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Effects of 9 Months of Resistance or Aerobic Training on Endothelial Function in Postmenopausal Women

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Title:
Effects of 9 Months of Resistance or Aerobic Training on Endothelial Function in Postmenopausal Women
Creator:
MERING, MARK CAMERON
Copyright Date:
2008

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Subjects / Keywords:
Blood ( jstor )
Endothelium ( jstor )
Exercise ( jstor )
Heart rate ( jstor )
Hormone replacement therapy ( jstor )
Plasmas ( jstor )
Protected health information ( jstor )
Strength training ( jstor )
Treadmills ( jstor )
Women ( jstor )
City of Daytona Beach ( local )

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University of Florida
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University of Florida
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Copyright Mark Cameron Mering. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
12/31/2005
Resource Identifier:
436098647 ( OCLC )

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EFFECTS OF 9 MONTHS OF RESI STANCE OR AEROBIC TRAINING ON ENDOTHELIAL FUNCTION IN POSTMENOPAUSAL WOMEN By MARK CAMERON MERING A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN EXERCISE AND SPORT SCIENCES UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Mark C. Mering

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This document is dedicated to the women who pa rticipated in the project and the graduate students who contributed to the completion of this project.

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ACKNOWLEDGMENTS A project of this duration and magnitude required a great deal of support and participation. First, this project would not have happened without the wonderful women who participated, so big thanks go to all the women who completed the study. I would like to thank my research partner, Kathy Howe, for all her dedication, time, and effort with making this project a success. Special thanks go out to Gary Pierce for his help and generosity with his time and skills which were vital for the studies completion. I owe a big thanks to Dr. Braith, first for allowing me a chance to attend the University of Florida under his tutelage and second for his guidance, support, knowledge and editorial talents, which were invaluable to completing this project. I also would like to thank my committee members, Dr. Stephen Dodd and Dr. Lesley White, for all their guidance, support, and time, not only with this project but throughout my time at the University of Florida. Finally, I would like to thank my family for their love and support. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Statement of Problem...................................................................................................1 Justification of Research...............................................................................................2 Research Question........................................................................................................3 Research Hypotheses....................................................................................................4 2 LITERATURE REVIEW.............................................................................................5 Nitric Oxide..................................................................................................................5 Estrogen Protection.......................................................................................................7 Flow Mediated Dilation................................................................................................8 Applanation Tonometry..............................................................................................10 Plasma Markers..........................................................................................................11 Previous Research on Women....................................................................................11 Summary.....................................................................................................................13 3 METHODOLOGY.....................................................................................................15 Study Design...............................................................................................................15 Subject Qualifications.................................................................................................16 Subject Recruiting......................................................................................................16 Group Assignments....................................................................................................17 Blood Drawing and Markers......................................................................................17 1-Rm Strength.............................................................................................................18 Applanation Tonometry..............................................................................................18 Flow Mediated Dilation (FMD)..................................................................................19 Aerobic Training.........................................................................................................19 v

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Resistance Training....................................................................................................20 Statistical Analysis......................................................................................................21 4 RESULTS...................................................................................................................22 Study Design...............................................................................................................22 Subject Characteristics................................................................................................22 Flow Mediated Dilation (FMD) Data.........................................................................23 Applanation Tonometry..............................................................................................24 Plasma Markers Data..................................................................................................27 Superoxide Dismutase (SOD).............................................................................28 8-Isoprostane.......................................................................................................29 C-Reactive Protein (CRP)...................................................................................30 Nitric Oxide.........................................................................................................31 5 DISCUSSION.............................................................................................................33 FMD Discussion.........................................................................................................33 Applanation Tonometry Discussion...........................................................................34 Plasma Markers of Oxidative Stress...........................................................................34 Superoxide Dismutase (SOD).............................................................................34 8-Isoprostane.......................................................................................................35 Plasma Markers of Endothelial Function...................................................................35 C-Reactive Protein (CRP)...................................................................................35 Nitrate/Nitrite......................................................................................................36 Limitations..................................................................................................................36 Future Experiments.....................................................................................................37 APPENDIX A STUDY QUESTIONAIRE.........................................................................................39 B INFORMED CONSENT............................................................................................43 LIST OF REFERENCES...................................................................................................54 BIOGRAPHICAL SKETCH.............................................................................................59 vi

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LIST OF TABLES Table page 4-1 Subject Characteristics.............................................................................................23 4-2 FMD Measurements.................................................................................................23 4-3 Changes in Hemodynamic Values after the Exercise Training................................25 4-4 Absolute Values of Applanation Tonometry Data...................................................25 4-5 Plasma Markers........................................................................................................28 vii

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LIST OF FIGURES Figure page 2-1 L-Arginine pathway (43)............................................................................................6 2-2 Action of estrogen (43)..............................................................................................8 4-1 Relative percent change in dilation and FMD of the brachial artery from pre to post in the aerobic trained group..............................................................................24 4-2 Relative percent change of change in dilation and FMD of the brachial artery from pre to post in the resistance trained group.......................................................24 4-3 Relative percent changes in augmentation index from baseline (T1) to post (T2)..........................................................................................................................26 4-4 Relative percent change in percent of augmentation index from baseline (T1) to post (T2)...................................................................................................................26 4-5 Relative percent change in augmentation index corrected for heart rate.................27 4-6 Relative percent change of total travel time of reflect wave....................................27 4-7 Relative percent change in SOD activity.................................................................29 4-8 Relative percent change in levels of 8-isoprostan....................................................30 4-9 Relative percent change in CRP levels....................................................................31 4-10 Relative percent change in nitrate/nitrite levels.......................................................32 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science in Exercise and Sport Sciences EFFECTS OF 9 MONTHS OF RESISTANCE OR AEROBIC TRAINING ON ENDOTHELIAL FUNCTION IN POSTMENOPAUSAL WOMEN By Mark Cameron Mering December 2004 Chair: Randy Braith Major Department: Exercise and Sport Sciences Cardiovascular disease (CVD), which includes heart disease, hypertension, coronary artery disease (CAD), peripheral vascular disease, and stroke, is the number one killer of women in America. This study was conducted to determine the effect of both resistance and aerobic exercise training protocols on endothelial function in postmenopausal women. Seventeen women aged 58.28+ 5.3 years completed the study. Participants were randomly assigned to one of three exercise groups: treadmill walking, a resistance training program consisting of nine exercise machines (Torso Arm, Chest Press, Seated Row, Overhead Press, Biceps Curl, Triceps Push down, Leg Extension, Leg Curl, and Abdominal Machine) or a resistance training program consisting of those nine machines plus the Leg Press and MedX Medical Lumbar Extension machine. The two resistance training groups were pooled together. ix

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Both groups were tested at baseline and at the conclusion of the study (9 month point) for flow mediated dilation (FMD) and applanation tonometry. Blood was also drawn at each of the time points and stored for later analysis of plasma blood markers. Two plasma markers of oxidative stress, Superoxide Dismutase (SOD) and 8-isoprostane and two plasma markers of endothelial function, C-Reactive Protein (CRP) and Nitrite/Nitrate were tested. We found that an aerobic protocol of treadmill walking twice a week at 65-80% of heart rate was stringent enough to cause an significant increase in FMD (p 0.05). However, the data from the applanation tonometry and the plasma blood markers were not significantly changed from baseline to post intervention in the aerobic group. The data from the resistance group were also significantly unchanged from baseline to post intervention. We can conclude from the data that aerobic training in postmenopausal women can improve endothelial function by increasing FMD. x

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CHAPTER 1 INTRODUCTION Statement of Problem Cardiovascular disease (CVD), which includes heart disease, hypertension, coronary artery disease (CAD), peripheral vascular disease, and stroke, is the number one killer of women in America according to the American Heart Association (2). CVD accounted for over 500,000 deaths in 2001 (2). CVD manifestations in women occur predominantly after menopause. In most women, symptoms of CVD are delayed until the women are postmenopausal because of the protective effect of ovarian hormones, most notably estrogen. It is hypothesized that estrogen protects the endothelium thus delaying CVD (46). Dysfunctioning endothelium is at the vanguard of a cascade of vascular events that lead to CVD. After menopause, natural or surgically induced, the incidence of CVD is comparable to age-matched men. Without endogenous production of ovarian hormones, the vascular protective benefits of estrogen appear to be totally lost (28, 41). This led to the practice of prescribing hormone replacement therapy (HRT) to sustain the cardiovascular protection of estrogen for women (22). HRT was also prescribed to reduce the symptoms of menopause, such as hot flashes and mood swings (22). HRT was thought to attenuate osteoporosis, reduce blood lipids, and confer vascular protection (5). However, the practice of prescribing the HRT was never clinically challenged until the Heart and Estrogen/Progesterin Replacement Study (HERS) and Women Health Initiative (WHI) study were conducted (5). WHI found that 1

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2 combined (estrogen+progesterin) or estrogen only oral hormone replacement therapy did not provide protective effects for the endothelium and CVD (5). Furthermore, HRT (estrogen+progesterin) increased the risk of breast cancer, stroke, and CHD (5). The verdict is still out on HRT consisting of only estrogen for women who had a hysterectomy. The only reported benefit of HRT found in the in WHI study was lower lipoprotein profile. Because of the increased cancer risk and lack of endothelial protection, the risks outweighed the benefits and the study was stopped early. HRT is no longer recommended as the standard of care to offset the loss of estrogen in healthy postmenopausal women (5, 18, 22, 38, 39). Justification of Research Because the practice of prescribing HRT to all postmenopausal women for relief of symptoms and cardioprotection was dramatically altered by the results of WHI, alternative interventions are in demand (5). Women who already have symptoms of heart disease may be switched to alternative drug therapies such as statin or antihypertensive drugs for protection (31). Women with postmenopausal symptoms may receive only short-term HRT. However, women who do not have CVD and do not want to take any medication for postmenopausal symptoms may be at increased risk for CVD. There is compelling evidence that age related damage to the endothelium can be attenuated with aerobic exercise in males (11, 37). However, there are few studies that have examined the effects of exercise on endothelial function in women, despite the fact that CVD is the number one killer of women in America (2). Endothelium-dependent vasodilation is a primary technique for evaluating endothelium function. The dependent vasodilation depends on the availability and release of nitric oxide (NO) from the endothelium in response to “shearing” effects of

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3 blood flow. Endothelium-dependent vasodilation is typically studied in the conduit and resistant vessels of the arm to assess systemic vascular health of a patient (12). In 1999, Rywik et al. (37) studied endurance training on older men ( 60 years of age) by examining endothelium-dependent brachial flow mediated dilation and reported that the endurance trained men (n=12) had augmented endothelium-dependent vasodilation compared to the sedentary group (n=23). In another cross sectional study, DeSouza et al. (11) showed that endurance trained men experience an age related decline in endothelium-dependent vasodilation. Moreover older sedentary men who initiated aerobic exercise programs restored endothelium-dependent vasodilation to levels that were observed in younger males (11). The mechanism responsible for improved vasoreactivity following endurance exercise training is the increase in available nitric oxide, likely stimulated by repeated bouts of shear stress (37). Central arterial stiffness is a predictor of cardiovascular risk, which can be assessed non-invasively with the technique of applanation tonometry. Central arterial stiffness is represented by the time delay of the reflected wave (T) and the augmentation index (AI). T is the travel time of the pulse wave’s round trip from the aorta, to the periphery, and back again. The AI represents the added work load that the left ventricle encounters from the reflected pressure wave. When AI is increased and T is decreased, the indices of LV work and O2 consumption are greater which increases the risk of having an adverse event such as a stroke, heart attack, or even death (14, 25, 32). Research Question The purpose of this study was to determine the effect of both resistance and aerobic exercise training protocols on endothelial function in postmenopausal women. We examined whether a nine-month program consisting of either resistance or endurance

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4 exercise performed 2 days/week would improve endothelium function. We performed flow mediated-dilation (FMD) tests on the brachial artery using high resolution ultrasound and Applanation tonometry to noninvasively assess aortic stiffness (AI and T). In addition, blood samples were collected to assess markers of oxidative stress, antioxidant defense and NO availability. Research Hypotheses We hypothesized that 1. Brachial artery FMD would increase in both the aerobic and resistance training groups. 2. Aortic AI would decrease and T would increase in both the aerobic and resistance training groups. 3. Nitric Oxide availability and superoxide dismutase (SOD) levels would be increased in both the aerobic and resistant training groups. 4. C-Reactive Protein (CRP) and the 8-isoprostane levels would decrease in both the aerobic and resistant training groups.

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CHAPTER 2 LITERATURE REVIEW Vascular tone refers to the degree of constriction experienced by a blood vessel relative to its maximally dilated state. The vascular endothelium is normally a one cell thick lining of the coronary artery walls that responds to physical or chemical stimuli to control vascular tone. Endothelium utilizes a variety of vasoactive regulators to maintain vascular tone. Dysfunctional endothelium is the precursor to coronary artery disease (CAD). A primary mechanism of endothelium dysfunction is the decreased bioavailability of nitric oxide (NO). With the decrease in the NO availability and protection, endothelium dysfunction may manifest through vasospasms, thrombus formation, hypertension, and atherosclerosis (12, 19). This review will focus on the role of endothelium on vascular health as well as factors that are known to contribute to vascular health. The review will also cover the techniques utilized for measuring vascular reactivity and markers of vascular health. Finally, the review will examine previous studies that support this line of research and to exemplify the lack of data in the female population. Nitric Oxide Nitric oxide (NO) was discovered in 1980, by Furchogott and Zawadski (19), through the finding that NO in the vascular endothelial cells of a rabbit was essential for the induced relaxation of the aorta. NO is a potent vasodilator which is continuously released from the vascular endothelium to maintain resting vascular tone. The effects of NO are balanced with various other endothelium vasoconstrictors and the sympathetic 5

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6 nervous system to keep a stable vascular tone (19, 44). The Larginine: nitric oxide pathway enables the endothelial cells to synthesize NO (Figure 1-1). Figure 2-1. L-Arginine pathway (43) The pathway utilizes the amino acid of L-arginine and the converting enzyme endothelial nitric oxide synthase (eNOS) to synthesize the formation of NO. NO has a half-life of less than four seconds. The NO diffuses to the vessel’s lumen and to the underlying smooth muscle cells, where the NO is used to signal intracellular cyclic guanosine-3’,5-monophosphate (cGMP) to mediated the biological effects of the NO and control vascular tone (19, 44). Endothelium dysfunction is categorized by the impaired ability of the vascular endothelium to stimulate vasodilation. This impairment initiates a cascade of events that leads to the development of atherosclerosis and other diseases such as diabetes, hypertension, hypercholesterolemia, or renal failure. The phenomenon of dysfunctional endothelium can be linked to the deficiency of local NO availability. The decreased NO availability may be a result of reduced expression of eNOS or the increased breakdown of eNOS-derived NO due to oxidative stress. Thus, the origin of endothelium dysfunction can be assessed by measuring the total nitrate/nitrite concentration or by the levels of oxidant stress/antioxidant protection present (12, 21, 44).

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7 Estrogen Protection Premenopausal women have a lower incidence of coronary artery disease than men. This is due to the production of hormones that protect the endothelium. Estrogen is thought to be a hormone that protects women from CAD. Estrogen effects on the body are triggered through either a genomic or a non-genomic pathway (Figure 1-2). The genomic path deals with the traditional view that estrogen affects transcriptional factors by binding to estrogen receptors (ER), ER-alpha and ER-beta located in the vascular smooth muscle cells (VSMC) and the endothelium cells. Estrogen can bind to one or both of the receptors causing the DNA to express more mRNA for eNOS, thus increasing the rate of translation of eNOS providing more eNOS for transportation through the MAP kinase path to the L-arginine pathway increasing the production of nitric oxide (NO). More NO production increases the endothelium-dependent vasodilation. In addition to increasing the production of NO, estrogen upregulates L-arginine availability and prostacyclins expression, while down-regulating the expression of endothelium-derived contracting factors such as endothelin-1 and angiotensin-II.

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8 Figure 2-2. Action of estrogen (43) The non-genomic path is independent of gene transcription. In this scenario the Maxi-K+ channel action utilizes the estrogen to induce cell hyperpoalrization through the decreasing of intracellular Ca2+ concentration and thus causing vasodilation of the endothelium through smooth muscle relaxation. Though estrogen can have an affect on the body through either pathway, the end result is always vasodilation (43, 46). Flow Mediated Dilation Endothelium-dependent flow mediated dilation (FMD) or, reactive hyperemia, can be measured using high resolution ultrasound imaging to non-invasively measure the diameter of the peripheral conduit arteries. Reactive hyperemia is caused by the transient increase in blood flow that occurs following a brief period of arterial occlusion. The magnitude of hyperemia is directly related to the duration of ischemia. The shear resulting from the increased blood flow on the endothelial cells activates the

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9 endothelium-dependent vasodilation pathway. This provides an increase in the release of NO causing the smooth muscle layer of the blood vessel to relax thus increasing the diameter of the blood vessel to allow for the increased blood flow without causing more stress on the cardiovascular system (10, 23). FMD testing is usually performed on people who have fasted and abstained from caffeine for 8-12 hours prior. The subject rested in a supine for a ten-minute period and then baseline two-dimensional images of the right brachial artery will be obtained approximately 2 cm above the antecubital fossa. A blood pressure cuff is placed proximal to the imaging transducer on the upper arm and inflated to suprasystolic pressure for exactly five minutes. With the release of the occlusion the vessel would be continuously imaged from thirty seconds to two minutes. Maximal brachial artery FMD diameter occurs generally about one minute after release of the occlusion. The equation for calculating percent FMD is FMD% = (FMD diameter max)/ (FMD baseline diameter) x 100. A healthy endothelium should have a flow-mediated dilation of 10% or more. A lower percentage indicates endothelial dysfunction and possible atherosclerosis (10, 24). In 2002, Corretti et al. (10) demonstrated that the optimal time for occlusion of the upper arm should be five minutes. There were increasing brachial artery diameter values for each additional minute of occlusion up to five minutes, but the five minute occlusion FMD and ten minute FMD were very similar. The researchers also showed that the upper arm was a better cuff position than the forearm because the reactive hyperemia response was greater with the upper arm occlusion and thus easier to study (10).

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10 Applanation Tonometry Applanation tonometry is a noninvasive technique to assess arterial stiffness, which is represented by the time delay of the reflected wave (T) and augmentation index (AI). AI is determined by the equation of AI=[(Ps-Pi)/(Ps-Pd)] where; Ps= peak of reflected wave, Pi= start of reflected wave, and Pd=baseline diastolic pressure. T is the total time of the pulse wave’s trip from the aorta, to the periphery, and back to the aorta. T with a smaller value indicates a faster velocity, which translates to stiffening of the periphery. A small T (or reflected wave time) correlates to an increased workload for the left ventricle (LV) because the LV has to eject the blood out of the aorta against the blood vessel’s natural resistance and against the added resistance from the early returning reflected wave. This early returning reflected wave increases the work and oxygen demand of the heart. The presence of healthier, more elastic, endothelium in the periphery results in a greater T value. AI is calculated using the amplitude of the reflected wave divided by the amplitude of forward traveling wave. An ideal AI would have a negative value. Stiff peripheral arteries are associated with a higher AI. With the increase of AI or decrease of T, there is an increase risk of incidence in stroke, CAD, and death (25, 32). Studying central aortic pressure is essential to determine CV health in females after the loss of hormonal protection with menopause. In 2001, Waddell et al. (45) reported that women had a larger age-related stiffening of large arteries due to their hormonal changes. Before menopause women had lower mean arterial pressures than men. The authors speculated that older women developed higher mean arterial pressure because they had lost the ability to produce protective gonadal hormones (45).

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11 Plasma Markers Investigating the effects of exercise on endothelium function by directly or indirectly measuring NO can be done with blood markers for NO availability or oxidative stress. The available levels of plasma NO can be estimated by measuring the nitrite/nitrate levels. Oxidative stress can be estimated in the plasma by measuring oxidative markers such as the levels of 8-isoprostane in the blood or by measuring the levels of plasma superoxide dismutase activity (SOD), an antioxidant. One can also study endothelium function or dysfunction with the inflammatory marker, C-Reactive Protein (3, 13, 16, 26). In 2001 Maeda et al. (26) trained eight sedentary men (20.3 .5 yr old) and examined how the levels of NO were affected. After the training period of 8 weeks on a cycling leg ergometer (70% VO2max for 1 hour 3-4 days/week), the levels of plasma NO were significantly elevated. The levels remained elevated at four weeks post training but returned to baseline levels at the eight-week mark. This study demonstrated that males can increase the availability of NO in the plasma with training and the adaptations have more than a transient benefit (26). In 2003, Edwards et al. (13) studied the effects of exercise on oxidative stress markers, antioxidant markers, and NO levels in men with CAD. Endurance exercise during a 12-week cardiac rehabilitation program lead to an increase of antioxidant (SOD) protection, an increase in available NO, and a decrease in the oxidant stress marker of 8-isoprostane (13). Previous Research on Women The majority of the research pertaining to exercise and endothelial function have been conducted using the male population. There have been only a few research studies

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12 that have dealt with the female population. Moreover, the existing studies were designed to assess the effect of hormone replacement on the endothelial function rather than the effects of exercise on the endothelium. In 1998 Tanaka et al. (42) studied women to determine how exercise may attenuate the age-related increase in central arterial stiffness. The women in the study were grouped as premenopausal sedentary or active and postmenopausal sedentary or active. The researchers measured the arterial stiffness using applanation tonometry. The study showed that postmenopausal sedentary women had higher PWV and AI compared to the premenopausal sedentary group. The authors concluded that in women, arterial stiffness increases with age and with the loss of hormonal protection. The study also showed that postmenopausal women who were active had a 30-50% lower PWV and AI levels than their sedentary age matched peers. These data suggest that females can decrease arterial stiffness with exercise comparable to the effects observed in older men (42). In a study published in 1999, Manson et al. (27) studied the effects of vigorous aerobic exercise versus a walking protocol. The authors concluded that a walking protocol was adequate to elicit cardioprotection. However, the study included both premenopausal and postmenpausal women thus allowing the confounding effect of estrogen to skew the data (27). Wong and Wong (47), in 1999, reviewed previous research studies and concluded that exercise was cardioprotective and elicited results comparable to HRT protocols. However, the study was flawed by grouping both premenopausal and postmenopausal women together thus the independent cardioprotective effect of exercise could not be determined (47).

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13 In 2001, McKechnie et al. (29) studied the effects of habitual physical activity on vascular reactivity in postmenopausal women. The authors eliminated the confounding factor of hormonal protection by only studying postmenopausal women and concluded that exercise was cardioprotective in older women. However, this study was a cross sectional study and the exercise was self-reported. The authors found that older women who participated in endurance training had healthier endothelium but they did not identify a threshold amount of exercise needed to restore endothelial function (29). Summary The available research on females suggests that endurance exercise improves endothelial function. However the available studies contain many compounding factors. For example, Manson et al. (27) and Wong et al. (47) did not group women based upon menopausal status. Thus the increases in endothelial function with endurance exercise could have been a result of the hormonal protection of estrogen and not from the exercise intervention. McKechnie et al. (29) eliminated the estrogen confounder by only studying postmenopausal women. However, this was not a training study and exercise volume was self-reported. Tanaka et al. (42) demonstrated that exercise decreased the arterial stiffness in premenopausal and postmenopausal women but the study design was cross-sectional. Thus further research to clarify the type and amount of exercise needed to elicit a healthier endothelium in postmenopausal women is warranted. Past research in males concluded that exercise is cardioprotective and aerobic exercise alone is protective (27, 29, 42, 47). No studies in either males or females, have examined if the cardiovascular protective effects of resistance training. We propose to design an exercise study that will

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14 compare effects of resistance versus aerobic training on endothelial function in postmenopausal women.

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CHAPTER 3 METHODOLOGY Study Design This study was randomized. All subjects were recruited from Gainesville and the surrounding areas through a newspaper ad ran in the Gainesville Sun, fliers, or by word of mouth. This study was conducted in conjunction with a study designed to determine the osteogenic efficacy of various mechanical loading protocols on bone density in women. The study was a nine month exercise training study involving 50-75 year old postmenopausal women assessing the effects of a progressive resistance training program. The postmenopausal women were assigned to one of three exercise protocols. One group exercised using nine pieces of exercise equipment typically found in commercial gyms. A second group used these same nine machines plus the MedX leg press and MedX medical lumbar extension machine. The resistance training regimen for the group with two extra machines represented our attempt to determine if these two machines had any additive benefit for the osteoporosis as had been previously reported by our Lab (4, 30). A third group, represented an endurance exercise group and subjects walked on a treadmill. The goal was to measure endothelial function in 10 subjects from the resistance trained groups and 10 subjects from the aerobic trained group. All testing was performed in the Center for Exercise Science at the University of Florida. All training was done at the Living Well Center at the University of Florida. Data was collected at study entry and after 9 months of exercise training. Laboratory measurements were performed at the same time of day in the same order for each subject. 15

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16 Subject Qualifications The following subjects qualified for the study: Female 50-75 years of age Postmenopausal for at least one year Still had their ovaries or had them removed after the age of 50 Currently not receiving hormone replacement therapy (HRT) or had been off HRT for at least six months Non-smokers Normotensive with resting blood pressure of less than or equal to 140/85 mm Hg No health problems prohibiting them from exercising Not currently engaged in routine exercise Had no history of cardiovascular disease or peripheral disease Were not on any medication that may alter study (e.g. Statin drugs) Subject Recruiting All the subjects who inquired about the study received a telephone call from study investigators explaining the study’s purpose, protocol and answered any initial questions. During the initial telephone interview, it was determined if the subject was qualified to participate and then the subject’s fitness level was assessed by questionnaire. A medical history was also obtained using a questionnaire (Appendix A). If no contraindications were determined during the initial telephone interview an initial laboratory visit was scheduled. In the initial laboratory visit, the benefits and risks were explained, any questions were answered, the participants filled out a health history, signed a inform consent (Appendix B), and baseline blood pressure measurement was taken in duplicate. Dr. Michael Fulton, M.D. (Director, Green Acres Orthopedic Center, Daytona Beach,

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17 FL.), evaluated the medical history forms and cleared the patients for participation in the study. Group Assignments The participants were randomized into one of two groups: aerobic training on the treadmill or resistance training on variable resistance machines. Once a patient was randomized into a group they were not able to change groups at any time. Group 1 performed a protocol consisting of 30-40 minutes of treadmill walking 2 days per week at an intensity of 65-85% of estimated heart rate reserve. Group 2 performed a resistance training protocol twice a week, but allowing at least 48 hours between visits. Blood Drawing and Markers Patients had 40 ml of venous blood drawn at study entry and after of 9 months of exercise training using sterile techniques by an experience lab technician. A 21 or 23 gauge needle was placed into a vein of the forearm. Blood was drawn into four 10cc vacutainers (two plasma and two serums). Plasma samples were refrigerated while serum samples were kept at room temperature for 15 minutes. All the samples were separated by centrifugation at 3000 RPM at 4 oC for 15 minutes. The serum and plasma were pipetted into polypropylene tubes and frozen at -80 oC until biochemical assays were performed. The plasma that was used for measuring markers of oxidative stress was stored with diethlenetriamine prentacetic acid and butylated hydroxytoluene at a concentration of .01 mmol/L. The plasma collected during the study would be used to measure markers for oxidative stress (8-isoprostane and C-Reactive Protein (CRP)), antioxidant defenses (superoxide dismutase activity (SOD)), and the release of nitric oxide (NO). By examining oxidative stress markers and a marker of antioxidant defense, we hope to

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18 correlate positive findings with an increase in brachial flow mediated dilation. By measuring plasma levels of NO were sought to provide a mechanistic basis of support for the changes in brachial artery FMD. 1-Rm Strength One repetition maximum (1-RM) strength testing was assessed on the Group 2 participants to establish the initial resistance loads for their strength training. The strength testing was conducted on six different machines. The machines that were used for the strength testing included the MEDX (Ocala, FL) chest press, leg extension, leg press, torso arm, seated row, and overhead press. The testing began with the subject performing light repetitions as a warm up and to teach correct lifting form. Then a weight comparable to 40-50% of body weight was chosen as a starting point for the two lower limb tests while 20-30% of body weight was chosen for the four upper body tests. The subject then rated the difficulty of the lift based on a scale of 1 to 10, with 10 being the most difficult. Resistance was then to be increased accordingly. Each 1-RM attempted was followed by two minutes of rest. The last weight that was successfully lifted throughout the full range of motion, while maintaining proper form and control, was the 1-RM. Most subjects achieved 1-RM in less than 5 attempts. Applanation Tonometry Applanation tonometry was performed as a noninvasive test of arterial stiffness. The SphygmacorTM Pulse Wave Analysis System was used. The patient refrained from drinking any caffeine 8-12 hours prior to the testing. The patient laid supine for 10-15 minutes prior to testing and they were wearing clothing that exposed the left arm. Blood pressure was taken in triplicate in the left arm using an automatic blood pressure monitor. Radial artery pulse wave analysis was assessed on the left arm. An aortic arterial

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19 pressure waveform was derived from the measured radial artery pulse waveform using the high fidelity ultrasound transducer. The SphygmoCor device utilized a generalized transfer function and various computations to synthesize a central aortic pressure wave and components of the wave. The augmentation index (AI), the percent of augmentation index (AI%), the augmentation correct for heart rate (AI@75), and the reflected wave travel time were recorded. Flow Mediated Dilation (FMD) Flow mediated dilation (FMD) of the brachial artery in the right arm was assessed using high resolution ultrasound. This was performed at study entry and after 9 months of training. The images were stored on a VHS videotape recorder for further analysis. The brachial artery diameter (BAD) was measured by using B-mode ultrasound images at end diastole. The BAD was imaged at resting baseline. Then a pneumatic cuff was inflated around the upper arm above the image site at 200 mmHg for five minutes. After the five minutes, the pressure was released from the cuff and flow mediated dilation did occur. Then the technician imaged the brachial artery diameter continuously for the next few minutes to capture the reactive hyperemia. The BAD measurements were performed at 45, 60, 75, 90, and 120 seconds following the release of the cuff pressure. Aerobic Training The participants in Group 1 performed treadmill exercise twice a week for the nine month period at the Living Well Center at the University of Florida. They walked at a speed that elicited a heart rate between 65-80% predicted heart rate reserve. They stayed in this heart rate zone for 30-40 minutes per session. The first few weeks they were closer to the 30 minutes exercise duration and a heart rate just above 65%. By the sixth week period, they were closer to the 80% of their heart rate and forty minute exercise

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20 duration. The subjects did not exceed 3.5mph speed to achieve their target heart rate. Rather, they increased the slope at which they walk to illicit a greater heart rate response. An exercise specialist monitored the subjects while they exercised and supervised any necessary equipment adjustments. Resistance Training All subjects in Group 2 performed resistance training. They trained at the Living Well Center at the University of Florida. They performed nine or eleven exercises twice a week, allowing at least 48 hours between sessions, for the nine month period. Each session started with five minutes of warm up on the treadmill. The initial resistance was 50% of their 1-RM. The subjects performed one set of 8-12 repetitions. The weight was increased by up to 5% after the subject could successfully complete two consecutive exercise sessions of 12 repetitions with proper form and control. The 12 repetitions were considered maximal effort. The subjects worked at their own pace and were able to take as little or as much time between each exercise machine. The following exercises were included in the nine resistance and the eleven resistance training protocols: 1. Leg Extension 2. Leg Curl 3. Torso Arm 4. Seated Row 5. Chest Press 6. Overhead Press 7. Bicep Curl 8. Triceps machine 9. Abdominal machine As stated before, this study was conducted in conjunction with an osteoporosis study to measure the effects of mechanical loading on bone density, so the resistance training group had two different protocols that were examined in the osteoporosis study. One of the resistance groups performed a regimen of variable resistance exercises, 2 days

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21 per week, on the nine machines listed. The other resistance trained group performed a regimen of variable resistance exercises, 2 days per week, on the nine listed machines plus exercised on the MedX clinical lumbar extension and leg press machines for a total or 11 machines. The subjects performed one set of up to 20 repetitions on the Isometric Lumbar Machine. When a subject had completed two consecutive training sessions of 20 repetitions, the weight was increased up to 5%. Each session was lead by a trained exercise specialist that monitored the subjects, set up the equipment, and taught proper form. Statistical Analysis The research design was a 2 X 2 (Exercise Group x Time) ANOVA with repeated measures on the second factor. The exercise group was either aerobic trained or resistance trained group. The time variable referred to the data collection times, pretest: before the treatment was administered vs. posttest: after the treatment was administered. A two-way repeated-measures ANOVAs were used to analyze the data in patients in either exercise group before and after exercise intervention. Statistical significance was set at p < .05.

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CHAPTER 4 RESULTS Study Design This study was conducted to determine the effects of aerobic and resistance exercise on vascular reactivity in postmenopausal women. A total of 43 subjects completed the nine month parent study involving osteoporosis. Of the 43 subjects, 17 were included in the present sub-study involving brachial measurements of endothelial function. Vascular studies were performed at study entry (T1) and after 9 months of exercise training (T2). Flow mediated dilation (FMD) scans, using high resolution ultrasound, examined brachial artery changes in baseline diameter (mm), absolute dilation (mm), and brachial FMD (%). Arterial stiffness analysis was conducted using Applanation Tonometry with a Sphygmacor Pulse Wave Analysis system to examine augmentation index (AI), percent of augmentation index (AI%), augmentation index corrected for heart rate (AI@75), and reflected wave travel time (Tr). Plasma markers for oxidative stress (8-isoprostane), inflammation (C-reactive Protein), antioxidant protection (Superoxide Dismutase activity), and Nitric Oxide (NO) availability (Nitrate/Nitrite levels) were also measured at T1 and T2. Subject Characteristics The subject characteristics at study entry and study compliance rates are shown in Table 4-1. There were no statistically significant differences in the descriptive characteristics between the subjects assigned to the two study groups. There were also no significant differences in exercise training compliance rates between the two groups. 22

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23 Table 4-1. Subject Characteristics Group Aerobic (n=6) Resistance (n=11) Age (yrs) 60.1.0 57.6.9 Height (in) 63.9.1 63.7.4 Weight (kg) 70.14.8 66.4.2 Compliance (%) 81.0.7 86.1.9 Values are means Standard Deviation. Flow Mediated Dilation (FMD) Data The means and standard deviations for the FMD measurements are listed in Table 4-2. There were no statistically significant differences in baseline diameter, absolute dilation, or brachial FMD between the study groups at study entry. FMD and absolute dilation were significantly increased (p<.05) in the aerobic group after the exercise intervention. Neither absolute dilation nor FMD were significantly increased in the resistance group after the exercise intervention. The Figures of 4-1 and 4-2 show the relative percent changes in absolute dilation (mm) and FMD. Table 4-2. FMD Measurements. Aerobic (n=6) Variable T1 T2 Baseline diameter (mm) 3.523771 .362633 3.529919 .35344 Absolute Dilation (mm) .302966 .051748 .41457 .083575 * Brachial FMD (%) 8.744695 2.174066 11.8637 2.633429 * Resistance (n=11) Variable T1 T2 Baseline diameter (mm) 3.530581 .264375 3.492387 .284127 Absolute Dilation (mm) .362613 .112826 .418036 .128109 Brachial FMD (%) 10.45574 3.788472 12.12792 4.222433 Values are expressed as means standard deviation. *P < .05

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24 Relative Changes in Aerobic Group0102030405060708090100Brachial DilationFMD %Percent Change (%) ** Figure 4-1. Relative percent change in dilation and FMD of the brachial artery from pre to post in the aerobic trained group. Values are means standard error. * p < .05 Relative Changes in Resistance Group0102030405060708090100Brachial DilationFMD %Percent Change (%) Figure 4-2. Relative percent change of change in dilation and FMD of the brachial artery from pre to post in the resistance trained group. Values are means standard error. Applanation Tonometry Hemodynamic variables are listed in Table 4-3. Brachial systolic, diastolic, and pulse pressures were not significantly different between groups at study entry. Aortic systolic, diastolic, and pulse pressures were also not significantly different between

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25 groups at study entry. There were no significant changes in either brachial or aortic hemodynamic variables after the 9 month exercise intervention in either group. Augmentation index (AI), percent of augmentation index (AI%), augmentation index corrected for heart rate (AI@75), and time of reflected wave (Tr) are shown in Table 4-4. Figures 4-3, 4-4, 4-5, and 4-6 present the relative changes (%) of each variable from T1-T2. Table 4-3. Changes in Hemodynamic Values after the Exercise Training. Aerobic Resistance Variable T1 T2 T1 T2 Heart rate (beats/min) 68.1667 7.250 68 6.573 65.385 6.838 62.462 6.385 Brachial SBP (mm Hg) 122 16.395 121.83 10.148 130.154 8.102 127.923 17.732 Brachial DBP (mm Hg) 77.667 5.854 78.667 3.445 79.385 5.378 80 11.328 Brachial PP (mm Hg) 44.333 12.612 43.167 10.543 50.769 5.862 47.923 9.853 Aortic SBP (mm Hg) 114.167 15.065 113 6.812 122.769 7.865 121.692 17.684 Aortic DBP (mm Hg) 78.5 6.156 79.5 3.619 80.307 5.437 80.923 11.514 Aortic PP (mm Hg) 35.667 10.132 33.5 6.285 42.462 6.022 40.769 9.782 Values are mean standard deviation. SBP = systolic blood pressure; DBP = diastolic blood pressure; PP = Pulse pressure. Table 4-4. Absolute Values of Applanation Tonometry Data. Aerobic (n=6) Variable T1 T2 AI (mmHg) 10.3333 5.6451 8.6667 1.6329 AI% 28.8333 8.5654 25.8333 5.7067 AI%@75 25.6667 8.1894 22.3333 5.0464 T (ms) 143 6.4807 147.1667 7.7049 Resistance (n=11) Variable T1 T2 AI (mmHg) 15 3.7417 14.5454 6.7729 AI% 34.5454 7.4211 35.1818 8.7271 AI%@75 29.2727 5.934491 28.9090 8.0803 T (ms) 140.5455 8.3111 140.9091 8.0803 Values are expressed as means standard deviation.

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26 Relative Change (%) in Augmentation Index-100-90-80-70-60-50-40-30-20-100Aerobic ResistancePercent Change (%) Figure 4-3. Relative percent changes in augmentation index from baseline (T1) to post (T2). Values are mean standard error. Relative Percent Change of AI%-50-40-30-20-1001020304050Aerobic ResistancePercent Change (%) Figure 4-4. Relative percent change in percent of augmentation index from baseline (T1) to post (T2). Values are mean standard error.

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27 Relative Change (%) of AI% Corrected for HR-50-45-40-35-30-25-20-15-10-50Aerobic ResistancePercent Change (%) Figure 4-5. Relative percent change in augmentation index corrected for heart rate. Values are mean standard error. Relative Change (%) in Reflected Wave Travel Time0510152025Aerobic ResistancePercent Change (%) Figure 4-6. Relative percent change of total travel time of reflect wave. Values are mean standard error. Plasma Markers Data Venous blood samples were drawn from 17 subjects at T1 and T2. The blood samples were immediately processed and stored at -80 degrees centigrade. All the samples were assayed in a single lot at the end of the study and the assays were done in

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28 duplicate. All the absolute values of the plasma markers of endothelium health are contained in Table 4-5. Table 4-5. Plasma Markers. Aerobic (n=6) Variable T1 T2 SOD (U/ml) 2.344 .322 3.038 .836 8-Isoprostane (pg/ml) 204.197 137.657 406.905 251.100 CRP (ng/ml) 3.504 1.227 4.173 2.953 Nitrite/Nitrate (M) 37.215 33.039 31.702 23.223 Resistance (n=11) T1 T2 SOD (U/ml) 2.553 .753 2.779 .797 8-Isoprostane (pg/ml) 345.203 251.231 202.967 123.679 CRP (ng/ml) 2.043 2.376 2.083 2.319 Nitrite/Nitrate (M) 23.438 15.386 38.774 26.844 Values are means standard deviation. T1 = baseline; T2 = post. Superoxide Dismutase (SOD) SOD at study entry was not significantly different between the aerobic and resistance groups. Plasma total SOD activity was not significantly increased in the aerobic group (2.344 .322 U/ml at baseline vs 3.038 .836 U/ml at 9 months). There were also no significant changes in the SOD activity in the resistance group (2.553 .753 U/ml at baseline vs. 2.779 .797 U/ml at 9 months). The relative change (%) of SOD activity is shown in Figure 4-7.

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29 Relative Change (%) in SOD Activity0102030405060708090100AerobicResistancePercent Change (%) Figure 4-7. Relative percent change in SOD activity. Values are means standard error. 8-Isoprostane There were no significant differences between 8-isoprostane values in the two groups at study entry. The baseline absolute values of 8-isoprostane were 345.203 251.231 pg/ml in the resistance group and 204.197 137.657 pg/ml. Plasma 8-isoprostane levels were not significantly different after exercise intervention in either group. The relative change (%) in 8-isoprostane level in both groups are displayed in Figure 4-8.

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30 Relative Change (%) in 8-isoprostane-150-100-50050100150200Aerobic ResistancePercent Change (%) Figure 4-8. Relative percent change in levels of 8-isoprostane. Values are means. C-Reactive Protein (CRP) There were no significant differences between CRP values in the two groups at study entry. Baseline values of CRP were 2.043 2.376 ng/ml in the resistance trained group and 3.504 1.227 in the aerobic trained group. There were no significant changes in plasma CRP levels in either the aerobic group or the resistance group following the exercise intervention. Relative change (%) of the CRP levels from baseline to post exercise are shown in Figure 4-9.

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31 Relative Change (%) in CRP Levels0102030405060708090100Aerobic ResistancePercent Change (%) Figure 4-9. Relative percent change in CRP levels. Values are in means standard error. Nitric Oxide There were no significant differences in nitric oxide values between the two groups at study entry. Baseline NO2 and NO3 levels were 23.438 15.386 M in the resistance group and 37.215 33.039 M in the aerobic group. Plasma nitrate and nitrite levels were not significantly changed in either the aerobic or resistance groups. The relative change (%) of the nitrate/nitrite levels from baseline to post exercise intervention are shown in Figure 4-10.

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32 Relative Change (%) in Nitrite/Nitrate Levels-60-40-20020406080100120Aerobic ResistancePercent Change (%) Figure 4-10. Relative percent change in nitrate/nitrite levels. Values are in means.

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CHAPTER 5 DISCUSSION Cardiovascular disease (CVD) is the number one killer of women in America (2). Since the premature termination of the Women Health Initiative in July 2002, the standard of care for cardiovascular disease prevention in postmenopausal women has undergone radical changes (5, 22). The old standard of prescribing Hormone Replacement Therapy (HRT) is no longer seen as advantageous. Consequently, alternative methods of cardioprotection are being explored. With this in mind, we investigated the affect of 9 months of aerobic and resistance training on endothelial function in postmenopausal women. FMD Discussion We hypothesized that brachial artery FMD would increase in both the aerobic and resistance trained postmenopausal women. The aerobic protocol utilized in this study was sufficient stimulus to elicit a significant increase in brachial FMD and absolute dilation. The increase in FMD (+ 3.11%, p< .05) and in absolute dilation (+.1116 mm, p < .05) were significant in the endurance training group. The FMD and absolute dilation in the resistance group were not significant (+.0554 mm). Edwards et al. (13) reported an increase in FMD of +3.3% in males with coronary artery disease after aerobic exercise training. Fuchsjager-Maryl et al. (15) also showed an increase in FMD of +3.2% with aerobic training in males with type 1 diabetes. Our increase in FMD of +3.11% was similar to those findings. 33

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34 Applanation Tonometry Discussion We hypothesized that Aortic AI and T, indices of arterial stiffness, would decrease and increase respectively in both the aerobic and resistance training groups. Pulse wave analysis in this study demonstrated that 9 months of aerobic or resistance training in postmenopausal women resulted in no significant changes in arterial stiffness as represented by augmentation index (AI), percent of augmentation index (AI%), augmentation index corrected for heart rate (AI%@75), and travel time of the reflected wave (T). Plasma Markers of Oxidative Stress Superoxide Dismutase (SOD) We hypothesized that plasma SOD activity level would increase in both the aerobic and resistance trained postmenopausal women. SOD activity is a marker of antioxidant protection an the amount of SOD activity in the plasma is crucial for prevention of free radical production, which can lead to a disease such as CAD (7). SOD catalyze the conversion of superoxide (a powerful free radical) to hydrogen peroxide and molecular oxygen, thus protecting the endothelium from being damaged from the free radicals in the blood (7). Plasma SOD activity was measured at baseline and following the exercise intervention. Plasma total SOD activity was not significantly increased in the aerobic group (2.344 .322 U/ml at baseline vs. 3.038 .836 U/ml at 9 months, p = .08). There were also no significant changes in the SOD activity in the resistance group (2.553 .753 U/ml at baseline vs. 2.779 .797 U/ml at 9 months). However, the data suggest a trend toward increased plasma SOD activity (p = .08) in the aerobic group.

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35 8-Isoprostane We hypothesized that plasma levels of 8-Isoprostane would decrease in both the aerobic and resistance trained postmenopausal women. 8-Isoprostane is a marker of lipid peroxidation. It has been found to disrupt and derange the endothelial cell barrier, thus causing dysfunctional endothelium. High levels of 8-isoprostane in plasma have been linked to cardiovascular disease (40). In this study, neither the aerobic nor resistance protocols produced significant differences in 8-isoprostane levels. We observed a paradoxical increase in 8-isoprostane, albeit statistically not significant, in the aerobic group. The elevated levels of 8-isoprostane could have been caused by the exercise session prior to the blood collection. Childs et al. (8) reported that muscle injury from eccentric exercise elevated the levels of 8-isoprostane for up to three days after insult. Plasma Markers of Endothelial Function C-Reactive Protein (CRP) We hypothesized that CRP levels would be decreased in both the aerobic and resistance trained postmenopausal women. CRP is a marker of inflammation that has been linked to predicting cardiovascular disease. CRP, in clinical applications, is a stronger indicator for cardiovascular disease than LDL cholesterol (1). CRP is clinically triaged into tertiles: low risk (CRP < 1 mg/L), medium risk (CRP 1 to 3 mg/L), and high risk (CRP > 3 mg/L). CRP values above 10mg/L are viewed as caused by acute infections or trauma and one should retest CRP levels at a future date (36). In this study, two subjects had CRP levels that were discarded because one of their CRP measurement levels registered above 10 mg/L, thus indicating that they were sick or just getting over an infection. There were no significant differences in CRP levels from baseline to post intervention in either the resistance or aerobic trained groups.

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36 Nitrate/Nitrite We hypothesized that plasma NO levels in the plasma would increase in both the aerobic and resistance trained postmenopausal groups. Nitric Oxide (NO) is a potent vasodilator, which is important in maintaining healthy endothelium. Endothelium dysfunction can be linked to the deficiency of NO availability. NO has a short half-life and readily breaks down in vivo to nitrite (NO2-) and nitrate (NO3-). Therefore, the best index of total NO availability is the sum of both nitrate and nitrite levels (6). In this study the nitrate/nitrite levels did not significantly change from baseline to post intervention in either group. Previous studies in postmenopausal women with controlled diet up to 48 hours prior to testing by Nikander et al. (33) and Konukoglu et al. (20) showed basal levels of NO to be 21 9.7 umol/l and 16.43 4.91 umol/L respectively. The values recorded in this study were higher than the previous studies values. Although the explanation for these disparate findings are unclear, one possibility is that dietary nitrate/nitrite was not controlled in the present study. Limitations This study was designed in conjunction with a study that was testing various resistance protocols and their effects on bone density in postmenopausal women. The exercise protocols were selected and tailored to gain maximum results for increasing bone density. The endothelium function measurements were performed on a random sub-sample of the parent study. The results of this study were also limited by a relatively small sample size. While the study protocol was designed to be a gradual increasing regimen that any postmenopausal women could perform, the women were not able to sustain the protocol for the entire 9 month period. The majority of the resistance group reported

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37 soreness and discomfort about 4-5 months into the program. For safety and comfort of the subjects, the resistance was lowered on the machines or training was halted until the soreness had dissipated. Subjects in the aerobic group also complained of musculoskeletal discomfort around the 6-8 month point and training regiment were altered accordingly. Reduced training intensity may have decreased the benefits of the exercise and could have altered the findings for this study. Indeed, the small observed increases in the inflammation marker (CRP) and lipid peroxidation marker (8-isoprostane) could possibly be attributed to muscle and joint soreness. Baseline NO levels were higher than previous studies in which diet was controlled before blood collection (20, 33). This suggests that the subjects in the present study may have been consuming a high nitrogen diet prior to the collection of the blood samples. Moreover, NO levels did not increase with an exercise intervention as reported by others (13, 26). The lack of increase in NO levels could be attributed to exercise frequency (2 x week). Alternatively, the subjects in the present study were highly selected and possibly possessed optimal NO levels. Maeda et al. (26) used a protocol of endurance exercise 3-4 days/week to increase NO levels and Edwards et al. (13) reported increase NO in CAD who trained 3 sessions/week. Future Experiments Cardiovascular disease (CVD), which includes heart disease, hypertension, coronary artery disease (CAD), peripheral vascular disease, and stroke, is the number one killer of women in America according to the American Heart Association (2). CVD accounted for over 500,000 deaths in 2001 (2). Endothelial dysfunction has been shown to be a known precursor to CAD, but research has been limited in studying ways to improve endothelial function in postmenopausal women.

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38 Our study focused on the measuring of brachial FMD to assess endothelial function. We based our findings off of Kuvin et al. (23), who recently showed that the presence of a healthy FMD (FMD 10%) correlated to the absence of CAD and good exercise tolerance. We assumed that any increase FMD correlated to decreased risk of CAD. Kuvin et al. (23) also demonstrated that subjects with 3 or fewer CAD risk factors (hypertension, hyperlipidemia, diabetes, fasting glucose levels, menstruating status, and family history) had a higher FMD than subjects with more risk factors. Future research could examine how an increase in FMD correlates with changes in the CAD risk factors. Future research should not solely relay on measuring endothelial function with non-invasive endothelial dependent FMD (EDFMD), caused by shear stress from brachial occlusion. This technique, although very effective in measuring endothelial function in humans, includes a limitation due to the uncontrolled nature of the evoked shear stress stimulus. With an uncontrolled stimulus from trial to trial, error is introduced that would increase the variation of the EDFMD measurements, thus preventing proper assessment of individual’s changes in EDFMD with intervention (35). Researchers can avoid this limitation, by controlling the brachial artery shear rate. The researcher should measure blood flow using the peak velocity plus the diameter of the brachial artery to assess endothelial function (35). The peak blood velocity would be measured using ultrasound Doppler immediately after release of cuff occlusion. By measuring the peak blood velocity, one can standardize the measurements by comparing the blood flow. If the two peak velocities are similar, then the stimuli for each shear stress are similar, thus the error of having different stimuli is removed (35).

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APPENDIX A STUDY QUESTIONAIRE N ame: ______________________________________ Date of Birth: ____________ Address: ______________________________ Phone number: (w) ___________ ______________________________ (h) ___________ Screening Blood Pressure Measurement : / Please answer the following questions: I. GENERAL HEALTH 1. Have you ever been told by a physician that you have Osteoporosis/Osteopenia? Y N 2. Have you ever been told by a physician that you have a heart condition? Y N 3. Have you or anyone in your immediate family had a heart attack, stroke, or Y N cardiovascular disease before age 50? If “yes,” please explain. _____________________________________________________________ 3. Have you ever been told by a physician that you have high blood pressure? Y N 4. Have you ever been told by a physician that you have high cholesterol? Y N 5. Have you ever been told by a physician that you have thyroid problems? Y N 6. Have you ever been told by a physician that you have kidney disease? Y N 7. Do you feel angina-like symptoms (pain or pressure in your chest, neck, shoulders, or arms) during or after physical activity? Y N 8. Do you ever lose your balance because of dizziness? Y N 9. Do you ever lose consciousness? Y N 10. Do you consider yourself to be generally healthy? Y N 39

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40 11. Please list all of the prescriptionmedication you are currently taking Medicine: Amount taken per day Medicine: Amount taken per day a. ____________ ________________ f. ___________ ________________ b. ____________ ________________ g. ___________ ________________ c. ____________ ________________ h. ___________ ________________ d. ____________ ________________ i. ___________ ________________ e. ____________ ________________ j. ___________ ________________ 12. Please list all of the over-the-counter medicines or supplements (including vitamins that you take regularly) Item: Amount taken per day Item: Amount taken per day a. ____________ ________________ g. ___________ ________________ b. ____________ ________________ h. ___________ ________________ c. ____________ ________________ i. ___________ ________________ d. ____________ ________________ j. ___________ ________________ e. ____________ ________________ k. ___________ ________________ f. ____________ ________________ l. ___________ ________________ II. REPRODUCTIVE STATUS 13. How long has it been since you reached menopause? ________________ 14. Do you still have your ovaries? _______ Y N a. If not, how old were you when they were removed? _______. 15. Have you ever been on hormone replacement therapy? Y N a. If so, are you still taking hormone replacement therapy? Y N

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41 a. If you have previously taken hormone replacement therapy, but have since stopped, when did you stop taking hormone replacement therapy? _________________ 16. Have you ever taken osteoporosis medications? Y N Which ones and for how long? ________________________________________ ______________________________________________________________ III. OSTEOPOROSIS/FRACTURE/BONE HEALTH SECTION 17. Please provide a list of bone fractures you have had within the past five years Bone: cause: (fall, accident, etc) Year _ ____________ __________________________________________ _ ____________ __________________________________________ _ ____________ __________________________________________ _ ____________ __________________________________________ 18. Did a doctor tell you that any of these fractures were due to Y N osteoporosis/osteopenia? 19. Is your diet low in dairy products? Y N 20. Do you take calcium supplements? Y N If so, how much per day? ______________________________ 21. In a typical week, how many alcoholic drinks do you consume? ________________________ 22. Do you drink coffee, tea, or cola products routinely? Y N About how much coffee, tea, or cola do you drink on an average day? _______________________________________________________

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42 IV. EXERCISE SECTION 23. How many times per week do you generally exercise? _____________________________ a. What type of exercise do you generally do? _____________________________ ______________________________________________________________________ b. In a typical week, how much time do you spend exercising? ______________________ 24. Do you have any joint problems that would prevent you from exercising? Y N 25. Do you have any lower back problems that would prevent you from exercising? Y N 26. Do you have any other medical condition that might prevent you Y N from exercising? (Diabetes, recent surgery, etc.) 27. Do you currently smoke? Y N 28. Are you a former smoker? Y N If so, how long has it been since you quit smoking? ________________________ 29. What is your current height and weight? Height _____ Weight _____ 30. Are there any other health related issues we should know about? _______________________ _ __________________________________________________________________________________ _ _ _______________________________________ _____________ YOUR SIGNATURE DATE Recommended for Study: ____ Not Recommended for Study: ____ _ __________________________ MEDICAL DIRECTOR Date: _________

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APPENDIX B INFORMED CONSENT Informed Consent to Participate in Research and Authorization for Collection, Use, and Disclosure of Protected Health Information You are being asked to take part in a research study. This form provides you with information about the study and seeks your authorization for the collection, use and disclosure of your protected health information necessary for the study. The Principal Investigator (the person in charge of this research) or a representative of the Principal Investigator will also describe this study to you and answer all of your questions. Before you decide whether or not to take part, read the information below and ask questions about anything you do not understand. Your participation is entirely voluntary. 1. Name of Participant. (“Study Subject”) _____________________________________________________________________ 2. Title of Research Study Resistance Exercise as Therapy for Postmenopausal Osteoporosis 3. Principal Investigator and Telephone Number(s) Randy W. Braith, Ph.D. (352) 392-9575 ext 1340 Michael N. Fulton, MD (386) 258-9502 Kathy Howe, MA (352) 392-9575 ext 1335 Mark Mering, BS (352) 392-9575 ext 1344 43

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44 4. Source of Funding or Other Material Support University of Florida 5. What is the purpose of this research study? In the United States, 50% of women suffer bone fractures after the onset of menopause. Of the more than 1.5 million fractures that occur each year in postmenopausal women, over 300,000 are fractures of the hip and 700,000 involve the spinal vertebra. The hip and vertebral fractures are caused by low bone thickness. No effective drug therapy exists to increase bone thickness in postmenopausal women. The purpose of this study is to determine the effects of different exercise programs on bone thickness. We believe that specific exercise programs will slow the loss of bone and, perhaps, even result in increased bone formation. Sixty healthy postmenopausal women will be included in the study. You are being asked to participate because you have experienced natural (non-surgical) menopause for at least one year and have not received hormone replacement therapy for at least six months. 6. What will be done if you take part in this research study? Participant Screening and Group Assignments : Prior to acceptance into the study, you will fill out a health questionnaire to determine your eligibility for the study. If you are accepted into the study, you will report to the Center for Exercise Science in the Florida Gymnasium at the University of Florida or to Medical Exercise Associates in Daytona Beach, Florida for a test that will measure your current bone thickness. At that time a 20 ml (two tablespoons) sample of blood will also be drawn from a vein in your forearm. You will also be given instructions on how to fill out a diet analysis worksheet so that we can determine your average daily intake of calcium. This visit will last approximately 1 hour. You will then be assigned to one of three 9-month exercise programs: Group #1 will perform 30-40 minutes of treadmill walking exercise 2 days per week at 65-80% of peak heart rate. Group #2 will perform weight-lifting exercises 2 days per week using 11 machines, including the clinical low-back machine and the leg press machine. Group #3 will perform weight-lifting exercises 2 days per week using 9 machines and will not include the clinical low-back machine and the leg press machine. Bone Thickness Testing : Individuals accepted into the study will report to the Center for Exercise Science at the University of Florida or to Medical Exercise Associates in Daytona Beach, Florida where they will undergo measurement of whole body and regional bone thickness. Participants will lie on their back or on their side while the X

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45 ray scanner performs a series of scans. This procedure will be performed under the supervision of a licensed X-ray technician and takes approximately 40 minutes. This test will be repeated after 4.5 and 9 months of exercise training to determine the effects of the exercise protocols on bone thickness. Participants will also be offered the opportunity for additional annual bone scans for a period of five years after the exercise program has ended. This will be done to document the long-term effects of the exercise program. Strength Testing : Prior to starting your 9-month exercise program, skeletal muscle strength will be measured in all study participants. This testing will be done at the Center for Exercise Science in the Florida Gym at the University of Florida or at Medical Exercise Associates in Daytona Beach, Florida. Muscular strength of your lower back will be measured on the MedX low-back machine at seven different positions (0, 12, 24, 36, 48, 60, 72 of low-back flexion). After completion of the low-back strength tests, your arm, leg, chest, shoulder and upper-back strength will also be determined by measuring the maximum amount of weight that you can lift one time. Arm and chest strength will be determined on a MedX chest press machine. During this test the arms are extended away from the chest while pressing against the maximum weight that can be resisted. Leg strength will be determined on a MedX knee extension machine and leg press machine. During this test you are seated and the lower leg is fully extended while lifting the maximum weight that can be resisted. Upper-back strength will be determined on a MedX pull down machine and seated row machine. During these tests you will pull the maximum weight you can. Shoulder strength will be determined using a MedX shoulder press machine. During this test you will push the maximum weight possible over your head. Strength testing visits will last approximately 2 hours. All strength tests will be repeated after 4 and 9 months of the exercise programs (weight training and treadmill walking groups). Treadmill Walking Program : The treadmill walking program will be available in 2 locations: (1) at the Center For Exercise Science in the Florida Gym at the University of Florida or (2) at Medical Exercise Associates, Daytona Beach, Florida. You will do all of your training and testing at the facility where you enter the study (either Gainesville or Daytona Beach). You will be asked to walk twice weekly on a treadmill for 30-40 minutes at a speed and elevation that causes the heart to beat at 65-80% of age-predicted peak heart rate. Peak heart rate will be estimated by subtracting your age from 220. During all treadmill sessions, your heart rate will be continuously monitored using a Polar heart rate monitor. You will begin the treadmill program gradually by walking for 20 minutes at 50% of estimated peak heart rate. Walking duration will increase by 5 minutes each week until you are walking continuously for 40 minutes. Treadmill speed and/or elevation will be gradually increased until you are walking at 70-80% of your estimated peak heart rate after 8 weeks. Treadmill speed will be capped at 3 mph, however, to avoid subjects having to walk too fast or jog. An exercise specialist will monitor you during all exercise-training sessions and supervise any necessary equipment adjustments. Each training session will begin with 5-10 minutes of light treadmill walking and will be followed by a 5-minute cool-down period on the treadmill. Each treadmill walking session will last approximately 1 hour. The duration of the treadmill walking program will be 9 months.

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46 Weight Lifting Programs : If you are assigned to either Group #2 or Group #3 you will be given a familiarization session with the MedX weight lifting machines. The weight lifting machines will be available in 2 locations: (1) at the Center For Exercise Science in the Florida Gym at the University of Florida or (2) at Medical Exercise Associates, Daytona Beach, Florida. You will do all of your weight training and testing at the facility where you enter the study (either Gainesville or Daytona Beach). Group #2 will be asked to perform weight lifting exercises 2 days per week on the following MedX machines: Leg Extension, Leg Curl, Lat Pull Down, Seated Row, Chest Press, Overhead Press, Biceps Curl, Triceps Pushdown, and Abdominal. These machines are designed to work all major muscle groups. Participants in Group #2 will also exercise on the clinical lumbar extension and leg press machines (total 11 machines). Group #3 will be asked to perform weight lifting exercises 2 days per week on the same MedX equipment as Group #2 but will not exercise on the clinical lumbar extension and leg press machines (total 9 machines). The initial resistance for each subject on each machine will be 50% of the participant’s peak strength. You will be asked to complete one set of 8 to 12 repetitions per machine. Participants in Group #2 will also be asked to complete one set of 20 repetitions on the lumbar extension machine. The final repetition should require all of your strength to complete. When 12 repetitions are achieved (or 20 on the lumbar machine), the training weight will be increased 5% at the next training session. An exercise specialist will monitor each participant during all exercise-training sessions. Each training session will begin with 5-10 minutes of treadmill walking and will be followed by a 5-minute cool-down period on the treadmill. Each weight lifting session will last approximately 1 hour. The duration of the weight lifting programs will be 9 months. Blood drawing: Each time you have a bone scan performed, we will draw 20 ml (2 tablespoons) of blood from a vein in your arm. We will use the blood samples to help determine if you are adding new bone, losing bone, or remaining the same. Additional blood samples will be drawn during the week prior to your 4-month and 9-month bone scans. These blood samples will be drawn both before and after one regularly scheduled exercise session. A total of 40 ml (4 tablespoons) will be drawn on these days. These blood samples will be used to determine changes in circulating levels of hormones that influence muscle size and strength. 7. What are the possible discomforts and risks? Strength Testing or Training . A potential source of discomfort to you will be from muscle soreness. This may occur 24-48 hours after a strength test or exercise session and usually the soreness goes away after a few days. Exercise on the MedX machines may cause you to experience some soreness in the muscles of the legs, arms and torso. Exercise on the MedX lumbar extension machine may cause you to experience some soreness in the low back muscles. There is a possibility that a muscle strain could occur which could halt training. In rare instances, some subjects may experience temporary discomfort from lap and leg restraints on the MedX lumbar machine. The risks

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47 associated with exercising on the MedX machines will be minimized by: a) practice on the machines with light weight to familiarize subjects with the testing/training process; b) increasing the exercise weight by only 5% when subjects are able to complete more than 12 repetitions (or 20 repetitions on the lumbar machine); c) supervising all exercise sessions with trained personnel. Weight lifting exercise could aggravate or worsen an existing medical problem that is known or unknown to you. You are encouraged to discuss participating in this study with your personal physician or clinician. Treadmill Walking . A possible source of discomfort to participants assigned to the treadmill walking group is muscle soreness. This may occur 24-48 hours an exercise session and usually the soreness goes away after a few days. The risk associated with treadmill walking will be minimized by having participants achieve an exercise heart rate no greater than 80% of peak heart rate and by having an exercise specialist supervise each of your treadmill sessions. Treadmill walking exercise could aggravate or worsen an existing medical problem that is known or unknown to you. You are encouraged to discuss participating in this study with your personal physician or clinician. Bone Scan . There is a small X-ray dosage during the bone thickness measurements. The effective radiation dose equivalent (HE) is about 2.5 uSv during a total body scan. This exposure is approximately 0.3% of the average annual per capita background radiation exposure in the U.S. Blood Drawing . Blood samples will be drawn from a forearm vein before you begin your exercise program and after 4 and 9 months of the exercise program. The risks of drawing blood from a vein include discomfort at the site of the injection; possible bruising and swelling around the injection site; rarely an infection; and, uncommonly, faintness from the procedure. This risk will be minimized by the use of sterile aseptic techniques. The principal investigator has 15 years of experience in drawing venous blood samples from human subjects. Throughout the study, the researchers will notify you of new information that may become available and might affect your decision to remain in the study. If you wish to discuss the information above or any discomforts you may experience, you may ask questions now or call the Principal Investigator or contact persons listed on the front page of this form. 8a. What are the possible benefits to you? Potential benefits to you include a free evaluation of your bone density, which would otherwise be very costly. You will also gain the experience of participating in a supervised exercise program. This experience may be useful to you in subsequent exercise programs. Also, you should experience an increase in muscle strength and/or

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48 bone thickness, which may reduce complications associated with postmenopausal osteoporosis. 8b. What are the possible benefits to others? The knowledge gained from this study may be used to reduce osteoporosis and its complications in other postmenopausal women. We may gain insight into what types of exercise programs are most effective in increasing bone density. We may also learn methods to predict changes in bone metabolism by looking at elements present in the blood. 9. If you choose to take part in this research study, will it cost you anything? All of the testing procedures and the exercise programs will be provided at no cost to you. 10. Will you receive compensation for taking part in this research study? No financial compensation will be given for participation in this study. 11. What if you are injured because of the study? If you experience an injury that is directly caused by this study, only professional ^ care that you receive at the University of Florida Health Science Center will be provided without charge. However, hospital expenses will have to be paid by you or your insurance provider. No other compensation is offered. 12. What other options or treatments are available if you do not want to be in this study? Participation in this study is entirely voluntary. You are free to refuse to be in the study, and your refusal will not influence current or future health care you receive at this institution.

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49 13a. Can you withdraw from this research study? You are free to withdraw your consent and to stop participating in this research study at any time. If you do withdraw your consent, there will be no penalty, and you will not lose any benefits you are entitled to. If you decide to withdraw your consent to participate in this research study for any reason, you should contact: Dr. Randy Braith at (352) 392-9575 extension 1340, or Dr. Michael Fulton at (386) 258-9502. If you have any questions regarding your rights as a research subject, you may phone the Institutional Review Board (IRB) office at (352) 846-1494. 13b. If you withdraw, can information about you still be used and/or collected? Yes, any information collected while in the study may be used, with your permission, in the final analysis of the data. 13c. Can the Principal Investigator withdraw you from this research study? You may be withdrawn from the study without your consent for the following reasons: (1) to protect your health or safety; (2) a change in medications that would affect the outcome of the study; (3) inability to continue the assigned exercise program. 14. How will your privacy and the confidentiality of your protected health information be protected? If you participate in this research, your protected health information will be collected, used, and disclosed under the terms specified in sections 15 – 24 below. If the results of this research are published or presented at scientific meetings, your identity will not be disclosed.

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50 15. If you agree to participate in this research study, what protected health information about you may be collected, used and disclosed to others? To determine your eligibility for the study and as part of your participation in the study, your protected health information that is obtained from you, from review of your past, current or future health records, from procedures such as physical examinations, x-rays, blood or urine tests or other procedures, from your response to any study treatments you receive, from your study visits and phone calls, and any other study related health information, may be collected, used and disclosed to others. More specifically, the following information may be collected, used, and disclosed to others: Complete past medical history to determine eligibility criteria listed in informed consent Records of physical exams 3-day Food Diary Results from bone mineral density tests Results from laboratory tests on blood samples to determine new one growth 16. For what study-related purposes will your protected health information be collected, used and disclosed to others? Your protected health information may be collected, used and disclosed to others to find out your eligibility for, to carry out, and to evaluate the results of the research study. More specifically, your protected health information may be collected, used and disclosed for the following study-related purpose(s): In the United States, 50% of women suffer bone fractures after the onset of menopause. Of the more than 1.5 million fractures that occur each year in postmenopausal women, over 300,000 are fractures of the hip and 700,000 involve the spinal vertebra. Most hip and vertebral fractures are caused by low bone thickness. No effective drug therapy is widely available to increase bone thickness in postmenopausal women. The purpose of this study is to determine the effects of different exercise programs on bone thickness. We believe that specific exercise programs will slow the loss of bone and, perhaps, even result in increased bone formation. Sixty healthy postmenopausal women will be included in the study. You are being asked to participate because you have experienced natural (non-surgical) menopause for at least one year and have not received hormone replacement therapy for at least six months. 17. Who will be authorized to collect, use and disclose to others your protected health information? Your protected health information may be collected, used, and disclosed to others by the study Principal Investigator ( Randy W. Braith, Ph.D. ) and his staff other professionals at the University of Florida or Shands Hospital that provide study-related treatment or procedures

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51 the University of Florida Institutional Review Board Randy Braith, Ph.D. Michael Fulton, MD Kathy Howe, MS Mark Mering, BS 18. Once collected or used, who may your protected health information be disclosed to? Your protected health information may be given to: the study sponsor (The University of Florida ) United States and foreign governmental agencies who are responsible for overseeing research, such as the Food and Drug Administration, the Department of Health and Human Services, and the Office of Human Research Protections Government agencies who are responsible for overseeing public health concerns such as the Centers for Disease Control and Federal, State and local health departments Malcolm Randall VA Medical center (Gainesville) 19. If you agree to participate in this research, how long will your protected health information be collected, used and disclosed? The Principal Investigators will use and disclose your protected health information until the end of the study. 20. Why are you being asked to authorize the collection, use and disclosure to others of your protected health information? Under a new Federal Law, researchers cannot collect, use or disclose any of your protected health information for research unless you allow them to by signing this consent and authorization. 21. Are you required to sign this consent and authorization and allow the researchers to collect, use and disclose (give) to others of your protected health information? No, and your refusal to sign will not affect your treatment, payment, enrollment, or eligibility for any benefits outside this research study. However, you cannot participate in this research unless you allow the collection, use and disclosure of your protected health information by signing this consent/authorization.

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52 22. Can you review or copy your protected health information collected, used or disclosed under this authorization? You have the right to review and copy your protected health information. However, you will not be allowed to do so until after the study is finished. 23. Is there a risk that your protected health information could be given to others beyond your authorization? Yes. There is a risk that information received by authorized persons could be given to others beyond your authorization and not covered by the law. 24. Can you revoke (cancel) your authorization for collection, use and disclosure of your protected health information? Yes. You can revoke your authorization at any time before, during or after your participation in the research. If you revoke, no new information will be collected about you. However, information that was already collected may be still be used and disclosed to others if the researchers have relied on it to complete and protect the validity of the research. You can revoke by giving a written request with your signature on it to the Principal Investigator. 25. How will the researcher(s) benefit from your being in this study? In general, presenting research results helps the career of a scientist. Therefore, the Principal Investigator may benefit if the results of this study are presented at scientific meetings or in scientific journals.

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53 26. Signatures As a representative of this study, I have explained to the participant the purpose, the procedures, the possible benefits, and the risks of this research study; the alternatives to being in the study; and how the participant’s protected health information will be collected used and disclosed: ________________________________ _____________________ Signature of Person Obtaining Consent and Authorization Date You have been informed about this study’s purpose, procedures, possible benefits, and risks; the alternatives to being in the study; and how your protected health information will be collected, used and disclosed. You have received a copy of this Form. You have been given the opportunity to ask questions before you sign, and you have been told that you can ask other questions at any time. You voluntarily agree to participate in this study. You hereby authorize the collection, use and disclosure of your protected health information as described in sections 14-24 above. By signing this form, you are not waiving any of your legal rights. __________________________________ _____________________ Signature of Person Consenting and Authorizing Date

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LIST OF REFERENCES 1. Alpha Diagnostic International. Human C-Reactive Protein (CRP): For Quantitative Determination of CRP in Human Serum. San Antonio, TX. 2. American Heart Association. Women Heart Disease and Stroke Statistics. http://www.americanheart.org/presenter.jhtml?identifier=4787 . last accessed October 10, 2004. 3. Aziz N, Fahey J, Detels R, and Butch A. Analytical Performance of a Highly Sensitive C-Reactive Protein-Based Immunoassay and the Effects of Laboratory Variables on Levels of Protein in Blood. Clinical and Diagnostic Laboratory Immunology, 2003. 652-657. 4. Braith R, Mills R, Welsch M, Keller J, and Pollock M. Resistance Exercise Training Restores Bone Mineral Density in Heart Transplant Recipients. Journal of the American College of Cardiology, 1996. Vol. 28(6). 1471-1477. 5. Brass LM. Hormone Replacement Therapy and Stroke Clinical Trials Review. Stroke, 2004. Vol. 35(Suppl I). 2644-2647. 6. Cayman Chemical. Nitrate/Nitrite Colorimetric Assay Kit. Ann Arbor, MI. 7. Cayman Chemical. Superoxide Dismutase Assay. Ann Arbor, MI. 8. Childs A, Jacobs C, Kaminski T, Halliwell B, and Leeuwenburgh C. Supplementation with Vitamin C and N-Acetyl-Cystiene Increases Oxidative Stress in Humans After an Acute Muscle Injury Induced by Eccentric Exercise. Free Radical Biology and Medicine, 2001. Vol. 31(6). 745-753. 9. Clarkson Peter, Montgomery HE, Mullen MJ, Donald AE, Powe AJ, Bull T, Jubb M, World M, and Deanfield JE. Exercise Training Enhances Endothelial Function in Young Men. Journal of the American College of Cardiology, 1999. Vol. 33(5). 1379-85. 10. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, and Vogel R. Guidelines for the Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery. Journal of the American College of Cardiology, 2002. Vol. 39(2). 257-265. 54

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55 11. DeSouza Christopher, Shapiro Linda, Clevenger Christopher, Dinenno Frank, Monahan Kevin, Tanaka Hirofumi, and Seals Douglas. Regular Aerobic Exercise Prevents and Restores Age-Related Declines in Endothelium-Dependent Vasodilation in Healthy Men. Circulation, 2000. Vol. 102. 1351-1357. 12. Drexler Helmut and Burkhard Hornig. Endothelial Dysfunction in Human Disease. Journal of Molecular and Cellular Cardiology, 1999. Vol. 31. 51-60. 13. Edwards DG, Schofield RS, Lennon SL, Pierce GL, Nichols WW, and Braith RW. Effect of Exercise Training on Endothelial Function in Men with Coronary Artery Disease. American Journal of Cardiology, 2004. Vol. 93. 617-620. 14. Edwards DG, Schofield RS, Magyari P, Nichols WW, and Braith RW. Effect of Exercise Training on Central Aortic Pressure Wave Reflection in Coronary Artery Disease. American Journal of Cardiology, 2004. Vol. 17. 540-543. 15. Fuchsjager-Maryl G, Pleine J, Wiesinger GF, Sieder AE, Quitten M, Nuhr MJ, Francesconi C, Francesconi M, Seit HP, Schmetterer L, and Wolzt M. Exercise Training Improves Vascular Endothelial Function in Patients with Type 1 Diabetes. Diabetes Care, 2002. Vol. 10. 1795-1801. 16. Goto C, Higashi Y, Kimura M, Noma K, Hara K, Nakagawa K, Kawamura M, Chayama K, Yoshizumi M, and Nara I. Effect of Different Intensities of Exercise on Endothelium-Dependent Vasodilation in Humans. Circulation, 2003. Vol. 108. 530-535. 17. Gulati M, Pandey DK, Arnsdorf MF, Lauderdale DS, Thisted RA, Wicklund RH, Al-Hani AJ, and Black HR. Exercise Capacity and the Risk of Death in Women. Circulation, 2003. Vol. 108. 1554-1559. 18. Herrington David, Espeland MA, Crouse JR, Robertson J, Riley WA, McBurnie MA, and Burke GL. Estrogen Replacement and Brachial Artery Flow-Mediated Vasodilation in Older Women. Arteriosclerosis, Thrombosis, and Vascular Biology, 2001. Vol. 21. 1955-1961. 19. Hunt BJ, Poston L, Schachter M, and Halliday A. An Introduction to Vascular Biology. Second edition. Cambridge University Press. 20. Konukoglu D, Serin O, and Yelke HK. Effects of Hormone Replacement Therapy on Plasma Nitric Oxide and Total Thiol Levels in Postmenopausal Women. Journal of Toxicology and Environmental Health, 2000. Vol. 60. 81-87. 21. Kuikka JT, Raitakri OT, and Gould KL. Imaging of the endothelial dysfunction in coronary atherosclerosis. http://link.springer.de/link/service/journals/00259/contents/01/00528/paper/s002590100528ch1 . last accessed on August 9, 2004.

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56 22. Kuller Lewis. Hormone Replacement Therapy and Risk of Cardiovascular Disease. Arteriosclerosis, Thrombosis, and Vascular Biology, 2003. Vol. 23. 11-16. 23. Kuvin JT, Patel AR, Sliney KA, Pandian NG, Rand WM, Udelson JE, and Karas RH. Peripheral Vascular Endothelial Function Testing as a Noninvasive Indicator of Coronary Artery Disease. Journal of the American College of Cardiology, 2001. Vol. 38(7). 1843-1849. 24. Lind L, Hall J, and Johansson K. Evaluation of Four Different Methods to Measure Endothelium-Dependent Vasodilation in the Human Peripheral Circulation. Clinical Science, 2002. Vol. 102. 561-567. 25. Lebrun CE, van der Schouw YT, Bak A, de Jong FH, Pols H, Grobbee DE, Lamberts S, and Bots ML. Arterial Stiffness in Postmenopausal Women: Determinants of Pulse Wave Velocity. Journal of Hypertension, 2002. Vol. 20. 2165-2172. 26. Maeda S, Miyauchi T, Kakiyama T, Sugawara J, Iemitsu M, Irukayama-Tomobe Y, Murakami H, Kumagi Y, Kuno S, and Matsuda M. Effects of Exercise Training of 8 Weeks and Detraining on Plasma Levels of Endothelium-Derived Factors, Endothelin-1, and Nitric Oxide, in Healthy Young Humans. Life Sciences, 2001. Vol. 69. 1005-1016. 27. Manson JE, Hu FB, Rich-Edwards JW, Colditz GA, Stampfer MJ, Willett WC, Speizer FE, and Hennekens CH. A Prospective Study of Walking As Compared With Vigorous Exercise in the Prevention of Coronary Heart Disease in Women. New England Journal of Medicine, 1999. Vol. 341. 650-658. 28. Matthews Karen, Kuller LH, Sutton-Tyrell K, and Chang YF. Changes in Cardiovascular Risk Factors During the Perimenopause and Postmenopause and Carotid Artery Atherosclerosis in Healthy Women. Stroke, 2001. Vol. 32. 1104-1111. 29. Mckechnie Ronald, Rubenfire M, and Mosca L. Association Between Self-Reported Physical Activity and Vascular Reactivity in Postmenopausal Women. Atherosclerosis, 2001. Vol. 159. 483-490. 30. Mitchell M, Baz M, Fulton M, Lisor C, and Braith R. Resistance Training Prevents Vertebral Osteoporosis in Lung Transplant Recipients. Transplantation, 2003. Vol. 76(3). 557-562. 31. Modena Maria, Bonetti Lorenzo, Coppi Francesca, Bursi Francesca, and Rossi Rossario. Prognostic Role of Reversible Endothelial Dysfunction in Hypertensive Postmenopausal Women. Journal of the American College of Cardiology, 2002. Vol. 40(3). 505-510.

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57 32. Nichols WW and Singh BM. Augmentation Index as a Measure of Peripheral Vascular Disease State. Current Opinion in Cardiology, 2002. Vol. 17. 543-551. 33. Nikander E, Metsa-Heikkila M, Tiitinen A, and Ylikorkala O. Evidence of a Lack of Effect of a Phytoestrogen Regimen on the Levels of C-Reactive Protein, E-Selectin, and Nitrate in Postmenopausal Women. The Journal of Clinical Endocrinology and Metabolism, 2003. Vol. 88(11). 5180-5185. 34. Patterson GC and Whelan RF. Reactive Hyperemia in the Human Forearm. Department of Physiology, The Queen’s University of Belfast, 1954. 35. Pyke K, Dwyer E, and Taschakovsky M. Impact of Controlling Shear Rate on Flow-Mediated Dilation Responses in the Brachial Artery of Humans. Journal of Applied Physiology, 2004. Vol. 97. 499-508. 36. Ridker P, MD. Clinical Application of C-Reactive Protein for Cardiovascular Disease Detection and Prevention. Circulation, 2003. Vol. 107. 363-369. 37. Rywik Tomasz, Blackman Marc, Yataco Alberto, Vaitkevicius Peter, Zink Richard, Cottrell Ernest, Wright Jeanette, Katzel Leslie, and Fleg Jerome. Enhanced Endothelial Vasoreactivity in Endurance-Trained Older Men. Journal of Applied Physiology, 1999. Vol. 87(6). 2146-2142. 38. Solomon Caren M.D., Dluhy Robert M.D. Perspective: Rethinking Postmenopausal Hormone Therapy. New England Journal of Medicine, 2003. 579-580. 39. Sorensen KE, Dorup I, Hermann AP, and Mosekilde L. Combined Hormone Replacement Therapy Does Not Protect Women Against the Age-Related Decline in Endothelium-Dependent Vasomotor Function. Circulation, 1998. Vol. 97. 1234-1238. 40. Stressgen Bioreagents. 8-iso-Prostaglandin F2 Elisa Kit. San Diego, CA. 41. Sutton-Tyrrell Kim, Lassila HC, Meilahn E, Bunker C, Matthews KA, Kuller LH. Carotid Atherosclerosis in Premenopausal and Postmenopausal Women and Its Association With Risk Factors Measured After Menopause. Stroke, 1998. Vol. 29. 1116-1121. 42. Tanaka H, DeSouza CA, and Seals DR. Absence of Age-Related Increase in Central Arterial Stiffness in Physically Active Women. Arteriosclerosis, Thrombosis, and Vascular Biology, 1998. Vol. 18. 127-132 43. Tostes RC, Nigro D, Fortes ZB, and Caralho MHC. Effects of Estrogen on the Vascular System. Brazilian Journal of Medical and Biological Research, 2003. Vol. 36. 1142-1158.

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58 44. Vallance P and Chan N. Endothelial Function and Nitric Oxide: Clinical Relevance. Heart, 2001. Vol. 85. 342-350. 45. Waddell TK, Dart AM, Gatzka CD, Cameron JD, and Kingwell BA. Women Exhibit a Greater Age-Related Increase in Proximal Aortic Stiffness than Men. Journal of Hypertension, 2001. Vol. 19(12). 2205-2212. 46. White Richard. Estrogen and Vascular Function. Vascular Pharmacology, 2002. Vol. 38. 73-80. 47. Wong S. and Wong J. Is Physical Activity as Effective in Reducing Risk of Cardiovascular Disease as Estrogen Replacement Therapy in Postmenopausal Women? International Journal of Nursing Studies, 1999. Vol. 36. 405-414.

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BIOGRAPHICAL SKETCH Mark C. Mering graduated from Michigan State University in 2001 with a double major, a Bachelor of Science in kinesiology and a Bachelor of Science with Lyman Briggs physical science. He spent the next year working as an exercise technician in a cardiology practice in Marquette, Michigan. Following a year of work experience, he returned to academia to obtain a master’s degree in exercise physiology. He has spent his past two and a half years completing a master’s degree in exercise physiology at the University of Florida focusing on cardiovascular physiology. He next plans to further his education by attending physician assistant school. 59