superscripts at individual time points have statistically different mean values (P<0.05) Change in Aspartate Aminotransferase (Post-Pre) 60 b 50 b 40 30 a Natural a Syn High 20 a AST (U/L) AST Syn Low 10 a 0 -10 SET 1 SET 2 Exercise Time Points

52 Figure 3.4: Average change in creatine kinase concentration for NAT (n=6; 4000 IU/d micellized RRR--tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac--tocopherol acetate) and SYN-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E supplemented horses calculated from post to pre for standard exercise tests performed before (SET1) and after (SET 2) six weeks of exercise conditioning. Change in Creatine Kinase (Post-Pre) 1200 1000 800

600 Natural 400 Syn High Syn Low 200 Creatine Kinase (U/L) KinaseCreatine 0 SET 1 SET 2 -200 Overall trt effect Exercise Time Points P=0.43

Figure 3.5: Average plasma protein carbonylation for NAT (n=6; 4000 IU/d micellized RRR--tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac--tocopherol acetate) and SNY-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E supplemented horses pre and post exercise tests performed before (SET 1) and after (SET 2) six weeks of exercise conditioning. * NAT horses had higher plasma protein carbonylation as compared to SYN-H and SYN- L (P<0.05). Plasma Protein Carbonyl Content 3.5 3 * 2.5 /mg)

2 Natural nmol ( 1.5 Syn High 1 Syn Low 0.5 Carbonylation 0 Pre SET1 Post SET1 Pre SET2 Post SET2 24hr Post SET2

53 Figure 3.6: Average total glutathione (GSH + GSSH) concentrations for NAT (n=6; 4000 IU/d micellized RRR--tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac-- tocopherol acetate) and SYN-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E supplemented horses at day 0, 7, 28 and 49 of treatment diets as well as pre and 2-hr post standard exercise tests and 24-hr post standard exercise test 2. Total Glutathione 1200 1000 800 600 Nat 400 Syn H

Glutathione (uM) 200 Syn L 0 Initial Pre SET 1 Post 3WK Pre SET2 Post 24 Post SET1 SET2 SET2 Time Point

Figure 3.7: Plasma malondialdehyde concentrations as measured by Thiobarbituric acid reactive substance (TBARS) in plasma for NAT (n=6; 4000 IU/d micellized RRR-- tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac--tocopherol acetate) and SYN-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E supplemented horses at pre and post standard exercise tests performed before (SET 1) and after (SET 2) six weeks of exercise conditioning. a,b: Treatments with differing superscripts at individual time points have statistically different mean values (P<0.05)

TBARS 2 b a,b 1.5 a

1 Nat

nmol/mL SynH 0.5 SynL

0 Pre SET 1 Post SET 1 Pre SET 2 Post SET 2 Time Point

54 Figure 3.8a and 3.8b: The percent change [(Pre-Post)/Pre *100] of total stride duration (swing + stance) for front and hind legs of horses supplemented with NAT (n=6; 4000 IU/d micellized RRR--tocopherol), SYN-H (n=6; 4000 IU/d synthetic all rac-- tocopherol acetate) and SYN-L (n=6; 1000 IU/d synthetic all rac--tocopherol acetate) vitamin E. Data for each standard exercise test was averaged to find overall change in stride. a,b: Treatments with differing superscripts at individual time points have statistically different mean values (P<0.05) Front Stride Duration Hind Stride Duration 0 (%Change) 0 (% Change) 1 1 -1 -1 -2 a -2 a -3 Nat Nat -3 -4 Syn High Syn High b -4 Percent change Syn Low Syn Low

-5 Percent Change b -6 b -5 b -7 trt effect -6 trt effect P=0.005 P=0.012

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46. Williams, C.A., Carlucci S.A. (2006) Oral vitamin E supplementation on oxidative stress, vitamin and antioxidant status in intensely exercised horses. Equine Vet J Suppl. 617-621.

47. Hosomi, A., Arita, M., Sato, Y., Kiyose, C., Ueda, T., Igarashi, O., ... & Inoue, K. (1997). Affinity for α‐tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS letters, 409(1), 105-108.

48. Mills, P. C., Smith, N. C., Casas, I., Harris, P., Harris, R. C., & Marlin, D. J. (1996). Effects of exercise intensity and environmental stress on indices of oxidative stress and iron homeostasis during exercise in the horse. European Journal of Applied Physiology and Occupational Physiology, 74(1-2), 60-66.

49. Gohil KI, Viguie CH, Stanley WC, Brooks GA, Packer LE. Blood glutathione oxidation during human exercise. Journal of Applied Physiology. 1988 Jan 1;64(1):115-9.

50. Chow, C. K., Reddy, K., & Tappel, A. L. (1973). Effect of dietary vitamin E on the activities of the glutathione peroxidase system in rat tissues. The Journal of nutrition, 103(4), 618-624.

60 CHAPTER 4

CONCLUSION

The results of this study support the hypothesis that the micellized RRR isomer of vitamin E was able to raise and maintain higher levels of serum -tocopherol throughout the duration of the six-week exercise protocol, whereas there was no significant difference between serum levels of synthetically supplemented horses. Therefore, according to the present study it is appropriate to conclude that the form of supplementation is more impactful than the amount given to horses. This is in agreement with other research that has investigated the effects of natural vs synthetic vitamin E supplementation (Fiorellino et al., 2012; Pagan et al., 2010).

Additionally, this study suggests that natural vitamin E supplementation may play a role in preventing muscle damage due to oxidative stress. Many of the parameters measured followed the inverse pattern of -tocopherol levels. Horses on natural supplementation (those with the highest serum vitamin E levels) showed less muscle damage as determined by significantly reduced levels of AST, TBARS and stride duration as compared to the two synthetically supplemented groups. Further, both the synthetic high and synthetic low groups demonstrated similar levels of muscle damage providing more evidence that the form of supplementation has a greater impact than the amount.

One explanation for this may be that the natural supplements are providing a greater dose of active vitamin E as there is significantly more RRR--tocopherol in these

supplements. This form has a significantly higher affinity to be bound to TTP and transported into circulation. Natural supplements are comprised of 100% RRR-- tocopherol while synthetic supplements encompass a mixture of eight -tocopherols

(12.5% RRR--tocopherol). The augmented levels of RRR--tocopherol available to the tissue may result in enhanced cellular protection.

In order to test this idea further and continue to find optimal vitamin E supplement protocols, future research may consider employing various levels of natural supplementation. Since it is thought the natural supplementation is providing more active

-tocopherol, a study investigating the effects of a lower dose of natural vitamin E versus low and high doses of synthetic supplements could provide more insight to the ideal vitamin E form and amount to supplement exercising horses. Additionally, supplementation for this study was only carried out for six weeks; a future study may consider the effects of supplementing horses for a longer period of time.

Vitamin E is the most commonly supplemented antioxidant for horses with a majority of equine feed companies including synthetic vitamin E additives in their formulated grains. Data from this study indicate superiority of natural supplementation, as opposed to the more commonly used synthetic version, in elevating blood levels of vitamin E, reducing oxidative stress, and preventing muscle damage resulting from exercise. This data should be considered by nutrition companies and performance horse owners aiming to enhance an animal’s athletic potential through vitamin E supplementation.

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72 APPENDIX

A. Thiobarbituric acid reactive substances (TBARS) Procedure

As an additional measurement of oxidative stress, levels of malonaldehyde

(MDA) were determined according to a TBARS centrifugation method described by Ahn et al. (1998) and Jo and Ahn (1998). Pretesting of this method was conducted to find the optimal dilution of plasma: water as this method describes samples as grams of meat opposed to ml of plasma. It was determined that at dilution of .25ml plasma: .75ml deionized (DI) water was ideal. Samples were analyzed pre and 2-hours post each SET, giving a total of 72 samples run. Each day, samples were randomly selected from each time point.

At least 24 hours prior to beginning the procedure a Thiobarbituric Acid/

Trichloroacetic Acid (TBA/TCA) solution was prepared to allow ample time for integration of these two reagents. 15% TCA (75g of TCA added to 500 ml of DI water) and 1.441g of TBA were added to a 500ml volumetric flask. This solution was kept on a stir plate until use. A 1,1,3,3-Tetraethoxypropane (TEP) solution was also prepared ahead of the procedure by adding 23.94 μl of TEP to 100 ml of DI water. This was kept in a freezer until use

On the days of the procedure, a daily TEP dilution was prepared for the standards by combining 1000 μl of the previously made TEP with 100 ml of DI water. Ten standards were prepared in duplicate with increasing concentrations of TEP combined with decreasing concentrations of DI water. This allowed for standard 0 to be 100%

73 water and 0% TEP, standard 5 to be 75% DI water and 25% TEP and standard 9 to be 0%

DI water and 100% TEP.

Once the plasma was thawed, it was transferred in triplicates of .25ml to 50 ml centrifuge tubes; .75ml of DI water was added to this for the plasma dilution. 50μl of

BHT was added to all tubes, followed by 2mls of TBA/TCA. All tubes were then briefly vortexed and incubated in a 90C water bath for 15 minutes. Tubes were then cooled in room temperature water for 10 minutes followed by a 15-minute centrifuge at 3000 x g.

Supernatant from each tube was transferred to a clean tube in order to leave the pellet undisturbed when reading samples. The absorbance of all tubes was then read using a spectrometer at 531 nm.

74 B. Protein Carbonylation Assay Procedure

Protein carbonyl content was analyzed in plasma using a protein colorimetric assay kit produced by Cayman Chemical (10005020, Ann Arbor, MI, USA). The kit included pre-measured hydrochloric acid, DNPH, TCA, guanidine hydrochloride, ethanol, and ethyl acetate. 126 total plasma samples were run over multiple days. Due to the capacity of the centrifuge, only 6 plasma samples were run at a time. Each day samples were randomly selected.

Before the assay began, some of the kit-supplied reagents were to be diluted. The supplied hydrochloric acid was added to 40 ml of HPLC-grade water to give 2.5 M HCl.

The supplied DNPH was dissolved in 10 ml of the prepared 2.5 M HCl. To prepare the

TCA solutions, the supplied TCA was added to 48 ml of HPLC-grade water, resulting in a 20% TCA dilution. 20 ml of the 20% TCA solution was added to 20 ml of HPLC-grade water for a 10% TCA dilution. The contents of the ethanol and ethyl acetate vials were combined for a 1:1 mixture of these chemicals. The guanidine hydrochloride was ready to use as supplied.

Plasma was thawed. Each sample was separated into two 2ml tubes containing

200 μl of plasma for a control tube and a sample tube. 800 μl of DNPH was added to the sample tube, while 800 μl of 2.5 M HCl was added to the control tube. All tubes were incubated in the dark for one hour and votexed every 15 minuets. 1 ml of 20% TCA was added to each tube which were then vortexed and incubated on ice for 5 minutes. Tubes were then centrifuged at 10,000 x g for 10 minutes at 4C. Supernatant was then removed

75 from each tube and the remaining pellets were re-suspended in 1 ml of 10% TCA. All tubes were again incubated on ice for 5 minutes. Tubes were centrifuged for an additional

10 minutes at 10,000 x g. The resulting supernatant was removed and the remaining pellets were suspended in 1 ml of an ethanol: ethyl acetate mixture. The pellets were then manually suspended, vortexed and centrifuged at 10,000 x g for 10 more minutes.

Discarding of supernatant, manually suspending the pellet in 1 ml of ethanol: ethyl acetate mixture and centrifuging for 10 minutes was repeated two additional times. The resulting supernatant was again removed and 500 μl of guanidine hydrochloride was added. The tubes were vortexed and centrifuged for a final 10 minutes at 10,000 x g. For analysis, 220 μl of supernatant from each sample and control tube were placed in two wells of a 96-well plate (each tube was run in duplicate). The absorbance was measured at 360 and 385 nm.

To calculate the protein carbonyl content as nmol/ml, the average was taken of the sample and control tubes for each plasma sample. The average of the controls was subtracted from the average of the samples giving the corrected absorbance. This value was then inserted into the following equation:

Protein Carbonyl = [(CA)/(0.011 μM-1)](500 μl/ 200 μl)

76 C. Diet and Treatment Groups

All horses were initially placed on a control diet of a formulated grain mixture with no added α-tocopherol for a two-week washout period. This period allowed horses to begin the treatment diets with similar baseline vitamin E levels. Horses were housed in stalls and given access to dry-lot turn out for approximately 6 hours per day. The dry lot ensured no horses were ingesting unaccounted for vitamin E via fresh pasture. During the washout period, each horse was offered 1.5% body weight (BW) in Russell bermudagrass hay and 0.5% BW in a 13% crude protein grain that would be used for the duration of the study.

Following the two-week washout, horses were placed on treatment diets for a one-week acclimation prior to beginning a 6-week conditioning program (Figure 3.1).

Treatment groups consisted of synthetic low (SYN-L, n=6, 559±60 kg, 11.8±2.6 yrs;

1000 IU/d synthetic all rac--tocopherol acetate), synthetic high (SYN-H, n=6, 550±30 kg, 12.0±3.9 yrs; 4000 IU/d synthetic all rac--tocopherol acetate) and natural (NAT, n=6, 547±62 kg, 11.2±3.9 yrs; 4000 IU/d micellized RRR--tocopherol) supplementation. For this acclimation period, horses received 0.5% BW of the formulated grain, top-dressed with supplemental vitamin E respective to their treatment group. NAT received 8mL of 500 IU/ml micellized RRR--tocopherol, SYN-H received 90.8g of

44.05 IU/g synthetic all-rac α-tocopherol, and SYN-L received 22.7g of 44.05 IU/g synthetic all-rac α-tocopherol. All horses also received 56.69g of grain top dressing once daily which provided 1.26mg biotin, 3005.1 IU vitamin D and 36099 IU vitamin A to

77 account for vitamins lacking in the grain. Horses also continued to receive 1.5% BW in hay. After the acclimation period, horses began a 6-week exercise conditioning program.

Grain was increased to 0.75% BW to order to account for higher energy demands. For the duration of the exercise program, each horse was given the vitamin E topdressing respective to treatment diet, vitamin premix topdressing and hay.

Grain was offered twice daily in stalls; once at 7:00am and once at 7:00pm.

Horses were weighed prior to the start of the project in order to calculate percent of BW for grain and hay. Horses were reweighed midway through the project to account for change in weight due to exercise and adjust grain and hay amounts.