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A Combination High in Antioxidant Foods’ Effects on Blood Antioxidant and Oxidative Stress Levels in Post-Menopausal Women. by Shelby Kloiber, B.S. A Thesis in Health, Exercise, and Sport Sciences Submitted in partial fulfillment of the requirements for the Degree of Master of Sciences Approved by Melanie A. Hart, Ph.D. Co-Chair of Committee Jacalyn McComb, Ph.D. Co-Chair Yoonjung Park, Ph.D. Peggy Gordon Miller Dean of the Graduate School August, 2011 Copyright 2011 Shelby Kloiber Texas Tech University, Shelby Kloiber, August 2011 Acknowledgements I would like to show my gratitude towards the individuals who supported and assisted me during research. Dr. Robert Sawyer for guiding me through the research, Will Martin, graduate student, for assisting me during my blood work and assay completion, Dr. Jamie Cooper for allowing me to use her lab facilities during the research, and to my parents Rick and Lydia Kloiber for their guidance and support during my entire education. I would also like to send appreciation to Dr. Joaquin Gonzales for his help and his direction with this project. ii Texas Tech University, Shelby Kloiber, August 2011 Table of Contents Acknowledgements ii Abstract vi List of Tables vii I. Introduction 1 Statement of the Purpose 4 Significance of Study 4 Hypotheses 5 Delimitations 5 Limitations 6 Assumptions 6 Definition of Terms 6 II. Review of Literature 8 Oxidative Stress 9 Free Radicals (ROS) 10 Antioxidants 11 Menopause and Oxidative Stress 12 Antioxidants and Oxidative Stress 13 iii Texas Tech University, Shelby Kloiber, August 2011 Women and Antioxidants 15 Summary 20 III. Methodology 21 Participants 21 Procedure 22 Hematocrit and Anthropometric data collection 23 Antioxidant capacity and oxidative stress data collection 24 Intervention protocol 25 Antioxidant capacity assay protocol 25 Oxidative stress assay protocol 26 Data Analysis 26 IV. Results 28 Oxidative Stress 28 Antioxidant Capacity 29 V. Discussion 31 Oxidative Stress 31 Antioxidant capacity 33 iv Texas Tech University, Shelby Kloiber, August 2011 Summary 34 References 36 A. Recruitment Materials 41 B. Informed Consent 44 C. Health History Survey 47 D. Physical Activity Survey 50 E. Dietary Recall 52 F. Banned Food List 55 G. Food Check-Sheet 56 v Texas Tech University, Shelby Kloiber, August 2011 Abstract Oxidative stress brought on by free radicals can lead to an increased risk of certain diseases such as heart disease and some cancers. Oxidative stress mediated damage can be reduced by scavengers, or antioxidants that can eliminate the high reactivity of free radicals by turning them into non-radical and nontoxic metabolites. Many scientists have investigated the effects of different kinds of foods (whole, liquid, or supplement) to measure the change in oxidative damage and antioxidant capacity. The purpose of this study is to examine the effects of two types of foods high in antioxidants on markers of oxidative stress and antioxidant capacity in postmenopausal women. Healthy post-menopausal women, (N=16) were divided into four groups (i.e., fruits, soymilk, fruits and soymilk and control). Oxidative stress and antioxidant capacity were measured before and after the intervention. Oxidative stress results indicated no significant differences. Antioxidant capacity results indicated a significant main effect for Test with the mean for the pre-test (M = 0.28 units/ml, SD = 0.15) being significantly lower than the mean for the post-test (M = 0.39 units/ml, SD = 0.23). The results from this study did not support the effectiveness of fruits and soymilk on the oxidative stress levels and antioxidant capacity in postmenopausal women. vi Texas Tech University, Shelby Kloiber, August 2011 List of Tables 1. Weight and BMI 28 2. MDA values pre and post 29 3. SOD values pre and post 30 vii Texas Tech University, Shelby Kloiber, August 2011 Chapter I Introduction Within the human body there are a number of chemical reactions that occur (Brooks, Fahey, & Baldwin, 2005). Most of the reactions are needed for the body to function appropriately. However, some of these reactions produce by-products that have the potential of being harmful to some of the body’s delicate tissues. One such byproduct is known as an oxidant. Oxidants are formed as a normal product of aerobic metabolism, but can be produced at elevated rates under pathophysiological conditions. These elevated rates may result in damage to cells. However, various compounds known as antioxidants protect biological systems against the potential harmful effects of processes or reactions associated with the presence of oxidants (Sies, 1997). An imbalance between oxidants and antioxidants in favor of the oxidants is known as “oxidative stress” (Sies, 1997). Oxidative stress biomarkers have been recognized as a factor in many acute and chronic diseases. Antioxidants can prevent or inhibit the effects of oxidation (i.e., oxidative stress) by decreasing localized oxygen concentrations so that increased oxidation is less likely to occur. This prevents some of the initial reactions of oxidation by scavenging free radicals that are capable of abstracting hydrogen from molecules. Numerous studies have been conducted examining the effects of antioxidants on various populations (Heitzer, Schlinizig, Krohn, Meinertz, & Munzel 2001; Seifried, Anderson, Fisher, & Milner, 2007; Svilaas et al., 2004). One benefit of antioxidants is that they have been found to reduce the damaging effects of oxidative stress on deoxyribonucleic acid (DNA) (Ryan-Borchers et al, 2006). 1 Texas Tech University, Shelby Kloiber, August 2011 This finding is of particular importance as increased levels of oxidative stress result in an imbalance of a body’s redox system. The redox system is important for oxygen homeostasis, the balance between antioxidants and oxidants. Redox reactions involve the transfer of electrons between two chemical species: compounds that lose electrons (oxidized) and those that gain electrons (reduced). If the homeostasis is not maintained, the cell becomes oxidatively stressed (Seifried et al., 2007). A disturbance in the homeostasis can result in toxic levels of oxidative stress that produce free radicals, which have been shown to damage all parts of a cell. Thus, oxidative stress may be involved in many different types of diseases such as coronary artery disease, stroke, arthritis, and cancer (Prior, 2003). Along with the increased risk of diseases, the cells of aging organisms can accumulate increased levels of oxidant-damaged nuclear DNA (Finkel & Holbrook, 2000), which may lead to mitochondrial DNA damage. This damage will potentially compromise the mitochondrial function; thus, releasing more reactive oxidative species (ROS) and increasing the cycle of more DNA damage. Termination of ROS can only be halted by the removal of intermediates (i.e., antioxidants) within a chain. After menopause, there is a decline in estrogen concentration (Ryan-Borchers et al, 2006). The sharp decline in endogenous estrogen production during menopause has, historically, resulted in hormone replacement therapy to alleviate certain risks (e.g., cardiovascular diseases and osteoporosis; Frankenfeld et al. 2003). Although many women engage in such therapy, others find this unnatural and/or are concerned with other health risks (Mingo, Herman, & Jasperse, 2000). Increasing antioxidant levels in food 2 Texas Tech University, Shelby Kloiber, August 2011 such as soy isoflavones can help alleviate the signs and symptoms in menopausal and postmenopausal women such as hot flashes (Han, Soares, Haidar, Rodrigues de Lima, & Baracat, 2002). Additionally, oxidative stress often increases after menopause; thus, further compromising a woman’s immune system. Many different antioxidants within a diet can help break this chain. Additional research is needed to examine the effect of antioxidants on oxidative stress in postmenopausal women. There are various sources of antioxidants such as fruits and vegetables (Prior, 2003), fruit and vegetable juices (Cilla et al., 2009), red meat, poultry (Djuric et al., 1998), soy products (Ryan-Borchers et al, 2006), and algae (Scoglio et al., 2009). Antioxidants found within these foods have the potential to inhibit oxidation within the cell. However without these antioxidants, the production of free radicals can set a chain reaction of events that will damage cells. The damage or destruction of cells because of the lack of antioxidants in a diet can result in many different diseases if antioxidants are lacking in the diet. A variety of approaches have been examined to determine the effects of antioxidant supplementation on oxidative stress (Seifried et al., 2007). Contradicting results have been found among these studies, because different amounts of antioxidant supplementation have been given during interventions. Antioxidants have been found to play a vital role in a person’s health (Seifried et al., 2007); however, the specific type, amount, and those that have an interdependent effect on each other are still being researched. 3 Texas Tech University, Shelby Kloiber, August 2011 Statement of the Purpose The purpose of the current study is to examine the effects of two types of foods high in antioxidant on markers of oxidative stress and antioxidant capacity in postmenopausal women. Significance of the Study An increase in oxidative stress levels as a result of menopause can lead to an increased risk for diseases such as coronary heart disease and cancer (Ryan-Borchers et al., 2006). Researchers have found positive effects within the body from foods high in antioxidants (Seifried et al., 2007). Whole food supplementation including meat products, fruits, vegetables, and walnuts were all shown to have a decrease in oxidative stress, which can reduce the risk of breast cancer and coronary heart disease (Djuric et al., 1998; McKay et al., 2010). Although there was a significant increase in antioxidant levels after a soymilk intervention, there were no significant changes in antioxidant levels with a fruit juice diet (Cilla et al., 2009; Ryan-Borchers et al., 2006). Grape powder, algae extract, and tablets containing large amounts of vitamin E are just a few of the antioxidant interventions that have been found to decrease oxidative stress, and increase well-being in postmenopausal women (Ryan-Borchers et al., 2006). Researchers (Zern et al., 2005) have used individual antioxidant supplementation to increase total antioxidant capacity (TAC). However, the influences of the combination of foods high in antioxidants have not been examined. Therefore, it is important to determine if there can be increased antioxidant levels because of a combination of antioxidants, such as increased fruit intake and soymilk. It is important to assess the 4 Texas Tech University, Shelby Kloiber, August 2011 effects of a combination of antioxidants in postmenopausal women, because an increased risk for certain diseases may occur due to high oxidative stress levels (Scoglio et al., 2009). Hypotheses The experimental design of this study will allow for the comparison of three antioxidant supplementations on antioxidant levels and levels of oxidative stress. There will be a soymilk intervention, a fruit intervention, an intervention that includes a combination of the two antioxidants, and a control group. Hypothesis I: All experimental groups will see an increase in antioxidant levels. Hypothesis II: All experimental groups will see a decrease in oxidative stress Hypothesis III: A combination of antioxidants will elicit a greater increase in antioxidant levels than a single supplementation. Hypothesis IV: A combination of antioxidants will elicit a greater decrease in oxidative stress markers than single supplementation. Delimitations This study was delimited to the following: 1. Females in amenorrhea for at least 12 months with climacteric symptoms of at least 4 months. 2. Women participating in hormonal treatments or therapies were excluded from the study. 5 Texas Tech University, Shelby Kloiber, August 2011 3. Participants had to have an absence of uterus dysfunctions, cardiovascular diseases, hypertension, or diabetes. 4. The antioxidant interventions were fruits, soymilk and a combination of the two. Limitations The following are potential limitations of the study: 1. The accuracy of the results was dependent on the participants adhering to the intervention protocols (i.e., consuming the proper antioxidants and the correct amount each day). 2. The participant’s truthfulness on the compliance check sheet. 3. A relatively low sample size may result in insufficient power to identify a significant effect. Assumptions The following assumptions were made during this study: 1. The daily food record was truthfully answered. 2. The participants adhered to the protocol intake of their assigned antioxidant group. 3. The participants purchased the appropriate food and/or beverages to complete the antioxidant supplementation. 4. Superoxide Dismutase (SOD) is an accurate detection for antioxidant levels. 5. Thiobarbituric Acid Reactive Species (TBARS) is a well-established method for screening and monitoring lipid peroxidation, an indication of oxidative stress. 6 Texas Tech University, Shelby Kloiber, August 2011 Definition of Terms 1. ROS – reactive oxygen species – a free radical with an unpaired electron that is potentially dangerous as they are likely to react indiscriminately with any molecule nearby including DNA (Brooks, Fahey, & Baldwin, 2005). 2. Postmenopausal is the point after which a woman has reached menopause or cessation of the menstrual cycle for one year (Robert-McComb, Norman, & Zumwalt, 2008). 3. SODs – superoxide dismutase- are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and this form a crucial part of the cellular antioxidant defense mechanism (McCord & Fridovich, 1969). 4. TBARS – Thiobarbituric Acid Reactive Substances- is a protocol that monitors lipid peroxidation, which is a well-established mechanism of cellular injury in both plants and animals and is used as an indicator of oxidative stress in the cells and tissue (Kosugi, Kojima, & Kikugawa, 1989). 7 Texas Tech University, Shelby Kloiber, August 2011 Chapter II Literature Review According to the World Health Organization, 605 million persons are currently 60 years of age or older (Kennedy, 2006), which is partially due to an increase in life expectancy. This increase in life expectancy is predominately as a result of better health care and preventative strategies, or overall wellness. Wellness is defined by Conrad (1994) as a conscious and deliberate approach to an advanced state of physical and psychological/spiritual health. One component of wellness is health, which is not suffering from disease, pain, or other defects (Hansen-Kyle, 2005). Living a healthy life can contribute to prolonged wellness and life expectancy. Some strategies include lifestyle changes such as engaging in regular physical activity and having a well-balanced diet. Health and health-promotion behaviors are frequently depicted as the good while disease and putatively disease-producing behaviors are seen as bad (Conrad, 1994). Infectious diseases of the early 1900s, such as pneumonia and tuberculosis, have been replaced by cardiovascular disease and cancers as the major causes of mortality (Conrad, 1994). Globally, chronic diseases such as obesity, type 2 diabetes, hypertension, coronary artery disease, and cancers are becoming more prominent (Kennedy, 2006). Many of these chronic diseases are preventable when individuals engage in healthy lifestyle behaviors. Engaging in physical activity has shown to increase the physiological component of healthy aging. Individuals who maintain physical conditioning and exercise regularly tend to have fewer medical problems than individuals who live a sedentary lifestyle. Along with physical activity, a nutritionally 8 Texas Tech University, Shelby Kloiber, August 2011 balanced diet high in protein and fiber has been found to be a factor that contributes to overall improved health (Hansen-Kyle, 2005). This improved health can lead to a higher quality of life, especially as one gets older. However as one ages, a number of diseases associated with an altered immune system, such as cardiovascular diseases, osteoporosis, and some cancers, may be prevented by a well-balanced diet. Micronutrients have been found to be associated with the reduction of physiological responses such as oxidative stress (Hansen-Kyle, 2005). Oxidative Stress Oxidative stress is defined as an imbalance between production of free radicals and reactive metabolites, so-called oxidants, and their elimination by protective mechanisms, referred to as antioxidative systems (Durackova, 2010). Three conditions that deal with the role of oxidative stress in aging include: 1) levels of molecular oxidative damage increase during aging; 2) a relatively longer life expectancy within and among species is associated with a correspondingly lower accrual of oxidative damage; and, 3) a prolongation of life-span by regimens such as caloric restriction in mammals is associated with the amelioration of oxidative damage (Sohal & Weindruch, 1996). Oxidative stress biomarkers have been recognized in many acute and chronic diseases, and increase throughout various phases in the human lifespan (Finkel & Holbrook, 2000). This finding is of particular importance as increased levels of oxidative stress result from an imbalance of a body’s redox system. The redox system is important for oxygen homeostasis; the balance between antioxidants and oxidants (Seifried et al., 2007). Redox reactions involve the transfer of 9 Texas Tech University, Shelby Kloiber, August 2011 electrons between two chemical species: compounds that lose electrons (oxidized) and those that gain electrons (reduced). If the homeostasis is not maintained, the cell becomes oxidatively stressed. This oxidative stress has been associated with several diseases, such as neurodegenerative diseases, diabetes mellitus, metabolic syndromes, skin and tumor diseases, and psychic impairments. Additionally, oxidative stress is associated with the aging process (Durackova, 2010). Currently, there is no possible way to measure ROS directly. Oxidative stress can be assessed by measurement of reaction products of oxidative damage. As noted previously, lipid peroxidation is the most studied reaction product for oxidative stress markers with a variety of techniques including thiobarbituric acid-reactive material assays, commonly known as TBARS (Betteridge, 2000). Free Radicals (ROS) Free radicals can be defined as any chemical species that contains unpaired electrons. Unpaired electrons increase the chemical reactivity of an atom or molecule (Betteridge, 2000). Free radicals, also known as reactive oxygen species (ROS), have long been assumed to have only negative functions in the organism. An increase in free radicals may lead to irreversible damage of mitochondrial DNA, membrane lipids and proteins; thus, resulting in mitochondrial dysfunction and ultimately cell death (Kowaltowski & Vercesi, 1999). Lipid peroxidation is perhaps the most extensively studied consequence of free radical attack. Reactive free radicals have the capacity to abstract a hydrogen atom from fatty acids, leaving behind an unpaired electron on the carbon. The remaining carbon undergoes molecular rearrangement resulting in 10 Texas Tech University, Shelby Kloiber, August 2011 conjugated dienes, which combine with oxygen and become radical. This production starts a chain reaction that continues until the substrate is consumed or the reaction is terminated by a chain-breaking antioxidant. This lipid peroxidation can have profound effects on cellular function (Betteridge, 2000). In summary, oxidative stress occurs when the balance between free radicals and antioxidative protection is disturbed leading to damage within the organism (Durackova, 2010). However, the effects of oxidative stress can be reduced when specific compounds are present. Antioxidants Various compounds known as antioxidants protect biological systems against the potential harmful effects of processes or reactions that can cause excessive oxidations (Durackova, 2010). Scavengers of free radicals (i.e., antioxidants) can eliminate the high reactivity of free radicals by turning them into non-radical and nontoxic metabolites. These antioxidants prevent oxidation by free radicals of biologically important molecules (Durackova, 2010). Antioxidant function can be described in terms of prevention, interception, and repair. The preventative function of antioxidation channels involves an attacking species by turning over or elimination of the whole cell, hence lowering the risk for further damage. The antioxidant function of interception involves transferring the radical function away from a more sensitive target sites (e.g., cell membrane ) to compartments of the cell in which an oxidative challenge would be less deleterious (e.g., cytosol). Lastly, repairing the damage of oxidation includes assisting multiple enzyme systems involved in DNA repair, lipolytic and proteolytic enzymes, that are capable of serving the functions of restitution or replenishment (Sies, 1997). The three major 11 Texas Tech University, Shelby Kloiber, August 2011 classes of antioxidant enzymes are the superoxide dismutases, catalases and glutathione peroxidases, and those involved in the transportion and elimination of reactive compounds. Antioxidant enzymes can be found in varying amounts in different subcellular sites and types of cell (Sies, 1997). Antioxidants such as vitamin E, βcarotene, and coenzyme Q are highly effective in interrupting the chain reaction associated with lipid peroxidation (Betteridge, 2000). Without the antioxidants, oxidative stress may impair the body’s immune function. Menopause and Oxidative Stress Menopause can lead to a decrease in estrogen production (Pick, 2005). Estrogen, an immune-modulating hormone is associated with proper functioning of the immune system. Because estrogen production decreases following menopause, the immune system of post-menopausal women may be compromised. The immune system encompasses an array of defenses that help to guard against the development of a number of diseases, some of them age-related (Ryan-Borchers et al. 2006). An increase in oxidative stress and a decrease in estrogen place postmenopausal women at increased risk for several diseases. Oxidative stress may be involved in many different types of diseases such as coronary artery disease, stroke, arthritis, and cancer (Prior, 2003). The damage to the cell as a result of decreased estrogen production combined with DNA damage from reactive oxygen species alters the mitochondrial physiology that may contribute to a greater cellular stress response, cell growth arrest, and subsequent apoptosis (Yakes & Van Houten, 1997). The life expectancy for women has increased leading to approximately 30 years of ceased production of estrogen from ovaries, and there is 12 Texas Tech University, Shelby Kloiber, August 2011 overwhelming evidence that indicates that estrogen has a protective effect against coronary artery disease (Robert-McComb et al. 2008). Based on these changes, postmenopausal women may benefit from a high antioxidant diet, which may reduce the amount of DNA damage brought on by the production of oxidative stress after menopause (Ryan-Borchers et al., 2006). Antioxidants and Oxidative Stress Some studies have examined antioxidant effects by assessing the diets of individuals. Most of these studies used a food recall or diary as opposed to a diet intervention or antioxidant supplementation to measure the effects of antioxidants (Djuric et al., 1998; Svilaas et al., 2004). In a study examining the effects of the consumption of various food groups in Norwegian adults on the total antioxidant capacity, antioxidant intake was determined by collecting a 7- day weighed dietary record (Svilaas et al., 2004). The results of the data analysis indicated that the total intake of antioxidants was significantly correlated with plasma lutein, zeaxanthin, beta carotene, and alpha carotene, all of which have strong antioxidant properties within the body. Although coffee consumption has been shown to increase plasma homocysteine, and is likely associated with a small increase in blood pressure after many years of consumption, the results of the dietary analysis showed coffee along with fruits such as berries were the greatest contributors to the total antioxidant intake. These antioxidants were found to be associated with a reduced risk of major chronic degenerative diseases. Another study by Prior (2003) examined the effects of fruits and vegetables on antioxidant levels. Specifically, Prior examined the amount of absorption between two 13 Texas Tech University, Shelby Kloiber, August 2011 different flavonoids, anthocyanins and flavonols. The effects of vegetables containing quercitin (e.g., onions) and fruits containing cyaniding glycosides (e.g., pears and dark fruits such as blueberries and blackberries) on cyaniding 3-glucoside were examined in a sample of rats. The major findings were a decrease in antioxidant levels and a measure of lipid peroxidation (i.e., TBARs) with dark berries. Additionally, an increase in antioxidant activity and a decrease in hydroperoxides, a measure of oxidative stress, were found with the consumption of onions. Tesoriere and colleagues (Tesoriere, Butera, Pintaudi, Allegra, & Livrea, 2004) studied the effects of cactus pear fruit on oxidative stress levels in healthy middle-aged men and women. Participants were given either 250g of cactus pear pulp or a vitamin C supplementation to be taken for 2 weeks. After a 6-week wash-out period participants received the other treatment. Blood samples were taken to measure hydroperoxides. The results indicated the cactus pear fruit intervention showed significant decreases in hydroperoxide, but no significant decrease was found with vitamin C supplementation. These results showed the antioxidant effect was due to the fruit and not the vitamin C in the fruit. Another study examined specific supplement antioxidant activity in both men and women (Cornell et al., 2001). The contents of the antioxidant supplementations included either capsules prepared with different types of antioxidants or a fluid formula with the same types of antioxidants. Formula 1 contained Zinc, Selenium, L-cysteine, Vitamin A and E, and beta-carotene. Formula 2 contained citrus flavanoids, Vitamin C and B-6, and Coenzyme Q10. The final formula contained all ingredients from formulas one and two. 14 Texas Tech University, Shelby Kloiber, August 2011 The type of supplementation and amount of antioxidants in the capsules and fluid were similar to that of an average amount of antioxidants consumed in a normal diet. The participants were asked to consume the supplementation every day during breakfast. Each participant’s treatment included the capsules, followed by a washout period, and ending with the fluid supplementation. Organic hydroperoxides within the blood serum were examined. The results indicated that low dosages of antioxidant supplementation given in fluid form showed the largest decrease in oxidative stress. Based on the studies summarized thus far, there is evidence that antioxidants in the diet, whether naturally consumed or supplemented, are associated with decreased levels of ROS. Women and Antioxidants The research examining the role of antioxidants in women is limited. Djuric and colleagues (1998) investigated the relationship between specific food intakes (e.g., meat, vegetables, and fruits) and the levels of oxidative DNA damage in female participants between the ages of 18 and 65 years who had at least one first-degree relative who had been diagnosed with breast cancer. Two groups were randomly assigned to a normal diet or diet low in fat (15% fat). Each group was given a list of appropriate foods to consume based on the group requirements and appropriate cooking methods for each of the foods. Dietary intake per day and the preparation method (e.g., raw, cooked, or high temperature cooked) were recorded. Analyses examined how individual food items and various combinations of the items related to DNA damage. The results showed a significant association of beef and pork with oxidative stress, and no association with cooked vegetables. Additionally, fish and poultry showed no or a negative association to DNA 15 Texas Tech University, Shelby Kloiber, August 2011 damage. Although there were many combinations with dietary options, a low fat diet (e.g., high in fruits, vegetables, fish and poultry) resulted in reduction in oxidative DNA damage. Thus, reduced oxidative DNA damage could decrease the possible cancer risks in women with a family history of breast cancer. Lycopene found in tomato products, was studied to identify its degree of DNA protection in women (Porrini & Riso, 2000). The researchers required the women to consume 25g of tomato puree daily for 14 days. Carotenoid concentrations and DNA strand breaks were analyzed before and after the intervention. The results indicated that tomato consumption increased plasma and lymphocyte lycopene concentrations, but betacarotene concentration increased only in plasma. These changes suggest that the tomato puree was taken up within the cell, which in turn increased the total antioxidant level significantly. The role of tomato puree in this study is shown to have benefits that protect against cancer and chronic diseases. These researchers concluded that small amounts of tomato puree might increase the resistance of lymphocytes to oxidative stress. Diets rich in antioxidants have been shown to reduce the risk of chronic degenerative diseases. Cilla’s (2009) research evaluated how the consumption of fruit beverages and the addition of milk and iron can affect the antioxidant status. Women in their twenties were assigned to one of three studies based on the consumption of different beverages that occurred twice a day for 3 weeks. In the first study the women consumed a fruit juice, while in the second study they consumed juice and milk, and the third study included the consumption of fruit juice, milk, and iron supplementation. Serum antioxidant capacity was measured. In the first two studies the fruit juice did not increase 16 Texas Tech University, Shelby Kloiber, August 2011 antioxidant capacity; however, there was an increase in superoxide dismutase (SOD) activity (measurement of antioxidant activity). SOD activity increased in the first two studies, and decreased with the iron supplementation initially then increased after 6 weeks. The induction of SOD activity and the complementary source of iron added to the drink in the third study lead to a conclusion that habitual consumption of fruit juice and iron can have beneficial effects against oxidative stress in women. This research was successful in finding new and creative ways to change a woman’s diet so she can reduce the risk of certain diseases. Another study used strawberries, spinach, and red wine as the antioxidant supplementation to determine if the total antioxidant level would increase (Cao, Russell, Lischner, & Prior, 1998). Diets of elderly women (N=8) were supplemented with 240 g of strawberries, 1250 mg of ascorbic acid, 294 g of raw spinach, and 300 ml of red wine. Blood samples were taken within 24 hours of consuming the supplement, and the total antioxidant levels were determined. Results were that the consumption of these four foods, which are rich in antioxidant phenolic compounds, can increase the serum antioxidant capacity in humans; thus, providing the potential to reduce the risk of some diseases associated with aging. Although this study was conducted on older women there was no menopause criteria, nor did they analyze markers of hormone/ hormone replacement therapy. Alternative plant products, specifically walnuts, have been shown to have high amounts of polyphenols (McKay et al., 2010). Healthy postmenopausal women, as well as men 50 years of age and older, were given 21-24g of raw walnuts per day. Although 17 Texas Tech University, Shelby Kloiber, August 2011 linoleic acid increased, which would be assumed to be a result of the consumption of walnuts, the lipid peroxidation only minimally affected antioxidant capacity. Overall the results concluded no significant increase in antioxidant levels. In another study researchers used grape polyphenol supplementation and its effects on the reduction of coronary heart disease (Zern et al., 2005). Lyophilized grape powder (LGP) has been reported to decrease triglyceride levels in animals. Zern and colleagues examined the effects of LGP on plasma lipids, lipoprotein metabolism, LDL oxidation, inflammation, and oxidative stress in twenty-four premenopausal and twenty postmenopausal women. The women were involved in a single-blind crossover study in which they consumed LGP or placebo for 4 weeks, a 3-week washout period, and finally the alternate treatment for 4 weeks. Lyophilized grape powder had no effect on triglyceride or HDL levels, glucose levels, IL-6, and C-reactive protein in both groups. However, LGP treatment decreased the tumor necrosis factor, cholesterylester transfer protein, and isoprostane in both pre- and postmenopausal groups. Isoprostane, formed from free radicals, was decreased showing a reduction in oxidative stress with LGP treatment. These results showed a reduction in major coronary heart disease risk factors that are typically elevated when women reach postmenopausal status. Based on the results of these two studies, further research is needed with controlled diets to determine the antioxidant capabilities of these and other products. Ryan-Borchers and colleagues (2006) examined the effect of isoflavones found in soy products such as soymilk or soy supplementation. The problem under investigation was to determine the effects of soy isoflavones on immune and oxidative markers in 18 Texas Tech University, Shelby Kloiber, August 2011 postmenopausal women. The participants included 52 healthy postmenopausal women. This double blind study randomly generated 3 groups; first group consumed cow’s milk and placebo (control), second group consumed soymilk and placebo, and third group consumed cow’s milk and isoflavones supplements. Participants were instructed to consume 706 ml of the specified milk per day as well as oral ingestion of supplement/placebo every day for 16 weeks. At the end of the 16 weeks, B cell populations, which heighten humoral immune response, were higher among women in both the soymilk and supplement groups than in the control group. Because of the stimulation of B cells from soy isoflavones, DNA oxidative damage in postmenopausal women was inhibited. Isoflavones intervention did not influence cytokine production, interleukin 2, interferon γ, TNF-α, lipid peroxidation, or CRP concentrations. Also, subjects receiving soymilk/supplementation did see a lower plasma 8-OHdg concentration than the control group suggesting a protective effect of soy isoflavones against oxidative stress in postmenopausal women. The studies that have been more successful with reduced oxidative stress with antioxidant consumption involve the intake of whole fruits and vegetables (Porrini & Riso, 2000; Prior, 2003; Svilaas et al., 2004). People who have an allergy to a fruit or vegetable, or those who are selective with their own diet need a different means to increase their antioxidant consumption. Scoglio and others (2009) researched the effects of a certain algae extract, Klamath, which is known to have antioxidant qualities. The aim of this investigation was to see the effects of a 2-month treatment with the Klamath algae extract on the general and psychological well-being of menopausal women, as well 19 Texas Tech University, Shelby Kloiber, August 2011 as on their oxidative stress status and level of antioxidants. Subjects participating in the study included twenty-one females between the ages of 47-54 years, amenorrhea for a year, no hormonal treatments, absence of uterus dysfunction, and no cardiovascular disease hypertension or diabetes. Subjects were given a Klamin tablet to be taken twice a day for two months. Results included a decrease in Malondialdehyde (MDA) as an index of lipid peroxidation and antioxidant levels were shown to increase. Menopausal symptoms evaluated before and after the study by the Green Scale were shown to decrease after treatment. Lastly, hormone plasma levels showed no significant change during the supplementation period. These researchers concluded that Klamin supplementation, a Klamath algae extract, is capable of reducing oxidative processes, and improving the well-being of menopausal women. The researchers proposed this alternative treatment for hormonal therapy as a way to overcome climacteric symptoms (Scoglio et al., 2009). Summary The previously reviewed studies generally indicated positive results with different types of supplementation and amounts increasing a person’s antioxidant levels. In most of the studies, whole foods, liquids, and supplementations were found to decrease the levels of oxidative stress (Cao, Russell, Lischner, & Prior, 1998; Cilla et al., 2009; Stampfer et al., 1993). The studies examining the role of antioxidants in postmenopausal women have typically found changes to oxidative stress markers, but most of the studies have examined the effect of only one type of antioxidant on measures of oxidative stress (Cornelli et al., 2001). The purpose of this current study was to see if there is a greater 20 Texas Tech University, Shelby Kloiber, August 2011 decrease in oxidative stress measures with a combination of antioxidants (i.e., increased levels of fruit/vegetables with soy supplementation). 21 Texas Tech University, Shelby Kloiber, August 2011 Chapter III Methodology It has been confirmed that an increased amount of antioxidant within the diet can reduce ROS levels and reduce the risk of diseases such as coronary heart disease and cancers (Ryan-Borchers et al., 2006). The purpose of the current study is to investigate the effects of a combination of antioxidants on levels of antioxidant levels and oxidative stress measures in post-menopausal women. Participants Recruitment of the participants by flyers and oral presentations (see Appendix A) took place at health and fitness clubs such as Body Works, Zach’s Gym, Freedom Fitness, Lifestyle Center, Zum Fitness, Fusion Fitness, and Studio 57. Local health care businesses, such as Grace Clinic and other gynecology practices were other locations for recruitment. Flyers were also placed within churches in the Lubbock area, and throughout the Texas Tech University campus and the Texas Tech University Health Sciences Center. There were also advertisement in the Daily Toreador, Tech Announce and the Lubbock Avalanche Journal. Participants signed informed consent (see Appendix B) in accordance with the committee of the use of human subject’s requirement. The participants (N=37) consisted of women who were given a health history survey (see Appendix C) to determine if they met the inclusion criteria. The inclusion criteria included: (1) currently in the postmenopausal phase whether naturally or surgically induced with cessation of menses greater than one year; (2) had a hematocrit level greater than 38% (i.e., not anemic); (3) weighed at least 110 pounds; (4) were not 22 Texas Tech University, Shelby Kloiber, August 2011 participating in any type of hormonal treatment, nor had been participating in hormone replacement therapy within the last 5 years; (5) had a BMI between 18 and 40; (6) nonsmoker for at least one year; (7) consumed two or more alcoholic drinks per day; and, (8) had no known uterus dysfunctions, cardiovascular diseases, hypertension, diabetes, gastrointestinal, renal disease, or HIV. There were 18 participants excluded based on the previously mentioned criteria. One participant was excluded because we were unable to collect a blood sample from the vein. The participants were asked to complete a physical activity survey (Appendix D) to insure that none of them were engaged in heavy physical activity. One additional participant was excluded because she regularly took a type of NSAIDS. Participants were then asked to complete a food recall record to help the researchers determine if the participant already had a high antioxidant diet in fruits and soymilk (Appendix E). Those who were found to consume a diet similar to the intervention were excluded from the research (n=1). The participants that were excluded from the study were thanked for their willingness to participate. One additional participant chose not to continue with the study after the initial meeting. Therefore, there were 16 participants who completed the study. Procedure All participants set up a meeting with the investigator at the participant’s availability. The first meeting consisted of completion of the written consent (Appendix B), the health history questionnaire (Appendix C), the physical activity survey (Appendix D), and the food recall (Appendix E). The participants that qualified for continuation with the study were then familiarized with the lab. These participants were given 23 Texas Tech University, Shelby Kloiber, August 2011 instructions on the procedures for the duration of the study. The study took place over a period of approximately 5 weeks. After the initial meeting, a one-week wash-out period where the participants refrained from foods on the Banned Food List (see Appendix F) took place. During the second and third meeting measurements (i.e., height, weight, heart rate and blood pressure) were taken, and blood draws were completed to obtain levels of antioxidant capacity and oxidative stress. The laboratories were in compliance with OSHA standards for blood collection procedures, and the investigators involved in blood collection procedures were appropriately trained. Pre- (i.e., second meeting) and post-dietary (i.e., third meeting) intervention testing was performed at the same time of day following an 812 hours fast. Heights and weights were obtained by a standard doctor’s scale. Body mass index (BMI) was calculated by weight (kg) divided by height (m2). Participant’s heart rates were measured using Polar Heart Rate Monitors, and blood pressure for each participant was obtained by a stethoscope and sphygmomanometer. Anthropometric measurements, blood draws, and a finger prick were repeated post-intervention (i.e., third meeting) following the same procedures and guidelines. Hematocrit and anthropometric data collection. Hematocrit levels were determined via blood collected by finger prick protocol. Alcohol pads were used to sterilize the finger site followed by a sterilized finger stick to draw the blood sample (2-3 drops) to determine hematocrit concentration. First, a finger is lanced and a small drop of blood is allowed to accumulate. Blood is drawn into a capillary glass tube, and one end of the blood filled tube is sealed with clay. The blood-filled tube is placed in a centrifuge 24 Texas Tech University, Shelby Kloiber, August 2011 and spun until the plasma and formed elements separate. The glass tube is moved across a reader board until the bottom of the red blood count column is at zero and the top of the plasma column is at 100. The percentage of red blood cells in the blood is determined by tracing the grid lines near the top of the red column in the tube to the scale (i.e., percentage; Hematocrit Test, 2011). If the participant weighed less than 110 pounds or if her hematocrit was less than 38% she was excluded from the study. The hematocrit levels, or the volume of red blood cells as a percentage of total blood volume, were assessed to exclude anemic participants. This was in accordance to the research guidelines of the Human Research Protection Office of Texas Tech University. Antioxidant capacity and oxidative stress data collection. Antioxidant capacity and oxidative stress levels were determined before and after the antioxidant intervention via blood samples that were taken during the second and third meetings. In preparation for the venipuncture procedure, the participant was asked to come having fasted for at least 8-12 hours. She was asked to refrain from any anti-inflammatory (NSAIDS) such as Ibuprofen because of blood thinning prior to the data collection. A phlebotomy chair and arm rests designed for blood draws was used during the blood draw procedure. Participants assumed a seated position in the phlebotomy chair. The participant’s nondominant arm was examined for the most prominent vein at the antecubital area of the elbow by a certified technician. After proper disinfectant by an alcohol pad, a tourniquet was positioned approximately 3-4 inches above the puncture site. The needle was angled 15-30 degrees with the surface of the arm. The needle was inserted through the skin and into the lumen of the vein. As the last tube is filling, the tourniquet was removed. The 25 Texas Tech University, Shelby Kloiber, August 2011 needle was removed from the arm swiftly, and gauze was pressed firmly to avoid formation of a hematoma. A total of 5 cc or about 5 ml per vial of blood was collected in three separate vials. This amount did not exceed the 550 ml limit specified be the research guidelines of the Human Research Protection Office at Texas Tech University Health Sciences Center (2007). Serum samples were separated by centrifugation and samples were frozen at -80° for later analysis. Intervention protocol At the second meeting participants were randomly assigned to one of four groups: a control group (C) who were asked to maintain their normal diet; daily soymilk consumption group (S); an increase in daily fruit consumption group (F); and, a combination of daily soymilk and increased fruits group (S+F). The S+F and S groups were instructed to consume a total of 706 ml of approved soymilk throughout the day. This amount was based on three dairy servings per day from the United States Department of Agriculture’s (USDA) MyPyramid (2011). The participants were asked to complete a daily check-off calendar to insure that they followed instructions (see Appendix G). Those participants who were in the F and S+F groups were instructed to increase their daily fruit intake to 5 or more servings per day based on serving sizes from the USDA’s MyPyramid. The participants were asked to choose a variety of fruits and check off the fruits from a daily check-off calendar along with given serving sizes (see Appendix G). All participants were informed of the importance of compliance with dietary requirements of the study for 4 weeks. 26 Texas Tech University, Shelby Kloiber, August 2011 Antioxidant capacity assay protocol Blood samples were assayed for superoxide dismutase (SOD) using the Superoxide Dismutase Assay Kit (Cayman Chemical Company, Ann Arbor, MI). A volume of 200 µl of diluted radical detector and 10 µl of standard were added to each designated standard well. Sample wells had 200µl of the diluted radical detector and 10 µl of sample in each well. Reactions were initiated by adding 20 µl of diluted xanthine oxidase to each well. The 96-well plate was covered and incubated on a shaker (LabLine Instruments Titer Plate Shaker; Melrose Park, Illinois) for 20 min at room temperature. The mixture was read by a spectrophotometer (Spectramax 384 Plus, Molecular Devices; Sunnyvale, CA) at 450nm. Standards of known SOD concentration were used to establish a standard curve. Oxidative stress assay protocol Blood samples were also assayed by the TBARs Assay Kit (Cayman Chemical Company; Ann Arbor, MI). A volume of 100 µl of each standard was added to each properly labeled 5ml vial followed by an additional 100 µl of sodium dodecyl sulfate (SDS) solution. The same procedure was followed for each sample. The vials were then swirled to mix the contents followed by the addition of 4 ml of color reagent. The vials were then capped and boiled in a water bath for 60 min. The vials were removed and immediately placed in an ice bath for 10 min. After removal from the ice bath, the mixtures were centrifuged for 10 min at 1600 g at 4°C. In duplicate, 150 µl of the mixture from each vial was added to a 96 well plate and read by a spectrophotometer 27 Texas Tech University, Shelby Kloiber, August 2011 (Spectramax 384 Plus, Molecular Devices; Sunnyvale, CA) at 535 nm. Standards of known TBARS concentration were used to establish a standard curve. Data Analysis The pretest data for oxidative stress and antioxidant capacity were analyzed using separate one-way ANOVAs to insure that the groups were equal at the beginning of the study. The data were then analyzed using two separate 4 X 2 (Group X Test) analyses of variance with repeated measures on the last factor. Alpha for this study was set at .05. 28 Texas Tech University, Shelby Kloiber, August 2011 Chapter IV Results The purpose of the current study was to examine the effects of two types of foods high in antioxidants on markers of oxidative stress and antioxidant capacity in postmenopausal women. The women were divided into three intervention groups (i.e., fruits, soymilk, and a combination of fruits and soymilk) and one control group. The oxidative stress was measured by the TBARS assay, and the antioxidant capacity was measured by the SOD assay. The mean heights, weights and BMI can be found in Table 1. Table 1. Means and standard deviations for weights and BMI. Weight (pounds) Pre Post 168.7±29.3 168.3±28.8 BMI Pre Post 28.3±3.7 28.2±3.7 Oxidative Stress The concentrations of MDA found using the TBARS assay were analyzed to determine the effects of the food high in antioxidants on oxidative stress. To determine if there was a difference among the groups, the pre-test data were analyzed using a one-way ANOVA. The results of the analysis indicated no significant differences among the groups, F (3, 12) < 1.0, p = .655. To determine the effects of the interventions, the data were analyzed using a 4 X 2 (Group X Test) ANOVA with repeated measures on the last factor. The levels of Group were the three intervention groups and the control group. 29 Texas Tech University, Shelby Kloiber, August 2011 The levels of Test were pre-test and post-test. The results of the analysis indicated no significant main effect for Group, F (3,12) < 1.0, p = .604 and Test F (1,12) < 1.0, p = .629. Additionally, the interaction was not significant, F (3,12) < 1.0, p = .539. The means and standard deviations for each group and test can be found in Table 2. Group Fruit Soymilk Fruit and Soymilk Control N 5 3 4 4 Pre-Test Post-Test 139.6 ± 136.5 47.4 ± 48.5 93.4 ± 95.0 207.4 ± 291.8 71.0 ± 76.0 22.2 ± 27.0 210.5 ± 337.5 52.4 ± 40.5 Table 2. Means and standard deviations for MDA (µM). Antioxidant Capacity The detection of superoxide radicals generated by xanthine oxidase and hypoxanthine found using the SOD assay were analyzed to determine the effects of the food high in antioxidants on antioxidant capacity. To determine if there was a difference among the groups, the pre-test data were analyzed using a one-way ANOVA. The results of the analysis indicated no significant differences among the groups, F (3,12) < 1.0, p = .680. To determine the effects of the interventions, the data were analyzed using a 4 X 2 (Group X Test) ANOVA with repeated measures on the last factor. The levels of Group were the three intervention groups and the control group. The levels of Test were pre-test and post-test. The results of the analysis indicated a significant main effect for Test F (3,12) = 5.180, p = .042. The mean for the pre-test (M = 0.28 units/ml, SD = 0.15) was significantly lower than the mean for the post-test (M = 0.39 units/ml, SD = 0.23). The results indicated no significant main effect for Group, F (3,12) = 2.377 p = .121. 30 Texas Tech University, Shelby Kloiber, August 2011 Additionally, the interaction was not significant, F (3,12) = 1.225, p = .343. The means and standard deviations for each group and test can be found in Table 3. Group N Pre-Test Post-Test Fruit Soymilk Fruit and Soymilk Control 5 3 4 4 0.24 ± 0.07 0.33 ± 0.12 0.35 ± 0.10 0.24 ± 0.27 0.29 ± 0.16 0.53 ± 0.19 0.53 ± 0.09 0.24 ± 0.31 Table 3. Means and standard deviations of units/ml SOD. 31 Texas Tech University, Shelby Kloiber, August 2011 Chapter V Discussion Free radicals can be defined as any chemical species that contains unpaired electrons. Free radicals, also known as reactive oxygen species (ROS), have long been assumed to have only negative functions in the organism. An increase in free radicals may lead to irreversible damage of mitochondrial DNA, and membrane lipids and proteins; thus, resulting in mitochondrial dysfunction and ultimately cell death (Kowaltowski & Vercesi, 1999). Oxidative stress biomarkers have been recognized in many acute and chronic diseases, and increase throughout various phases in the human lifespan (Finkel & Holbrook, 2000). Scavengers of free radicals (i.e., antioxidants) can eliminate the high reactivity of free radicals by turning them into non-radical and nontoxic metabolites. These antioxidants prevent oxidation by free radicals of biologically important molecules (Durackova, 2010). The purpose of this study was to examine the effects of two types of foods high in antioxidant on markers of oxidative stress and antioxidant capacity in postmenopausal women. The diet manipulations included increasing the servings of fruit to five per day, adding soymilk or both. Oxidative Stress It was hypothesized that adding more fruits that are high in antioxidants, soymilk and a combination of these two would decrease the levels of oxidative stress in postmenopausal women. The data did not support this hypothesis, as no change in any of the groups was indicated in the levels of oxidative stress. These results are similar to those in Engleman (Engleman, Alekel, Hanson, Kanthasamy, & Reddy; 2005) who had 4 32 Texas Tech University, Shelby Kloiber, August 2011 groups of different amounts of isoflavone and phytate. These soy protein isolates were taken at 40g/day and showed no significant decreases in oxidative stress marers. There have been studies that have found reduced levels of oxidative stress by increasing foods or supplements with antioxidants. Increasing the consumption of whole fruits and vegetables (Porrini & Riso, 2000; Prior, 2003; Svilaas et al., 2004) has resulted in the most decrease in oxidative stress. More specifically, in relation with studies on postmenopausal women who typically have an increased rate of oxidative stress production, can be reduced with diets high in antioxidants. Many of the studies (RyanBorchers et al., 2006; Scoglio et al., 2009) concluded that the reduction of oxidation because of the increase in antioxidant levels reduce the risk of diseases such as cancer and CHD as well as increasing the well-being of women. Ryan-Borchers (2006) used a soymilk intervention similar to the current study in regards to 706mL soymilk, however their study laster 16 weeks. It is possible that the current study was not long enough to see effects on oxidative stress levels. Scoglio (2009) found significant decreases in oxidative stress with a 2 month intervention of a 0.8g tablet of algae extract. These studies show evidence that a diet including foods high in antioxidants such as fruit and/or soymilk can significantly increase blood antioxidant levels and decrease oxidative stress levels. It is possible that in the current study, the fruit consumption of five servings a day was not met, or a variety of the given fruit list was not consumed. Also, it is possible that those in the soymilk and combination group consumed less than the required three cups per day. The soymilk brand and flavor was not monitored, and could have increased or decreased the antioxidant availability within the body. The non-significant changes in 33 Texas Tech University, Shelby Kloiber, August 2011 oxidative stress and antioxidant levels between groups could be because of a cap of antioxidant absorption in the body. Oxidative stress in the blood was measured by the TBARs assay. Although this assay has been used in studies that have found significant changes in oxidative stress (Kasapoglu & Ozben, 2001) it should be noted that this assay has been found to be nonspecific as the level of TBARs seen in the blood can be increased by other chemical reactions occurring in the body not specific to reactive oxygen species (Vollaard, Shearman, & Cooper, 2005). It is currently unclear the certain amount of food products and specific types of antioxidants are needed to decrease the risk for certain diseases, however, the guidelines for this study were taken from the Food and Drug Administration recommended daily intake for fruit and dairy products (MyPyramid, 2011). Guidelines for the participants in this study were given a check off list to remind them of at least five servings of fruit a day and/or three servings of soymilk per day. The participants’ check list of consumed foods indicated a compliance rate of 96.8% in the fruit group, 81% in the soy milk group, and 92.3% for the combination group. No significant results for the current study could be a result of the decreased compliance rate along with the 4-week length of the study. Antioxidant Capacity It was hypothesized that adding more fruits high in antioxidants, soymilk and a combination of these two would increase the antioxidant capacity in postmenopausal women. The data did not support this hypothesis, as no change in any of the groups was indicated in the antioxidant capacity. These results are similar to McKay (2010) who 34 Texas Tech University, Shelby Kloiber, August 2011 found no significant increase in antioxidant capacity in the cross-over study of either 21g/day or 42g/day of raw walnuts. Insignificance in our results could be a result of the compliance rate, and the possibility of the wash out week directions not properly being followed. However examining the data, indicated the means were moving in the expected directions. The means of the control group from pretest to posttest were essentially unchanged, while the means for the three control groups increased from pretest to posttest. It would be expected that if a larger sample size was examined these results of the SOD analyzes would produce a significant interaction. Additionally, a longer intervention period could possibly allow for a greater time for the antioxidant capacity to increase. Diets rich in antioxidants have been shown to reduce the risk of chronic degenerative diseases. Cilla’s (2009) research indicated the consumption of fruit beverages and the addition of milk and iron can positively affect the antioxidant status. The study did use the same assay, SOD, to measure antioxidant capacity; however the study lasted longer than the current study at a total of 12 weeks. Another study used strawberries, spinach, and red wine as the antioxidant supplementation to determine if the total antioxidant level would increase (Cao, Russell, Lischner, & Prior, 1998). Results found that the consumption of these four, which are rich in antioxidant phenolic compounds, can increase the serum antioxidant capacity in humans and help reduce the risk of some diseases associated with aging. Compared to the current study, the assay used to determine antioxidant capacity was not similar and the study lasted 10 weeks. During specimen collection, Cao (1998) blood samples as well as urine samples were 35 Texas Tech University, Shelby Kloiber, August 2011 taken to measure possible changes hourly during test consumption day. In another study researchers used grape polyphenol supplementation and its effects on the reduction of coronary heart disease (Zern,et al., 2005). The study was similar to the current study with an intervention of 4 weeks. These results showed a reduction in major coronary heart disease risk factors that are shown to be elevated when women reach postmenopausal status. Summary The results of this study indicate that consumption of foods high in antioxidants such as fruits and/or soymilk increased, though not statistically significant, blood antioxidant levels measured by superoxide dismutase, but the levels of oxidative stress did not change. Combining the fruit and soymilk, combination group, showed no significant difference compared to the single food of fruit or soymilk. It is apparent in the results that diets high in antioxidant rich food, specifically fruits and soymilk, did not reduce oxidative stress after a 4 week time period as measured by TBARs. It is apparent from other studies (Cao et al. 1998; Prior 2003; Tesoriere et al 2004; Zern et all 2005) that a diet high in antioxidant foods plays a large role in oxidative stress compared to a non-intervention group. More research is necessary to further investigate the specific types of fruit and soymilk, and amount of each needed to show a greater reduction on oxidative stress. Also, studies combining two different types of foods rich in antioxidants other than fruits and soymilk should be researched further. In conclusion, the results of the current study showed no significant differences among groups and between groups on TBARs measuring oxidative stress and SOD 36 Texas Tech University, Shelby Kloiber, August 2011 measuring total antioxidant capacity. Antioxidant levels for each group showed a significant main effect for mean pre antioxidant levels compared to post measures. Lastly, using a blood assay for measuring blood antioxidant levels and oxidative stress levels may not be sensitive or reliable enough to look at the change of antioxidant and oxidative stress properties between our studies’ groups. According to the results, a diet of five servings of fruits high in antioxidant and/or a daily intake of 3 cups of soymilk do not significantly increase antioxidant levels. 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Grape polyphenols exert a cardioprotective effect in pre- and postmenopausal women by lowering plasma lipids and reducing oxidative stress. The Journal of Nutrition, 135, 1911-1917. 41 Texas Tech University, Shelby Kloiber, August 2011 Appendix A Recruitment Materials SCRIPT USED TO RECRUIT VOLUNTEERS FOR THE The effects of a combination of foods high in antioxidants on blood antioxidant and oxidative stress levels in post-menopausal women PROTOCOL Drs. Melanie Hart, Jacalyn McComb, and Yoonjung Park, and Shelby Kloiber, graduate student in the Department of Health, Exercise and Sport Sciences are conducting a study for which they are interested in seeking volunteers. The purpose of this study is to determine if there is a greater benefit in consuming a combination of sources of antioxidants compared to just one type of food source in postmenopausal women. Participation in this study will require three visits to the Exercise Physiology Laboratory housed within the Exercise Sciences Center on the Texas Tech campus. The first visit will include a complete explanation of the study protocol, completion of the medical history questionnaire and consent form, and familiarization with the testing equipment and procedures. At the end of this meeting you will take home a no food list. We ask that you do not consume anything that is on this last for the week before your second visit. You will have a food diary to be completed during this week as well. This visit will last about 20 min. Visit 2 will consist of blood collection at a vein in the elbow. We will also gather measurements such as height, weight, BMI, blood pressure, heart rate, and hematocrit levels by finger prick. At the end of this visit you will take home the list of foods/liquids along with the daily servings determined by the USDA. This will allow you to keep track of the required diet you shall complete depending on the group you are randomly placed. Half way through the intervention the research group will contact you for updates and any questions you may have. The third and final visit will include the last blood draw and measurements. The second and third visit will last about an hour. You will be asked not to consume any non-steroidal anti-inflammatory medications (such as ibuprofen or Aleve (Naproxen)) 2 days prior to the blood draws. You will be asked to fast 8-12 hours before each blood draw. Recruitment includes the completion of a food recall record that will allow us to exclude those who already consume a diet high in antioxidants. Recruitment for this study is limited to postmenopausal women, healthy subjects who are not participating in 42 Texas Tech University, Shelby Kloiber, August 2011 hormonal therapy. (Cessation of menses > 1 year) Participants need to be willing to participate in a diet high in antioxidant rich food/liquid. Anyone willing to participate must be free of any known disease, and a non-smoker. Those interested cannot consume more than 2 alcohol drinks per day and do not engage in heavy exercise. There is absolutely no penalty if you should decline to participate. Participation in this study is completely voluntary and participants are free to quit the study at any time. Thank you for your attention and time. Newspaper Announcement Are you a postmenopausal woman not taking part in hormone replacement therapy? The Health, Exercise, & Sport Sciences department is asking adult females who are in their postmenopausal stage to join in a research study examining the effects of a combination of foods high in antioxidants. The study will be three (3) meetings, and each visit will last less than an hour. Women will be asked to add to their normal diet five or more servings of fruit every day and/or three servings of soymilk every day. Measurements taken at the last two meetings include height, weight, body mass, blood pressure, heart rate, and hydration levels. Blood will be taken to measure antioxidant and oxidative stress levels. Your taking part in this study will be voluntary and greatly appreciated. You may say no or stop your involvement at any time during the study. If you are interested and would like to see if you can take part in the study or want more information contact Shelby Kloiber in the Department of Health, Exercise, and Sport Sciences at (806) 787-5070 or by email at [email protected] Thank you for your interest, Shelby Kloiber Graduate Teaching Assistant at Texas Tech University [email protected] 43 Texas Tech University, Shelby Kloiber, August 2011 The Department of Health, Exercise, and Sport Sciences at Texas Tech University is studying the effect of a high antioxidant diet on total antioxidant and oxidative stress levels Study includes: -Total of three (3) visits Study Involves: -Each visit takes -Blood draws during visits Less than 1 hour two and three -Consuming ≥ 5 fruit -Measurements taken: servings Height, weight, BMI, hydration, soymilk a and/or ~ 3cups of Heart rate, blood pressure Shelby Kloiber day for 4 weeks [email protected] 44 806-787-5070 Texas Tech University, Shelby Kloiber, August 2011 Appendix B Informed Consent You are being invited to participate in the research project entitled: The effects of a combination of foods high in antioxidants on blood antioxidant and oxidative stress levels in post-menopausal women. The people responsible for this research project are: Drs. Melanie Hart, Jacalyn McComb, and Yoonjung Park, and Shelby Kloiber, graduate student in the Department of Health, Exercise and Sport Sciences at Texas Tech University, (806) 742-3371. I. Purpose and explanation of the study The purpose of this research is to determine if there is a difference in markers of oxidative stress and antioxidant capacity in individuals with single or a combination of food source interventions. Oxidative stress occurs when substances called free radicals occur in large amounts and cause damage to cell DNA. Postmenopausal women have a decrease in estrogen, lowering their immune function, increasing their risk for disease caused by these free radicals. Antioxidant capacity is the body’s ability to take free radicals out of the system. You will be asked to complete the following procedures: 1. Insertion of a needle in a vein at the elbow for blood collection at the beginning and end of the research project. 2. Completion of one week of following no consumption from a No food list, and 4 weeks of a specific diet and intake record. All blood draws and measurements will be conducted in the Exercise Physiology Lab at Texas Tech University Exercise Sciences Center. The first visit will include a complete explanation of the study protocol, completion of the medical history questionnaire and consent form, and familiarization with the testing equipment and procedures. You will also be asked to start recording a food diary for one week, and follow a list of foods not to consume (wash out period). This visit will last about 20 min. Visit 2 will consist of blood collection at a vein in the elbow. We will also gather measurements such as height, weight, BMI, blood pressure, heart rate, and hematocrit by finger prick. At the end of this visit you will take home the list of foods/liquids along with the daily servings determined by the USDA. Fruit consumption includes 5 or more fruits and/or 706mL of soymilk. Food and liquid will be purchased by the participant. The food check off list will allow you to keep track of the required diet. Half way through the intervention the research group will contact you for updates and any questions you may have. The third and final visit will include the last blood draw and measurements. At this point you are no longer required to following the intervention. 45 Texas Tech University, Shelby Kloiber, August 2011 Before you undergo the diet intervention, you must certify to the researchers that you are in good health to the best of your knowledge. You will fill out a medical history questionnaire that will be reviewed by trained professionals prior to undergoing the intervention and blood work. Based on the medical history you may be disqualified from the study because of increased risk to you during the diet intervention. Consequently, it is important that you provide complete and accurate responses to the interviewer and recognize that your failure to do so could lead to possible unnecessary health problems during the procedures. You will have a blood draw of about 3 tubes at 5 ml each (about 1-2 teaspoons each) taken from a needle that will be inserted in a vein at your elbow to determine markers of free radical activity and antioxidant activity. There will be a blood draw at the second and third meeting. You will be asked not to take anti-inflammatory drugs (such as ibuprofen and Aleve (Naproxen)) 3 days prior to, and during the blood draws. Lastly, you will be asked to come in fasting 8-12 hours before each blood draw. II. Risks There exists the possibility of adverse changes during the intervention. These changes could include diarrhea, constipation, minor GI discomfort such as bloating and cramping, hyperglycemia, hypoglycemia, and gastro-esophageal reflux. Some minor discomfort may be experienced with a needle prick for the collection of blood samples and hematocrit testing. There is the possibility of infection at the needle prick site; however this is greatly minimized by following strict aseptic technique. In the event of any problems such as physical, psychological, financial, etc, Texas Tech University or the Student Health Center may not be able to treat your injury. You will have to pay for treatment from your own insurance. The university does not have insurance to cover such injuries. More information about these matters may be obtained from Dr. Kathleen Harris, Associate Vice President for Research, (806) 742-3884, Room 203 Holden Hall, Texas Tech University, Lubbock, Texas 79409. III. Benefits to be expected The results of this research may or may not benefit you. Potential benefits relate mainly to a possible decrease in your risk for developing chronic diseases. Those who are increasing their fruit intake will meet their requirement for fruit servings per day. Those who are increasing their soymilk consumption will meet their requirements for dairy servings per day. IV. Confidentiality and use of information All information obtained from these procedures will be treated as privileged and confidential and will consequently not be released or revealed to any person without your written consent. By signing this form, you are agreeing to the use of any data recorded 46 Texas Tech University, Shelby Kloiber, August 2011 for research or statistical purposes so long as it does not provide facts that could lead to your identification. A numeric code will be used as an identifier for statistical purposes and these will be deleted upon completion of the analysis. The information contained in the medical history form will be maintained with complete confidentiality during the course of the study and destroyed upon completion of the study. Any other information obtained, however, will be used only by the program staff to evaluate your diet intervention. Any information that can lead to your identification will be kept in the exercise physiology laboratory in the Exercise Science Center in a locked file cabinet, with access controlled by Drs. McComb and Park, and Shelby Kloiber. V. Inquiries and freedom of consent Dr. Hart, Dr. McComb, Dr. Park, or Shelby Kloiber will answer any question that you may have about this study. For questions about your rights as a participant or about illness caused by this research, you should contact TTU Institutional Review Board for the Protection of Human Subjects, Office of Research Services, Texas Tech University, Lubbock, Texas 79409. Or you can call (806) 743-3884. Participation in this research project is voluntary and refusal to participate involves no penalty or loss to which you may be entitled and you may discontinue participation at any time without penalty or loss of benefits. By signing this form, you are acknowledging that you have read this document in its entirety and that the investigator reviewing this document has made certain that you understand it. ____________________________________ Date____________ Participant’s signature This consent form is not valid after December 31, 2011. 47 Texas Tech University, Shelby Kloiber, August 2011 Appendix C Health History Survey MEDICAL HISTORY QUESTIONNAIRE Demographic Information Last Name __________________________ First Name __________________________ Middle Initial ________________________ Date of Birth ________________________ Sex _______________________________ Home Phone ________________________ Address ____________________________ City ______________________________ State ______________________________ Zip Code ____________________________ Work Phone ________________________ Family Physician ____________________ The following questions are to ensure that you are healthy enough to participate in the intervention in this study, and are for your protection. Section A 1. When was the last time you had a physical examination? *2. If you are allergic to any medications, foods, or other substances, please name them. 3. If you have been told that you have any chronic or serious illnesses, please list them. 48 Texas Tech University, Shelby Kloiber, August 2011 4. Give the following information pertaining to the last time you were hospitalized. Reason for hospitalization: Month and year of hospitalization: Hospital: City and State: 5. Are you affected with hemophilia? *6. Have you participated in any type of hormone replacement therapy in the last five years? If yes, explain_____________________________________ *7. Please list the date of your last menstrual cycle. _____________________________ Section B For questions 1-13 have any of the following situations happened during the past 12 months? 1. Has a physician prescribed any form of medication for you? Yes___ No ___ If yes, were the medications for a cardiovascular condition? Yes ___ No ___ 2. Has your weight fluctuated more than a few pounds? Yes ___ No ___ If yes, did you attempt to bring about this weight change through diet or exercise? Yes___ No___ 3. Have you experienced any faintness, light-headedness, or blackouts? Yes___ No ___ If yes, what were the circumstances? 4. Have you occasionally had trouble sleeping? Yes ___ No ___ 5. Have you experienced any blurred vision? Yes ___ No ___ 49 Texas Tech University, Shelby Kloiber, August 2011 6. Have you any severe headaches? Yes ___ No ___ 7. Have you experienced any temporary change in your speech pattern, such as slurring or loss of speech? Yes ___ No ___ 8. Have you felt unusually nervous or anxious for no apparent reason? Yes___ No___ *9. Have you smoked cigarettes? Yes___ No ___ If yes, how often? For questions 10-14 are you currently experiencing any of the following situations? *10. Have you ever been told that your blood pressure was abnormal? Yes ___ No ___ 11. Have you ever been told that your serum cholesterol or triglyceride level was high? Yes ___ No ___ *12. Do you have diabetes? Yes ___ No ___ ___ Dietary means If yes, how is it controlled? (Check One) ___Insulin injection ___Oral medication ___Uncontrolled 13. How often would you characterize your stress level as being high? (Check One) ___ Occasionally ___Frequently ___Constantly *14. Have you ever been told that you have any of the following illnesses? Yes ___ No___ __ Cardiovascular Disease __Uterus dysfunctions __ Hypertension __ Gastrointestinal disease __ Renal disease __ HIV Section C BMI= body weight (kg)/height (m2) = ___________ Is your BMI within 18-40? Yes ___ No ___ Exclusion Criteria 50 Texas Tech University, Shelby Kloiber, August 2011 Questions with an asterisk * can exclude the person from the study unless clarified or cleared by a physician. Other questions will help the researchers determine if the subject does not fit the apparently healthy criterion. 51 Texas Tech University, Shelby Kloiber, August 2011 Appendix D Physical Activity Survey General Practice Physical Activity Questionnaire Date_____________ Name _________________ 1. Please tell us the type and amount of physical activity involved in your work. Please mark one only a. _______I am not in employment (e.g. retired, retired for health reasons, unemployed, fulltime carer etc.) b. _______I spend most of my time at work sitting (such as in an office) c. _______ I spend most of my time at work standing or walking. However, my work does not require much intense physical effort (e.g. show assistant, hairdresser, security guard, childminder, etc.) d. _______My work involves definite physical effort including handling of heavy objects and use of tools (e.g. plumber, electrician, carpenter, cleaner, hospital nurse, gardener, postal delivery workers, etc.) e. _______My work involves vigorous physical activity including handling of very heavy objects (e.g. scaffolder, construction worker, refuse collector, etc.) 2. During the last week, how many hours did you spend on each of the following activities? Please answer whether you are in employment or not. None Physical exercise such as swimming, jogging, aerobics, football, tennis, gym workout, etc. Cycling, including cycling to work and during leisure time Walking, including walking to work, shopping, for pleasure, etc. Housework/childcare 52 Some but less than 1 hour 1 hour but less 3 hours than 3 hours or more Texas Tech University, Shelby Kloiber, August 2011 Gardening? DIY 3. How would you describe your usual walking pace? Please mark one only _______ Slow pace (i.e. less than 3 mph) _______ Steady average pace _______ Brisk pace _______ Fast pace (i.e. over 4 mph) 53 Texas Tech University, Shelby Kloiber, August 2011 Appendix E Dietary Recall This questionnaire will give us information about your eating habits. There are no “right” or “wrong” answers. Accurate and thoughtful responses will allow us to pinpoint your eating habits. • Use the past month as your standard for how you eat. • Recall the times during the day when you ate, and what you had. • Include snacks and “nibbles” as well as meals and beverages. • If you ate out regularly or traveled, remember to include those foods too. • Be sure to answer every item on this form. If you did not eat a food listed below— or ate it less than once a week — write a “0” in the space provided. Please do not leave blanks. Part I. We want to know how often you ate certain foods. For each of the foods listed, please indicate how many servings per week you usually ate in the past month. (If you ate a food less than once a week, write a “0” in the space provided.) Food Amount YOU eat/month Typical Serving Size Fresh fruit 1 whole piece or ½ cup cut-up Dried fruit ½ cup Fruit juice ½ cup or 4 ounces Food Amount YOU eat/month Typical Serving Size Cooked vegetables ½ cup Raw vegetables 1 cup Food Amount YOU eat/ month Red meat (beef, pork, ham, veal, lamb) Typical Serving Size 4 ounces (size of a deck of cards) 54 Texas Tech University, Shelby Kloiber, August 2011 Dried beans, split peas, or lentils Food ¾ cup cooked Amount YOU eat/month Typical Serving Size Chocolate or candy bar 1 regular candy bar Alcoholic drinks 1 drink, 1 can of beer, 1 glass of wine Soymilk ½ cup or 4 ounces Soy products (please give food type, amount consumed and how many times you consumed the soy product per week) _________________________________________________________ Part II. We want to know the different types of fruits and vegetables consumed in the last month. For each of the foods listed, please indicate how many servings per week you usually ate in the past month. (If you ate a food less than once a week, write a “0” in the space provided.) Please place a number in the blank cell that indicates how many times you have consumed this food in the past month. If the food is not mentioned please list the food and amount of times consumed per month in others section. Apple Apricot Blackberry blood orange Blueberry boysenberry Cactus Pear Cantaloupe Cherry cranberry grapefruit Green grape guava Honeydew melon kiwi lemon Lime mango artichokes arugula asparagus avocadoes broccoli rabe broccoli brussel sprouts celery cucumbers endive green beans cabbage green peppers leafy green (kale) leeks lettuse okra peas 55 Texas Tech University, Shelby Kloiber, August 2011 nectarine Orange papaya Peach Pear Persimmon pineapple Plum Pomegranate prunes purple grape Raspberry starfruit Strawberry tangerine Watermelon kumquat Litchis rambutan spinach zucchini cauliflower mushroom onions parsnips potatoes white corn beets red peppers red potatoes tomatoes squash sweet corn yellow pepper eggplant carrots red onion sweet potato Others: _____________________________________________________________________________ 56 Texas Tech University, Shelby Kloiber, August 2011 Appendix F Banned Food List During the first week of this study, we ask you to refrain from the foods below. Multivitamins Alcohol Tea Coffee Orange Pomegranate Blueberry Blackberry Cherry Soy Products Beans Chocolate Vegetables orange in color Dark green leafy vegetables Tomatoes 57 Texas Tech University, Shelby Kloiber, August 2011 Appendix G Daily Food Check-Sheet Soymilk daily check-off DATE 15-Jan 16-Jan 17-Jan 18-Jan 19-Jan 20-Jan 21-Jan 22-Jan 23-Jan 24-Jan 25-Jan 26-Jan 27-Jan 28-Jan 29-Jan 30-Jan 1-Feb 2-Feb 3-Feb 4-Feb 5-Feb 6-Feb 7-Feb 8-Feb 9-Feb 10-Feb 11-Feb 12-Feb *dates subject to change Soymilk Brand: Amount Total amount per day: 706 mL Can by split up into 2 or 3 servings 58 Texas Tech University, Shelby Kloiber, August 2011 Fruit List ( dates subject to change) Fruit Apple Apricot Blackberry blood orange Blueberry boysenberry Cactus Pear Cantaloupe Cherry cranberry grapefruit Green grape guava Honeydew melon kiwi lemon Lime mango nectarine Orange papaya Peach Pear Persimmon pineapple Plum Pomegranate Serving 1 small 1/2 cup 1/2 cup 1 small 1/2 cup 1/2 cup 1 cup 1 cup 1/2 cup 1/2 cup 0.5 large 15 3/4 cup 15-Jan 16Jan 1 cup 1 whole 3/4 cup 2 whole 1/2 small 1 small 1 large 0.50 1 large 1 mediu m 2 1 cup 2 large 1/3 59 17Jan 18Jan 19Jan 20-Jan 21-Jan Texas Tech University, Shelby Kloiber, August 2011 prunes purple grape Raspberry starfruit Strawberry tangerine Watermelon kumquat Litchis rambutan whole 3 dozen 1/2 cup 2 large 6 berries 2 small 1 1/4 cup 5 10 1/3 cup 60