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Effect of Black Cohosh on Biochemical Markers of Bone Remodeling in Postmenopausal Women

Permanent Link: http://ufdc.ufl.edu/UFE0021928/00001

Material Information

Title: Effect of Black Cohosh on Biochemical Markers of Bone Remodeling in Postmenopausal Women
Physical Description: 1 online resource (118 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: black, bone, cimicifuga, menopause, postmenopausal, serum
Nursing -- Dissertations, Academic -- UF
Genre: Nursing Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Postmenopausal osteoporosis affects more than eight million American women leading to an imbalance in bone remodeling, resulting in fragile brittle bones which are susceptible to fracture. There are more than 1.5 million fractures annually, many resulting in increased morbidity and mortality. Every year more than $14 billion is spent on health care for osteoporosis-related fractures. Estrogen replacement has long been known to decrease the bone remodeling that occurs during menopause. Postmenopausal women not taking estrogen replacement are not only at risk for bone loss, but often experience many of the vasomotor symptoms of menopause. The dietary supplement black cohosh is used by many postmenopausal women to decrease or eliminate most of the vasomotor symptoms of menopause. The current mechanism of action of black cohosh is unclear but it is possible that it may have an estrogenic effect on bone. The purpose of this study was to evaluate the effect of black cohosh on bone metabolism, and ultimately to see if black cohosh had an effect on bone that was similar to estrogen. Forty-eight healthy postmenopausal women were recruited for this study. Forty six women completed the study: 23 women in the black cohosh group and 23 in the placebo group. Participants were randomized into a double-blind, placebo-controlled clinical trial evaluating the effect of a standardized 40mg dose of black cohosh given orally once daily. All of the women had serum samples drawn at the onset of the study and again after 12weeks of study medication. Serum samples were analyzed for serum C-terminal telopeptide, a marker of bone resorption; and serum osteocalcin, a marker of bone formation. Analysis of covariates (ANCOVA) conducted on the results of the bone biochemical markers revealed that there was no statistically significant difference in either bone resorption or formation after 12 weeks of black cohosh therapy. In addition, an ANCOVA was conducted to compare onset and conclusion blood pressures, as hypotension is a documented side effect of black cohosh. Results showed that black cohosh had no statistically significant effect on either systolic or diastolic blood pressure. Since estrogen is well-known to decrease serum markers of bone resorption and formation, these findings suggest that, under the conditions of this clinical study, black cohosh lacks an estrogenic effect on bone remodeling.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Jessup, James V.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021928:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021928/00001

Material Information

Title: Effect of Black Cohosh on Biochemical Markers of Bone Remodeling in Postmenopausal Women
Physical Description: 1 online resource (118 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: black, bone, cimicifuga, menopause, postmenopausal, serum
Nursing -- Dissertations, Academic -- UF
Genre: Nursing Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Postmenopausal osteoporosis affects more than eight million American women leading to an imbalance in bone remodeling, resulting in fragile brittle bones which are susceptible to fracture. There are more than 1.5 million fractures annually, many resulting in increased morbidity and mortality. Every year more than $14 billion is spent on health care for osteoporosis-related fractures. Estrogen replacement has long been known to decrease the bone remodeling that occurs during menopause. Postmenopausal women not taking estrogen replacement are not only at risk for bone loss, but often experience many of the vasomotor symptoms of menopause. The dietary supplement black cohosh is used by many postmenopausal women to decrease or eliminate most of the vasomotor symptoms of menopause. The current mechanism of action of black cohosh is unclear but it is possible that it may have an estrogenic effect on bone. The purpose of this study was to evaluate the effect of black cohosh on bone metabolism, and ultimately to see if black cohosh had an effect on bone that was similar to estrogen. Forty-eight healthy postmenopausal women were recruited for this study. Forty six women completed the study: 23 women in the black cohosh group and 23 in the placebo group. Participants were randomized into a double-blind, placebo-controlled clinical trial evaluating the effect of a standardized 40mg dose of black cohosh given orally once daily. All of the women had serum samples drawn at the onset of the study and again after 12weeks of study medication. Serum samples were analyzed for serum C-terminal telopeptide, a marker of bone resorption; and serum osteocalcin, a marker of bone formation. Analysis of covariates (ANCOVA) conducted on the results of the bone biochemical markers revealed that there was no statistically significant difference in either bone resorption or formation after 12 weeks of black cohosh therapy. In addition, an ANCOVA was conducted to compare onset and conclusion blood pressures, as hypotension is a documented side effect of black cohosh. Results showed that black cohosh had no statistically significant effect on either systolic or diastolic blood pressure. Since estrogen is well-known to decrease serum markers of bone resorption and formation, these findings suggest that, under the conditions of this clinical study, black cohosh lacks an estrogenic effect on bone remodeling.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Jessup, James V.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021928:00001


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EFFECT OF BLACK COHOSH ON BIOCHE MICAL MARKERS OF BONE REMODELING IN POSTMENOPAUSAL WOMEN By ALICE PETERS CARLISLE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Alice Peters Carlisle 2

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To my husband Andrew Mitchell Ca rlisle, who supported me without fail. Without his love and support, none of this would have been possible. 3

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ACKNOWLEDGMENTS I would like to thank my committee chair, Dr. James V. Jessup, for his guidance, patience, and continual encouragement. I appreciate him taking on a nurse midwife, even though he wasnt always sure what to do with my midwifery research ideas. I thank Dr. Thomas Wronski for taking on a nursing doctoral student, and teaching me almost everything I now know about bones. I appreciate his extensiv e knowledge of bone metabolism and all the time he spent educating me. I thank Dr. Sharleen Simpson for her extensive knowledge of womens health and for being with me on yet another nursing research committee. I thank Dr. Saunjoo Yoon for her assistance with my study design and for supporting my dietary supplement research. I thank Linda Reilly CNM for giving me a work schedule that allowed me to go to all of my classes and for her unwavering support. In a ddition, I thank all of the midwives, the office manager (Linda Gerds), and all th e wonderful staff at Midwives of Ocala for their patience and understanding of my many absen ces, strange schedule, and nast y moods. Huge thanks go to Hilary Morgan, a true friend and a constant sour ce of encouragement. Even though we started our PhD program at the same time and she gr aduated a year ahead of me, she never stopped helping and encouraging me. I thank Leslie Th orne and Elaine Kaplan, both good friends and great recruiters for my study. I thank my family (husband Andy, son Chris, and daughter Jennifer) for their unwavering love and support during this long and often stressful PhD program. None of this would have been possible without the three of them behind me. Lastly, I thank my lord God for walking with me every step of the way. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 LIST OF ABBREVIATIONS.......................................................................................................... 9 ABSTRACT...10 CHAPTER 1 INTRODUCTION................................................................................................................. .12 Statement of the Problem....................................................................................................... .12 Rationale for Further Research...............................................................................................18 Research Purpose....................................................................................................................20 Research Hypotheses............................................................................................................ ..20 2 REVIEW OF THE LITERATURE........................................................................................21 Introduction................................................................................................................... ..........21 Bone Formation and Structure................................................................................................22 Bone Cells...............................................................................................................................27 Bone Remodeling...................................................................................................................28 Role of Cytokines in Bone Remodeling.................................................................................32 Role of Estrogen in Bone Remodeling...................................................................................34 Effect of Calcium on Bone Remodeling.................................................................................37 Postmenopausal Osteoporosis................................................................................................39 Biochemical Markers of Bone Remodeling...........................................................................42 Effect of Calcium and Vitamin D Supplementation on Biochemical Bone Markers in Postmenopausal Women.....................................................................................................48 Role of Black Cohosh ( Cimicifuga Racemosa )......................................................................49 Adverse Effects of Black Cohosh...........................................................................................55 Standardization of Black Cohosh...........................................................................................56 Future Research on Black Cohosh..........................................................................................58 3 METHODS...................................................................................................................... .......65 Research Design.....................................................................................................................65 Sample....................................................................................................................................65 Measures.................................................................................................................................67 Operationalization of the Variables........................................................................................68 Procedure................................................................................................................................69 5

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Laboratory Tests Protocol...............................................................................................70 Medication Protocol........................................................................................................71 Statistical Analysis........................................................................................................... .......72 Ethical Considerations............................................................................................................72 4 ANALYSIS AND RESULTS.................................................................................................76 Demographic Statistics......................................................................................................... ..76 Research Question.............................................................................................................. ....80 5 CONCLUSIONS AND DISCUSSION..................................................................................87 Discussion of the Findings..................................................................................................... .88 Research Hypotheses............................................................................................................ ..91 Strengths of the Study......................................................................................................... ....92 Limitations of the Study....................................................................................................... ..93 Design Limitations..........................................................................................................93 Statistical Analysis Limitations.......................................................................................96 Conclusions.............................................................................................................................96 Recommendations for Future Research..................................................................................98 Implications for Clinical Practice...........................................................................................98 APPENDIX A Demographic Information Questionnaire.............................................................................100 B Medical History Questionnaire.............................................................................................101 C Certificate of Analysis..103 D Investigational New Drug Number......104 List of references..........................................................................................................................106 BIOGRAPHICAL SKETCH.......................................................................................................117 6

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LIST OF TABLES Table page 2-1. Role of cytokines in bone resorp tion and the influence of estrogen......................................64 3-1. Research Design for a two group study in cluding an experimental group and a placebo group. ...............................................................................................................................74 3-2. Timeline for study participants.......................................................................................... ....74 4-1. Means and Standard Deviations for Age, BMI, Years Postmenopause and Blood Pressure....................................................................................................................... .......82 4-2. Frequency and Percentage on Education...............................................................................83 4-3. Analysis of Covariates on Systolic a nd Diastolic Blood Pressure after Controlling for Age and BMI......................................................................................................................84 4-4. Analysis of Covariates on CTX and OC Levels after Controlling for Age and BMI............85 4-5. Means and Standard Devia tion of Serum Osteocalcin and Cterminal Telopeptide at the Onset and Conclusion of the Study....................................................................................86 7

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LIST OF FIGURES Figure page 2-1 Human femur.. 61 2-2 Haversian system 61 2-3 Osteoclast 62 2-4 Postmenopausal osteoporosis theoretical model. 63 2-5 Black cohosh mechanism of action theoretical model 64 3-1 Conceptual framework of the relationship of the research variables.. 76 8

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LIST OF ABBREVIATIONS BMI Body mass index BMD Bone mineral density CTX C-terminal telopeptide DOH Department of Health DEXA Dual energy x-ray absorptiometry ER Estrogen receptor FDA Food and Drug Administration FSH Follicle stimulating hormone IL Interleukin IRB Institutional Review Board LH Luteinizing hormone LMP Last menstrual period NIH National Institutes of Health NOF National Osteoporosis Foundation NTX N-terminal telopeptide OC Osteocalcin OPG Osteoprotegerin ORS Office of Research Support PTH Parathyroid hormone RANKL Receptor activator of NFB ligand TGF Transforming growth factor beta TNF Tumor necrosis factor alpha 9

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF BLACK COHOSH ON BIOCHE MCIAL MARKERS OF BONE REMODELING IN POSTMENOPAUSAL WOMEN By Alice Peters Carlisle May 2008 Chair: James V. Jessup Major: Nursing Sciences Postmenopausal osteoporosis affects more than eight million American women leading to an imbalance in bone remodeling, resulting in fragile brittle bones which are susceptible to fracture. There are more than 1.5 million fr actures annually, many resulting in increased morbidity and mortality. Every year more th an $14 billion is spen t on health care for osteoporosis-related fractures. Estrogen replacement has long been known to decrease the bone remodeling that occurs during menopause. Postmenopausal women not taking estrogen replacement ar e not only at risk for bone loss, but often experience many of the va somotor symptoms of menopause. The dietary supplement black cohosh is used by many postmenopausal women to decrease or eliminate most of the vasomotor symptoms of menopause. The current mechanism of action of black cohosh is unclear but it is possible that it may have an estrogenic effect on bone. The purpose of this study was to evaluate the effect of black cohosh on bone metabolism, and ultimately to see if black cohosh had an effect on bone th at was similar to estrogen. Forty-eight healthy postmenopa usal women were recruited for this study. Forty six women completed the study: 23 women in the bl ack cohosh group and 23 in the placebo group. Participants were randomized into a double-blind, placebo-controlled clinical trial evaluating the 10

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effect of a standardized 40mg dose of black c ohosh given orally once daily. All of the women had serum samples drawn at the onset of the st udy and again after 12wee ks of study medication. Serum samples were analyzed for serum C-termin al telopeptide, a marker of bone resorption; and serum osteocalcin, a marker of bone formation. Analysis of covariates ( ANCOVA) conducted on the resu lts of the bone biochemical markers revealed that there was no statistically significant differen ce in either bone resorption or formation after 12 weeks of black cohosh therapy. In addition, an ANCOVA was conducted to compare onset and conclusion blood pressures, as hypotension is a documented side effect of black cohosh. Results showed that black cohosh had no statistically significant effect on either systolic or diastolic blood pre ssure. Since estrogen is well-known to decrease serum markers of bone resorption and formation, these findings suggest that, under the conditions of this clinical study, black cohosh lacks an estroge nic effect on bone remodeling. 11

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CHAPTER I INTRODUCTION Statement of the Problem Osteoporosis is a condition in which bone loss oc curs leading to bone that is fragile and at increased risk for life-threatening fractures. Osteoporosis is a major health condition which by current estimates affects over 10 million Americans, 80% of them postmenopausal women (National Osteoporosis Foundation, 2005). In add ition, 34 million Americans have osteopenia, or low bone mass, which puts them at increased risk for developing osteoporosis and related fractures in the future. Every year osteoporosis is estimated to be responsible for more than 1.5 million fractures, including 300,000 fractures of the hip, 700,000 fractures of the spinal vertebrae, and 250,000 fractures of the wrist (N ational Institutes of Health, 2005). Costs associated with osteoporosis and the resulting fractures are staggering, with estimates in the year 2002 at $18 billion (NOF, 2005). In addition, fractures often result in a change in lifestyle and quality of life, with hip fractures resulting in nursing home admissions, impairment in mobility, and death. The National Institutes of Health (NIH, 2005) estimates th at 1 out of 5 patients with a hip fracture dies in the y ear after the fracture. Loss of bone mass, resulting in weakened bones w ith a potential for fracture, is thought to be from an imbalance in the bone remodeling pr ocess. Bone remodeling is the process of rebuilding new bone and repairing existing bone. Remodeling occurs throughout the skeleton at many different sites, and is ongoing throughout the life span (Jee, 1988). Bone remodeling begins with bone resorption, the removal of old bone, and is completed with bone formation, the laying of new bone matrix. Bone loss, due to an imbalance in remodeling where resorption occurs at a higher rate then formation, occurs approximately 10 years earlier and occurs two times faster in women than in men (Jee). The loss of endogenous estrogen which occurs in the 12

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postmenopausal period results in a change in the remodeling process causing an increase in bone resorption and a smaller increase in bone formati on, resulting in an imba lance in the remodeling process. Menopause is broadly defined as the final mens trual period and the termination of ovarian function, and is made retrospec tively after the woman has been without a menstrual period for one year (Dawood, 2000). The postmenopausal peri od is the time in a womans life after the menopause has been completed. Recent estimates are that nearly 46 million women in the United States are experiencing or completing the menopause transition, and that number is expected to increase as the population ages (The North American Menopause Society, 2001). Menopause is a natural multisystem transition that usually takes 3 to 5 years to complete and is often referred to as the perimenopause or the climacteric. As a woman enters the menopause transition there is a d eclining level of estr adiol, the predominant female estrogen produced by the theca and gra nulosa cells of the female ovary (Jones & DeCherney, 2005). Vasomotor symptoms such as hot flashes, hot flushes, and night sweats as well as insomnia, forgetfulness and irritability are all well known symptoms of the menopause (Whiteman, Staropoli, Benedict, Borgeest, & Flaws, 2003). In addition to the vasomotor symptoms associated with the menopausal years, the decrease in estrogen also causes an imbalance in the bone remodeling process which results in incr eased resorption and a smaller increase in formation. Increased bone remodeling causes a loss of bone mineral density and an increased fragility of the bones, specifically cancellous bone, resulting in an increas ed risk of fractures (Akesson, 2003). As a result of living longer, women will spend approximately one-third of their lives in the postmenopausal period, and therefore increase their risk of osteoporosis and fractures. 13

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Hot flashes, a defining characteristic of th e menopause, are the primary reason women in this phase of their life seek me dical attention. It is estimat ed that 40% to 70% of early menopausal women experience hot flashes, near ly 4 to 5 million women in the United States alone, and may continue to experience them fo r up to 30 years after the menopause transition (Whiteman et al., 2003). Hot flashes are th e most common reason that women try estrogen therapy, and research has shown that estrogen re placement therapy alleviates up to 80% -90% of the vasomotor symptoms of menopause (Shana felt, Barton, Adjei, & Loprinzi, 2002). Synthetic estrogens were discovered in 1938 and by the 1950s clinical studies were developed to test their effectiveness and use in postmenopausal women (Ferguson, 2004). In addition to the relief of vasomotor symptoms, estrogen was shown to help prevent bone loss by inhibiting osteoclastic activity, stimulating co llagen synthesis, and decreasing osteoporosisrelated hip and vertebral fractur es by 50% and 90% respectively (Notelovitz, 2003). Estrogens are able to move in and out of cells by simp le diffusion and their ac tion is dependent on the presence of an estrogen receptor in the nucleus of the targeted cell (Barrett, 2005). Bord and colleagues (2001) found the presence of estrogen receptors in both cortical and cancellous bone. The degree of the effect of estrogen on the bone varies with the dosage and route of administration of the estrogen used. Estrogen replacement therapy was widely accepted as a treatment for menopausal symptoms, and as a method of preventing the development of osteoporosis. The Womens Health Initiative Study, published in 2002, revealed an incr eased risk of developing breast cancer, stroke, non-fatal myocardial infarction, pulmonary embolus, and deep vein thrombosis. The positive results of the study revealed a decrease in the risk of oste oporosis-related fractures and colorectal cancer, both statistically significant results. 14

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The publication of the Womens Health Ini tiative Study, and the resulting media frenzy, spurred postmenopausal women to discontinue their estrogen, and to look into other alternatives with their health care providers (Writing Group fo r the Women's Health Initiative Investigators, 2002). Several other treatment options are available fo r osteoporosis. These include the use of the selective estrogen receptor modulators (SERMs) the bisphosphonates, parathyroid hormone, calcitonin, and the phytoestrogens. These treatments, like estroge n, are aimed at countering the imbalance in the bone remodeling process. All of the above treatments work to decrease bone resorption except parathyroid hormone, which increases bone formation (Akesson, 2003). Selective estrogen receptor modulators (SERMs) such as Raloxifene and Tamoxifen are commercially available with a prescription and are taken daily. Tamoxifen is primarily used to treat breast cancer, not osteopor osis, though it has been shown to have a beneficial effect of decreasing bone resorpti on (Notelovitz, 2003). The disadvantage associated with Tamoxifen is that studies have shown that it has a detrimental effect on th e endometrium of the uterus. Raloxifene has been shown to prevent postmenopausal bone loss and reduce vertebral fractures by 30%-50%, as well as reducing biochemical markers of bone resorption and formation (Notelovitz). Bisphosphonates such as Alendrona te, Risedronate, and Ibandr onate are all commercially available with a prescription, a nd have the convenience of dail y, weekly, and monthly dosing. Bisphosphonates mechanism of action is that th ey prevent bone resorption by binding to hydroxyapatite, inhibiting osteoc last activity and preventing osteoblast apoptosis (Eqbal, Inzerillo, Moonga, & Zaidi, 2003). The advantage of the bisphosphonates is that studies have shown a decrease in bone reso rption through the use of biochemical bone markers and dual 15

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energy X-ray absorptiometry (DEXA) scans an d a reduction in the fr acture rate. The disadvantages are the side eff ects such as esophageal irritatio n, the low rate of intestinal absorption, and the fasting state that is necessa ry both before and after dosing (Akesson, 2003). Parathyroid hormone, commercially available by prescription as Teriparatide, is the only approved osteoporosis treatment which increases bone formation. Low intermittent doses of parathyroid hormone can cause growth hormone to increase, stimulate osteoblast activity, and reduce osteoblast apoptosis, thereby increasing bone mineral density (Notelovitz, 2003). Teriparatide has been shown to reduce new ve rtebral fractures by 65%-69% and non-vertebral fractures by 53% (Akesson, 2003). The disadvantag e of Teriparatide is that it must be administered by subcutaneous (SC) injection daily. Calcitonin is commercially available as both a nasal spray and by SC injection. The most common is Miacalcin, a nasal spray commercially av ailable by prescription. The mechanism of action of calcitonin is that it decreases bone remodeling by interfer ing with the attachment of the osteoclast to the bone surface, and it interferes with the ability of the osteoclasts ruffled border to function properly (Mehta, Mal ootian, & Gilligan, 2003). Calcitoni n has also been shown to be beneficial in controlling bone pain that occurs in seve ral diseases in addition to osteoporosis, such as osteoarthritis, bone can cer, and Pagets disease (Mehta, Malootian, & Gilligan). The disadvantage of calcitonin is th at is produces nasal symptoms such as discharge, congestion, bleeding, and sores. Phytoestrogens are plant compounds available as dietary supplement s that have estrogenlike properties and are available commercially without a prescription. Intere st in phytoestrogens has increased as women are looking for more natura l alternatives without th e risk of serious side effects. Numerous herbal products are available commercially without a physicians 16

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prescription, and the sale of herbal products is a multibillion dollar business in the United States. In the United States alone there was a 380% incr ease in the use of herbal supplements from 1990 to 1997, with spending in 1997 on herbal suppleme nts estimated at 5.1 billion dollars (Mahady, 2001). In the United States it is estimated that Americans pay out of pocket $10.3 billion annually for a variety of alternat ive therapies such as acupuncture, massage, chiropra ctic services and herbal dietary supplements (Eisenberg et al., 1993). It is only within the last twenty years that clinical research has evaluated the effec tiveness and potential harmful effects of dietary supplements. Phytoestrogens are plant derived supplements that have estrogen-like properties. The phytoestrogens usually consists of three main classes: the isofla vones, lignans, and the coumestans (Turner, Rickard, Spelsbery, & Sibonga 2003). Phytoestrogens are believed to exert their estrogen-like effect by binding to th e two subtypes of estrogen receptor, ER and ER but preferentially to the ER and producing an estrogen like eff ect in the body similar to, but less potent than, estradiol (Kuiper et al., 1998). Several studies evaluating the effect of phytoestrogens on the primary complaint of the menopause, the hot flash, have shown conflicting results. The difficulty with evaluatin g the effect of the phyt oestrogens is that phytoestrogen products are often unr egulated, specifically in the United States, and may contain numerous different ingredients su ch as two or more phytoestrogens and a variety of fillers. Several phytoestrogens belonging to the isof lavones category such as genistein and daidzein found in soy products, have been found to have bone protective properties in ovariectomized rats, an animal model for postm enopausal bone loss (Setchell, 1998). A study comparing the soy product genistein with Raloxi fene and estradiol in vitro on cultured rat bone marrow cells showed all products decreased th e number of osteocla sts present with no 17

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statistically significant difference in osteocla st number between the groups (Sliwinski, Folwarczna, Janiec, Grynkiewicx, & Kuzyk, 2005). Research continues on the many variations of soy products currently available as research ers look for alternative methods for treating and preventing osteoporosis. Black cohosh is a perennial plant found in th e eastern United States and Canada and is formally called Cimicifuga racemosa or Actaea racemosa Black cohosh was first mentioned in writing in 1749 by Carl von Linne as a treatment for female problems such as dysmenorrhea and menopausal complaints (Upton, 2002). Black cohosh has been used extensively in Germany for many years, and more recently across the globe for the relief of the hot flashes associated with menopause. It has been suggest ed that since the he rb has the estrogenic activity necessary to relieve hot flashes, it may also have a sim ilar estrogenic activity on bone, a decrease in bone remodeling. Several studies using rat models and biochemical markers of bone resorption and formation have shown promising evidence th at black cohosh can reduce bone remodeling (Niblein & Freudenstein, 2003; Seidlova-Wuttke et al., 2003; Wuttke et al, 2003). Wuttke and colleagues (2006) found that the use of black cohosh in postmenopausal women in Germany induced an increase in osteoblast activity and further studies were r ecommended. A review of the literature shows a need for more randomized double blind, placebo-controlled clinical trials of the herb black cohosh to evaluate the e ffect it has on bone metabolism and remodeling in humans. Rationale for Further Research Osteoporosis is a condition which research has shown is treatable and possibly preventable. It does not have to be the end result of the aging process for postmenopausal women. Current pharmacological treatments exist which can prev ent the bone loss that occurs with osteoporosis, and the resulting fractures. Estrogen, bisphosphonates, and SERMs are 18

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currently well accepted treatment options. However, women are also looking for more convenient, natural and less ri sky treatment options such as over the counter dietary supplements. Americans are using more dietary supplements, however little clinical evidence exists to substantiate claims made by the companies ma rketing these products. Recently, black cohosh has been shown clinically to relieve the vaso motor symptoms of the menopause. The exact mechanism of action of black cohosh remains unclear. However, it has been shown that phytoestrogens do bind with estrogen receptors in the targeted cell, and produce estrogenic-like activity. It is possible that black cohosh may activate an unknown receptor to produce the positive effect on vasomotor symptoms. Bone has estrogen receptors and may be a site for the estrogen-like effect of black c ohosh to exert an influence. Fe w studies have been done which address the effect black cohosh may have on bone remodeling and most are in animals. Only one published study known to this researcher has addressed the possible effect on bone remodeling in postmenopausal women and further te sting was called for. It is clear that well designed studies are needed to address the e ffect of black cohosh on bone remodeling. In conclusion, millions of women are using ove r the counter dietary supplements and black cohosh is one of the most popular supplements in postmenopausal women. Women often look for a more natural option when attempting to trea t and deal with the symptoms associated with menopause. It is well known by health care providers that bone remodeling increases in postmenopausal women after estr ogen withdrawal. However, few postmenopausal women are concerned with their bone health. Bone h ealth should be an important issue for both postmenopausal women and their health care providers. Postmenopausal women have been shown to have an increased and an imbalanced bone remodeling which causes a loss of bone 19

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mass. This loss of bone mass which results in fragile bones makes women more prone to fractures of the hip, vertebrae, and wrist. The increased morbidity and mortality of osteoporotic fractures is unacceptable if treatm ent options exist. However, as previously stated the women themselves are looking for altern ative options. It is imperativ e that herbal products undergo more rigorous testing, and the results be made available to women so they may make an informed and educated decision. Future research into black cohosh should investigate the effect on bone health, long term usage, and adverse effects. Research Purpose The purpose of this study was to determine if a standardized (2.5% tr iterpene glycoside) commercial preparation of the herb black cohosh will alter bone remodeling in the postmenopausal female. Bone remodeling is a two step physiologic process which includes bone resorption and bone formation, and can be measur ed with commercially available biochemical bone markers. A biochemical marker of bone re sorption, serum C-terminal telopeptide (CTX), and a biochemical marker of bone formation, seru m osteocalcin, were measured at the initiation of the study and again after 12 weeks of th erapy with the black cohosh supplement. Research Hypotheses Hypothesis 1: Postmenopausal women taking a sta ndardized 40 mg (2.5% triterpene glycoside) dose of an oral black cohosh supplement daily for 12 weeks will show a decrease from baseline in the level of serum C-terminal telopeptide, a biochemical marker of bone resorption. Hypothesis 2: Postmenopausal women taking a sta ndardized 40 mg (2.5% triterpene glycoside) dose of an oral black cohosh supplement daily for 12 weeks will show a decrease from baseline in serum osteocalcin, a biochemical marker of bone formation. 20

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CHAPTER 2 REVIEW OF THE LITERATURE Introduction In the United States it is estimated th at over ten million Americans suffer from osteoporosis, with 80% of those Americans being postmenopausal women (National Osteoporosis Foundation, 2005). In addition, appr oximately 28 million more Americans are estimated to have low bone mass, which puts them at increased risk for developing osteoporosis (National Institutes of Health, 2000). The estimat ed costs associated with osteoporosis and the resulting fractures in 2002 were $18 billion, and that number is expected to increase as Americans grow older and live longer (NOF, 2005). The risk of a woman developing osteoporosis and experiencing a fracture increas es during the postmenopausal years, and with advancing age. Nearly 30 million American women are menopausal, and that number is expected to rise as large nu mbers of women born during the baby boom generation are entering and completing the menopause transition (The roux & Taylor, 2003). Osteoporosis is the underlying cause for over 1.5 million fractures a year including fractures of the hip, vertebrae, and wrist (NOF, 2005). Fractures that result from osteoporosis are associated with increased morbidity and mortality, with statistics reveal ing 20% of Americans with a hip fracture will die within one year, 28% will requir e long-term care, and 50% will be unable to walk without the assistance of a walker (NIH, 2005). These alarming figures present the need for future research to thoroughly understand the pathophysiology of osteoporosis, deve lop better treatments, and fina lly to better educate health care providers and the public about the risk factors and lifestyle choices which can prevent, or accelerate, the bone loss that oc curs with osteoporosis and it s precursor osteopenia. The fractures of osteoporosis and the resulting increas e in morbidity and mortality can and should be 21

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prevented. Osteoporosis and bone fractures do not necessarily have to be a consequence of aging. Bone is living dynamic connectiv e tissue which has the ability to constantly repair and rebuild itself. It also provides structure for the body, acts as a reservoir for important minerals such as calcium and phosphorus, and contains specia lized tissue in its core for the production of blood cells essential for human life (Ferguson, 2004). Bone tissu e responds to a variety of stimuli, both internal and external. Therefore treatments for a bone condition such as osteoporosis are varied. Lifest yle changes such as exercise, appropriate diet, discontinuing smoking and heavy alcohol use, and supplementing the diet with the appr opriate vitamins and minerals are all external stimuli proven to affect bone tissue. In ternal stimuli such as hormones, cytokines, and mechanical stress also affect bone tissue. To fu rther osteoporosis research an understanding of the physiology of bone, the pat hogenesis of postmenopausal osteoporosis and the current and future treatment options is necessary. Bone Formation and Structure The human body is composed of 206 bones whic h come in two different architectural forms. Cortical or compact bone forms the outer layer of all bones and accounts for 80% of the human skeleton. Cortical bone is dense and solid, and is the main component of long bones such as the humerus and femur (Barrett & Barrett, 200 5). Trabecular or cancellous bone accounts for the remaining 20% and is present in the interior of the long bones and the vertebrae. Trabecular bone is not solid and dense, but is instead composed of a series of lattices and arches (Barrett & Barrett). Though trabecular bone accounts for only 20% of the bone in the human skeleton, the lattice like structure allows fo r a higher surface to volume ratio, and a bone turnover rate much higher than cortical bone (Barrett & Barrett). 22

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Bone is developed through a process calle d ossification, and there are two types of ossification: intramembranous and endochondral (S aladin, 2004). The flat bones of the skull, parts of the mandible, and the cl avicle or collarbone are formed by intramembranous ossification (Jee, 1999). The bones of the arms, legs, vertebrae, and the pelvic bones specifically a large majority of cancellous bone, are formed by endochondral ossification. Intramembranous ossification occurs without form ation of a cartilage scaffold prior to mineralization, whereas endochondral ossification utilizes a hyaline cartilage model for formation (Jee, 1988). Intramembranous ossification begins duri ng the embryonic period when mesenchymal cells develop into a sheet of soft tissue with a large supply of blood vessels. Cells from this sheet of mesenchymal connective tissue differentiate in to osteogenic cells and form a collection of trabeculae (Saladin, 2004). The osteogenic cells continue to develop near the newly forming trabeculae and become osteoblasts. The osteoblasts deposit osteoid tissu e, and the trabeculae begin to connect and develop the primary s pongiosa, or cancellous bone (Jee, 1988). The osteoblasts continue to deposit bon e within the primary spongiosa, filling in the spaces, and the initial cancellous bone becomes compact or cortical bone. The endosteum is formed when the mesenchymal cells and the inactive osteoblasts on the endosteal inner surface become the endosteum. The periosteum is formed when th e connective tissue remaining on the outer surface condenses and becomes fibrous (Saladin). The large supply of blood vessels within the cancellous bone will eventually become the ha ematopoietic tissue of the bone marrow (Sims & Baron, 2000). Endochondral ossification begins with the form ation of a cartilaginous model or scaffold of the future bone. Mesenchymal cells develop into chondroblasts which secrete a cartilage-like matrix. The chondroblasts become surrounded by newly formed matrix and then become 23

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chondrocytes. The chondrocytes, or cartilage cells, continue to gr ow and eventually the matrix calcifies (Sims & Baron, 2000). Calcification begins in the center of the cartilage scaffold and progressively moves to the ends of the bone as row after row of layered chondrocytes deposit a matrix which then calcifies. After calcification of the cartilage matrix, blood vessels bring osteoclasts and osteoblasts into the center of the cartilage sca ffold which replace the cartilage with woven bone. This woven bone will then be replaced with lamellar bone, the main form of bone in the adult skeleton (Jee, 1999, Sims & Baron). Eventually two architecturally and functionally different types of bone are formed in the human skeleton, cortical or comp act and trabecular or cancellous. Cortical bone accounts for 80% of the bone of the human skeleton and cancellous bone accounts for only 20% of the skeleton (Barrett & Barrett, 2005). Cortical and cancellous bone are functionally different, with cortical bone providing protection of the soft internal organs and mechanical support, and cancellous bone providing the metabolic functions (Marks, Jr. & Hermey, 1996). Architecturally, cortical bone is solid and dense and cancello us bone is composed of lattices, arches, and plates. Cortical bone forms the outer su rface of bone and is solid and dense. The majority of cortical bone is in the shaft of the long bones. Cortical bone is composed of bone units called osteons, which consist of a central Haversian canal and surrounding lamellae (Jee, 1988). In cortical bone the osteon is 200 m in diameter and 10-20 mm l ong with surrounding concentric lamellae 3-7 m thick (Athanasiou, Zhu, Lanctot, Agrawal, & Wang, 2000). Cortical bone accounts for 33% of bone surface and has a slower bone turnover rate then cancellous bone (Jee, 1999). 24

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Cancellous bone is in the inte rior portion of bone and consis ts of a series of connecting trabeculae which form cavities that are then o ccupied with bone marrow (Jee, 1988). Cancellous bone has a lattice-like appearance, is very porou s, and accounts for 67% of bone surface. This lattice-like structure and the larger bone surf ace accounts for a higher surface to volume ratio, eight times higher than cortical bone (Jee, 1999). Since bone re modeling takes place on the bone surface, cancellous bone has a higher turnover rate. Trabeculae are usually approximately 100300 m thick and spaced approximately 300-1,500 m apart (Athanasiou, Zhu, Lanctot, Agrawal, & Wang, 2000). Bones do not contain cortical and cancellous bone equally. Prime examples are the long bones such as the femur, humerus, and the ulna compared with the vertebrae of the spine. A long bone consists of three distinct areas: diaphysis, meta physis, and the epiphysis. The diaphysis is the central shaft of the bone and is composed primarily of cortical bone. The two ends of the bone, the epiphyses, are composed pr imarily of cancellous bo ne with a covering of cortical bone. The metaphysis, the portion of the bone which connects the epiphysis to the diaphysis, is also primarily cance llous bone with a cortical bone shell (Jee, 1999). The ulna, a long bone in the forearm, is 92% cortical bone an d 8% cancellous bone comp ared with the spinal vertebrae which is 62% cortical and 38% cancellous bone (Jee). All bone, whether cortical or cancellous, is co mposed of multiple layers of lamellae, and a system of connecting channels and canals. Lame llated bone is composed of collagen fibers that are arranged in either a parallel (cancellous) or concentric (cortical) pattern, which assures the highest density of collagen per unit, making lamellated bone mu ch stronger than woven bone (Sims & Baron, 2000). Lamellated bone replaces woven bone at approximately two to three years of age (Jee, 1999). 25

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In cortical bone there are three distinct la mellae patterns: circular circumferential, and interstitial (Jee, 1988). Circul ar rings of lamellae surround a cen tral canal called the Haversian canal containing nerves and vessels responsible for carrying nutrients from the bone marrow and periosteum to cortical bone. Connecting one Haversian canal to another are canals called Volkmanns canals, which are differentiated from Haversian canals by their lack of surrounding lamellae (Jee). Lacunae, small spaces containing an osteocyte, are consistently spaced throughout the lamellated tissue and are connected by channels called canaliculi. This arrangement of lacunae, canals, and channels make it possible for the bone tissue to have access to a constant supply of essential nutrients and fluids The entire system is referred to as either an osteon or Haversian system. Ci rcumferential lamellae are on th e outer and inner surfaces of cortical bone beneath the perios teum and endosteum, respectively, and can extend uninterrupted around the Haversian systems. Interstitial lamell ae are remains of circular and circumferential lamellae which fill the spaces between the Haversian systems (Jee). Cancellous bone consists of trabecular packets and interstitial lamell ae. The trabecular packet is comparable to the osteon or Haversian system. The trabecular packets are sheets of parallel lamellae layered one atop the other form ing a series of interconnected trabeculae. Interstitial lamellae fill the spaces in the trab eculae packet. The spaces between the trabecular packets are occupied by bone marrow. Osteocyt es receive nutrients and fluid via diffusion through canaliculi which extend from the osteoc yte to the trabecular surface (Jee, 1988). Bone is composed of approximately 60% mineral, 30% collagen matrix, and 10% water (Athanasiou et al., 2000). Bone mineral, in the form of small crystals, is situated in and around the collagen fibers and is primarily hydroxyapati te. Hydroxyapatite, an inorganic salt composed primarily of calcium, phosphorus, and a hydroxide, is responsible for the hardness and strength 26

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of the bone (Jee, 1999). Mineralization occurs when hydroxyapatite crystals attach to the collagen matrix and are bound together by specific binding proteins, developing into mature bone (Ferguson, 2004). Mature bone is the main reservoir for calcium in the human body, with 99% of the calcium found in bones (Ferguson). Th e organic matrix consists of type I collagen and several other non-collagenous pr oteins. Ninety percent of th e organic material of bone is type I collagen and the remaining ten percen t are the non-collagenous proteins such as osteocalcin, osteonectin, a nd osteopontin (Jee). Bone Cells Four types of bone cells are most recognized as being important to bone metabolism. The four cells are: bone lining cells osteoclasts, osteoblasts, and os teocytes (Jee, 1988). Bone lining cells are attached to mature inactive bone, and ar e thought to be derived from osteoblasts that have become inactive. They are flat elongated cells attached directly to the bone surface. Their precise role on the cell surface is unclear, but thes e cells are thought to play a role in calcium homeostasis (Jee). Osteoclasts are large, multinucleated bone cel ls which are responsible for bone resorption (Jee, 1988). Osteoclasts are derived from haemat opoietic stem cells which originate from the phagocytic cell line (Ferguson, 2004). Osteoclasts line the surface of bone, though they are not actively resorbing bone all the time. Activation of osteoclasts must occur, and several substances such as parathyroid hormone, vitamin D, and pr ostaglandins have been proven to activate osteoclasts to begin resorption (Ferguson). The osteoclast forms a seal on the bone surface called the sealing zone, allowing the ruffled border to begin resorption of bone, forming a crater called a Howships lacuna (Jee). Resorption of calcified bone and creation of the Howships lacuna is possible because the osteoclast is equipped with specialized sodium pumps, ion exchangers, and specialized lysosomal enzymes (Sims & Baron, 2000). After the formation of 27

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the Howships lacuna, the osteoblasts move in to begin the bone formation phase of the remodeling process. Osteoblasts are the cells res ponsible for bone formation and they secrete a bone matrix which contains collagen and mucopolysaccharid es (Ferguson, 2004). Type I collagen accounts for 90% of the bone matrix secreted by the osteoblast. Osteoblasts secrete numerous noncollagen proteins such as osteocalcin, osteone ctin, and prostaglandins, with osteocalcin the predominant protein. Osteocalcin is responsib le for one percent of the bone matrix and potentially plays a role in calcium binding and mineralization (Sims & Baron, 2000). Serum osteocalcin is currently used as a biochemical mark er for osteoblast activity. As with osteoclasts, osteoblasts also contain receptors for substanc es such as parathyroid hormone, vitamin D, prostaglandins, glucocorticoids, and insulin (J ee, 1988). Osteoblasts are cuboidal in shape, attached to the bone surface in a single layer, and possess microtubules allowing the osteoblasts to communicate with each other and receive nutrients essential for cell life (Ferguson). Osteocytes are mature bone cells and originat e from osteoblasts. A single osteocyte is present in a lacuna surrounded by newly formed bone, and communi cates with other osteocytes via gap junctions (Jee, 1988). The osteocyte has a number of channels wh ich allow the flow of nutrients, fluids and wastes among several oste ocytes (Ferguson, 2004). In addition, osteocytes are strain sensors and when a bone is strained the osteocyte is thought to send a message to the osteoblasts on the surface to begi n forming bone (Saladin, 2004). Bone Remodeling Bones are constantly changing to meet the needs placed on them by humans. The process of building new bone and repairing existing bo ne is called remodeli ng. Remodeling occurs throughout the skeleton at many diff erent sites and is ongoing. Bone remodeling is the job of a specialized group of cells called bone remodeli ng units, which are present on the surface of 28

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trabecular bone and in resorption tunnels in cortical bone (Jee, 1988). The major purpose of the bone remodeling unit is to replace the old bone w ith new, through resorption of the old bone and formation of new bone. Bone remodeling should be in constant balance with formation equaling resorption. When the balance of bone remodeling is skewed in one direction or the other, disease processes occur, such as osteoporosis when resorption exceeds formation and osteopetrosis where formation exceeds resorption (Key & Ries, 2002). Bone remodeling occurs differently in cortical bone than trabecular bone. Cortical bone remodeling is done through a resorption tunnel with in bone. The osteoclasts form a cone shaped resorption tunnel by removing the existing bone and Haversian system. The resorption tunnel is composed of three zones: cutting zone, reversal zone, and closing zone (Jee, 1988). The cutting zone is composed of a layer of osteoclast s which form a space called a resorption cavity. Immediately behind the cutting zone is the reversal zone. The reversal zone is an area where the osteoclasts have completed the process of resorp tion, but formation has not yet started (Jee). The resorption tunnel is then closed by a layer of osteoblasts forming a new layer of mineralized bone tissue, thereby closing the reso rption tunnel. A new Haversian system with new bone tissue has now replaced the old Haversian system (Jee). In trabecular bone the resorption process is faster and occurs on the bone surface (Ferguson, 2004). There are two proposed t ypes of trabecular bone remodeling, one where formation does not begin until resorption is comp leted, and another in which formation begins while resorption is still occurring (Jee, 1988). In trabecular bone remodeling a pit or lacuna is produced on the bone surface by the osteoclasts, then osteoblasts replace the osteoclasts, and finally new bone is formed (Riggs & Melton, 1992). 29

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Remodeling begins when the osteoclasts have been activated and are in place on either the trabecular bone surface or in the interior of cortical bone. The exact mechanism for the activation of osteoclasts and the site selected fo r the remodeling process remains unclear. It is hypothesized that microfractures in the bone alte r the osteocytes around the fracture, and these osteocytes send a signal to the osteoclast pr ecursors (Vaananen, 2005). It has also been suggested to work in the opposite manner with he althy osteocytes sendin g a signal to inactivate osteoclasts, and when this signaling mechanism stops the osteoclasts begin to move toward that area to begin the remodeling process (Vaananen). The answer may be a combination of factors or one specific factor, but fu rther studies are called for. Once a signal has been received, osteoclasts move toward the area for remodeling and a resorption zone is established. Three separate do mains are recognized in os teoclasts: the ruffled border, the sealing zone, and a f unctional secretory domain. The ruffled border is the actual area where the breakdown of the hydroxyapatite surface and the collagen occurs, and its formation is the first step in the resorpti on process (Vaananen, 2005). The s ealing zone is responsible for anchoring the osteoclast to bone. The functional secretory domain is responsible for ridding the cell of the byproducts of bone degradation. The byproducts of degradation are moved through the cell by endocytosis and transported by a transc ytotic pathway, where they are then secreted into the extra-cellular fluid (Wang, Miller, Kopeckova, & Kopecek, 2005). Once a sealing zone and a ruffled border are established HCL acid is released onto the surface of the bone and resorption begins. In order for the osteoclasts to have a steady supply of HCL, proton pumps and chloride channels are present in the cell membrane. The hydrogen ion is provided via the carbonic anhydr ase reaction converting carbonic acid to H+ and bicarbonate. The bicarbonate is exchanged for chloride throu gh a bicarbonate/chloride exchanger located in 30

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the membrane of the osteoclast. Mitochondria l-provided ATP pumps the H+ ion toward the ruffled border where it combines with the chloride ion forming HCL. The HCL then begins the process of breaking down the hydroxyapatite surf ace and collagen fibers (Vaananen, 2005). The byproducts of bone resorption must be remove d from the cell in order for resorption to continue. Cathepsin K, a protease, is respons ible for digesting the calcium, phosphate and collagen fibers produced by the resorption process. These byproducts are then removed from the cell and secreted into the extra-ce llular fluid (Wang et al., 2005). Once resorption has been completed the osteocla sts disappear leaving a reversal zone. The edges of the resorption pit are then smoothed over and a cement line is formed (Jee, 1988). The next step is new bone formation a nd that is the function of the os teoblasts. Bone formation is a multi-step process and the first step is the formation of the bone matrix, the osteoid. The osteoblasts secrete a number of substances in the production of the bone matrix. These substances include osteocalcin, type-I procollagen peptides, a nd bone specific alkaline phosphatase (Srivastava et al., 2005). After the bone matrix is deposited, mineralization begins and is completed in two steps: primary and s econdary mineralization. Primary mineralization occurs approximately 5-10 days after hydroxyapati te crystals are deposited between the organic matrixes. Secondary mineralization is a much slower, gradual process and increases the bone mineral density by increasing the number of hydroxya patite crystals and th e crystal size, but not the volume of the new bone (Boivin & Meunier, 2002). Bone formation and bone resorp tion are usually well balance d, or coupled in the adult providing they are healthy, have good nutrition, are active, and exer cise. Bone remodeling is a continuous process and necessary for normal bone growth and repair. Bone mass in males and females, peaks at approximately 30 years of age. Normal healthy adults will usually lose a small 31

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amount of bone throughout their lives, a bout 0.4% per year after age 30. However, postmenopausal women have increased bone loss secondary to an imbalance in bone remodeling, and lose approximately 1% to 2% of bone a year for the first 5-8 years if there is no intervention to prevent bone loss (Srivastava et al., 2005). Bone loss occurs when bone resorption exceeds bone formation. This is exactly what occu rs in the condition known as postmenopausal osteoporosis. The Role of Cytokines in Bone Remodeling Cytokines are small size proteins secreted by several different type s of cells such as macrophages, leukocytes and mast cells (Salad in, 2004). Cytokines have many roles in the human body, with the regulation of bone metabolism being only a small part of their function. Interleukins, tumor necrosis factor and transforming growth factor are the cytokines most often involved with bone metabolism. Receptor activator of NFB (RANK) and receptor activator of NFB ligand (RANKL) are members of the tumor necrosis factor (T NF) family, and play a part in osteoclast differentiation and osteoclast resorption respectively (Eqbal, Inzerillo, M oonga, & Zaidi, 2003). RANKL is produced by osteoblasts and stroma l cells and is necessary for osteoclast development. RANKL stimulates bone resorptio n by interacting with th e RANK receptor on the surface of the preosteoclast. RANKL can also bind to the decoy receptor osteoprotegerin, which inhibits osteoclast formation and bone reso rption (Khosla, 2001; Reddy & Roodman, 2004). Osteoprotegerin, a glycoprotein produced by os teoblasts, blocks the differentiation of the osteoclast by antagonizing RANKL and interfering with the signa ling pathway necessary for cell differentiation (Blair, Robinson, & Zaidi, 2005). A fine balance between the effects of RANKL and osteoprotegerin (OPG) is necessary to co ntrol the bone resorption process. Several substances have been identified as having either a stimulatory or inhi bitory effect on RANKL 32

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and OPG. TNF IL-1, and vitamin D increase the expres sion of RANKL. Parathyroid hormone (PTH) and glucocorticoids increa se the expression of RANKL a nd decrease the production of OPG, which promotes osteoclastogenesis and bon e resorption (Khosla, 2001; Rubin et al., 2002). TGFincreases production of OPG and stimulates RANK, and estrogen increases production of OPG and interferes with RANKL signaling, th ereby inhibiting osteoc lastogenesis (Khosla; Rubin et al.). TNF and are both potent stimulators of bone resorption, are produced by osteoblastlike cells, and their release appears to be in fluenced by the presence of estrogen (Reddy & Roodman, 2004). Turner and co lleagues (1994) found that estroge n inhibited the release of TNF in cultured bone cells in vitro. When es trogen was reduced, osteoclast numbers were increased, and when estrogen was replaced the osteoclast number decreased. Interleukin 1 (IL-1) and Interleukin 6 (IL-6) are potent stimulators of bone resorption, produced by a variety of cells such as monocytes, macrophages, and stromal cells, and their effects appear to be mediated by estrogen (Reddy & Roodman, 2004). Experiments done on transgenic mice in which the IL-6 gene was elim inated showed that mice without the IL-6 gene showed no bone loss after removal of the ovaries as opposed to the normal mice which showed considerable bone loss after ovariectomy (Turne r, Riggs, & Spelsberg, 1994). IL-6 stimulates osteoclast differentiation, and its expression may be stimulated by IL-1, parathyroid hormone (PTH), TNF and vitamin D, while estroge n inhibits its expression (Eqb al et al., 2003). IL-1 is produced by a number of cells incl uding osteoclasts, and it stimula tes an increase in osteoclast precursors as well as stimulating mature os teoclasts to begin bone resorption (Reddy & Roodman). Il-4 inhibits oste oclast activity there by decreasing bone resorption; however some studies show that high levels of IL-4 may also inhibit bone form ation (Reddy & Roodman). IL-7 33

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is a potent cytokine responsible for increasing the osteoclast population in the absence of estrogen. Weitzmann and colleagues (2002) found that in the absence of estrogen, IL-7 had an increased presence in the bone marrow resultin g in an increase in osteoclastogenesis. Transforming growth factor (TGF) is secreted by both osteobl asts and osteoclasts, and is an osteoblastic stimulator and an osteoc lastic inhibitor (Reddy & Roodman, 2004). TGFworks in an autocrine manner to increase osteobl ast cell differentiation and proliferation, inhibit osteoclast proliferation, and i nduce osteoclast apoptosis throu gh the protein osteoprotegerin (Eqbal et al., 2003). Turner and colleagues ( 1994) reported that the presence of estrogen increased the levels of TGFin cultured bone cells. Reddy and Roodman reported that very low levels of TGFincreased osteoclast formation and bone resorption in cu ltured bone cells. Role of Estrogen in Bone Remodeling Three forms of estrogen are present in the female human body: estradiol, estrone, and estriol. The most potent form of endogenous estroge n in the female is estradiol, which is carried by the blood and produced mainly by the theca and gr anulosa cells of the do minant follicle in the female ovary through steroidogenesis of cholesterol (Jones & DeCherney, 2005). As a woman ages the pool of potential follicles diminishes, a nd the primary source of estradiol is lost. The endocrine changes that occur as a woman ages include decr easing ovarian estrogen production and increased pituitary follicle stimulating hormone This endocrine transition is referred to as the menopause or the climacteric. Menopause is de fined as the cessation or the absence of the menstrual period for one year; however menopause is actually a multisystem transition that occurs in the lives of all middle aged women (Greendale, Lee, & Arriola, 1999). The average age of menopause is 48 to 55 years of age. Curre nt estimates for the year 2000 are that there are 46 million postmenopausal women in the United States, and that number is expected to reach 50 million in the year 2020. In 1998 it was estimated that there are more than 477 million 34

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postmenopausal women worldwide and that number is expected to reach over one billion by the year 2025 (The North American Menopause Societ y, 2001). As the life expectancy of women increases, a large majority of women will spe nd one quarter to one third of their lives in menopause. During the menopause years the potent estrogen estrad iol is lost, and the primary estrogen becomes estrone, a much weaker estrog en with no discernable estrogenic effects (Jones & DeCherney). It has long been understood that estradiol has protective effects on bone, and the loss of these protective effects in the menopausal years leads to an increase in bone loss and potentially osteoporosis. Estrogen plays a vital role in the preserva tion of bone by maintaining a balance between bone resorption and formation, and by protectin g cancellous bone from excessive remodeling (Turner et al., 1994). Estrogen also regulates many cytokines that are responsible for osteoclast formation and activation such as IL-1, IL-6, IL-7, TNF, and RANKL and exogenous estrogen replacement has been shown to decrease the production of these cytokines (U emura et al., 2005). To understand the effect of estrogen on bone it is necessary to understand how estrogen enters and affects a target organ. Estrogens have the ability to move in and out of cells by simple diffusion, but the action of estrogen is dependent on the presence and acti vation of an estrogen receptor in the nucleus of the targeted cell (B arrett, 2005). There are two estrogen receptor subtypes, ER and ER which are distinctive from each other and located on different chromosomes (Bord, Horner, Beavan, & Compston, 2001). Bord and colleagues found that both ER and ER were present in human bone, with ER present in higher concentrations in cortical than cancellous bone, and ER present in higher concentrations in cancellous bone than cortical bone. The presence of distinctive and entirely different ERs suggests th at each receptor may have a different function in the estrogen modul ation of bone remodeling. Once the hormone has 35

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bound to a specific estrogen receptor in the nuc leus, the receptor then becomes a specific transcription factor which will bi nd with a specific hormone response element (HRE) resulting in gene transcription (Igarashi, 2005). Estrogen receptors, both ER and ER have been identified in the three most important bone cells: osteoclasts, osteoblasts, and osteocytes (Vaananen & Harkonen, 1996). Estrogen is necessary to main tain bone mass. The presence of estrogen maintains a balance between bone resorption and formation by suppressing bone remodeling. Estrogen has been demonstrated to decrease osteoclast fo rmation, activation, and lifespan and has been suggested to increase osteobl ast formation (Riggs, Khosla, & Melton, 2002). The loss of circulating estradiol, the most potent estrogen, ca uses an estrogen deficiency which then causes an imbalance in bone remodeling. Without es trogen a rapid loss of bone occurs when bone resorption is markedly increased and bone formation is onl y slightly increased. Riggs and colleagues found that when estrogen levels decreased in menopause there was an increase in bone biochemical markers, however the increase was not equal. Biochemical markers of bone resorption increased by 90% at menopause; however biochemical markers of bone formation only increased by 45%, half as much. This inequa lity leads to a remodeling imbalance which in turn leads to bone loss, structural weakness, and perforation of th e bone, specifically the cancellous bone (Vaananen & Harkonen, 1996). The substantial decrease in circulating estr adiol decreases the regulatory effect that estrogen exerts over cytokines. Cytokines have b een shown to increase os teoclast activity and promote bone resorption. The most potent cy tokines in bone resorption IL-1, IL-6, and TNFhave been shown to be modulated by endogenous estrogen (Pacifici, 1998). The removal of the regulating effect of estrogen on cytokines in creases their expressi on, therefore increasing 36

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osteoclast differentiation and activation. This in turn increases a skewed form of bone remodeling with resorption exceeding formation. This imbalanced bone remodeling leads to a loss of bone resulting in more fragile bones whic h are prone to fracture. This condition is commonly called postmenopausal osteoporosis. Effect of Calcium on Bone Remodeling Calcium is the most abundant mineral in the human body with 99% of the bodys calcium stored in the bones (Ferguson, 2004). Calcium is necessary for bone strength, nerve and muscle conduction, and the maintenance of a regular he artbeat (Lyon & Sutton, 1993). Calcium is the primary mineral of hydroxyapatite, an inorganic salt which is responsible for the hardness and strength of bone (Jee, 1999). Calcium is rele ased from the bone during resorption, and then replaced in the form of hydroxyapatite by the osteoblasts during forma tion (Parfitt, 1993). Serum levels of calcium are regulated by two hormones: parathyroid hormone and calcitonin. Parathyroid hormone is secreted when serum levels of calcium are low and calcitonin is secreted when serum levels of calcium ar e high (Barrett & Barrett, 2005). Vitamin D is a fat soluble vitamin that is pr imarily stored in the body fat. The primary form of vitamin D is the dihydroxylated metabolite of vitamin D3, Calcitrol (1,25(OH)2D3). Vitamin D is synthesized in the sk in when the skin is exposed to ultraviolet light, mainly in the form of sunlight. Vitamin D acts mainly on th e kidney, bone and intestin e to regulate serum calcium under the influence of parathyroid hormone (Barrett & Barrett, 2005). Vitamin D promotes absorption of calcium from the intestin e, increases calcium reabsorption in the kidney, and has both an indirect and di rect effect on bones (B arrett & Barrett). Th e immediate direct effect of vitamin D is to obtai n calcium from the bones by promo ting the development of mature osteoclasts, thereby increasing bone resorption. The indirect effect of vitamin D is the enhanced absorption from the intestine and reabsorption from the kidney, creating an overall surplus of 37

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serum calcium, promoting storage of calcium in the bone, and bone mineralization (Barrett & Barrett). Aging decreases the intestinal absorption of both di etary calcium and vitamin D, resulting in decreased available stores and the necessity for mobilizing calcium from other stores, mainly the bones (Lyon & Sutton, 1993). In addition, advan ced age limits or prevents women from being outdoors, resulting in decreased vitamin D stores. The deficiency of both calcium and vitamin D increases net bone loss, resulting in a loss of bone mineral dens ity and increased risk of fractures. Dawson-Hughes and colleagues (1990) studied the effect of calcium supplementation in postmenopausal women in a double-blind, placebo-contro lled trial. It was noted that calcium supplementation showed no difference in bone loss from the spine in women who were less than five years postmenopausal. Women who were six or more years postmenopausal showed less bone loss than women on placebo. In 1997 DawsonHughes and colleagues looked at the effect of calcium and vitamin D supplementation in men and women over age 65 and found reduced bone loss in the spine and femoral neck, as well as a reduced incidence of nonvertebral fractures. Riggs and colleagues (1998) investigated the effect of long term calcium supplementation in postmenopausal women and found that supplement ation decreased the age-related increase in serum parathyroid hormone and bone resorption, and decreased bone loss. A meta-analysis done by Kanis (1999) revealed that calcium supplemen tation slows bone loss but is not effective at rebuilding bone, and the effect is more pronounced in older women than women at the beginning of menopause. It is generally accepted that supplementation with calcium and vitamin D will not prevent postmenopausal bone lo ss in trabecular bone but can sl ow age related bone loss in cortical bone (Dawson-Hughes, 2000). Flynn (2003) concluded in a meta-analysis that calcium 38

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and vitamin D supplementation reduced the inci dence of nonvertebral fractures and reduced morbidity in elderly men and women. Pr ince and colleagues (2006) found that calcium supplementation with calcium carbonate reduced the risk of an osteoporosis -related fracture in women who were at least 80% compliant with the medication regimen. However, this large study (n=1460) showed that the majority of the women in the study were not compliant with the recommended dosage of calcium carbonate due to the side effects. Therefore this is not suggested as a form of single therapy for the prev ention of osteoporosis-related fractures. It is currently recommended that wo men not on estrogen replacement take calcium 1500 mg/day and Vitamin D 400 IU/day (Goldfeder, 2005; Luckey, 1999; Lyon & Sutton, 1993). Postmenopausal Osteoporosis Menopause is a natural midlife transition that all older wo men will experience in their lifetime. Currently in the United States th ere are an estimated 46 million women who are postmenopausal (North American Menopause Soci ety, 2001). It is we ll known that menopause is due to a loss of ovarian function causing a decrease in circulat ing levels of estradiol, the most potent female estrogen. This loss of estroge n causes an imbalance in the bone remodeling process which favors resorption over formation. This imbalance causes a loss of bone which increases the fragility of th e remaining bone. The increasingly fragile bone becomes more susceptible to fractures, specifically in cancellous bone of the hip a nd vertebrae. This disease in which bones are susceptible to fracture due to a d ecrease in bone mass and strength secondary to loss of endogenous estrogen is known as postmenopausal osteoporosis. Adult human bone is constantly being rem odeled. In adulthood, 25 percent of cancellous bone is remodeled every year while only 3 perc ent of cortical bone is remodeled yearly (Manolagas & Jilka, 1995). The imbalance in bone remode ling that occurs in menopause therefore affects cancellous bone more than cor tical bone. Beginning in the fourth or fifth 39

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decade of life, with the average age of menopa use 48-55 years, bone loss accelerates, sometimes as much as ten times the amount of loss that occurs in premenopausal women (Manolagas & Jilka). Bone loss in cancellous bone weakens the plates and promot es the alteration of plates to the less substantial rods (Raisz, 1988). The weakened more fragile bone is then susceptible to the fractures that characteri ze postmenopausal osteoporosis. In the first few years of menopause circulating estradiol levels have been shown to drop to 10-15 percent of the premenopausal level (Riggs Khosla, & Melton, 2002). This decrease in circulating estrogen causes a loss of the regulat ory effect that estroge n has on bone remodeling. Bone remodeling increases, however it is not th e balanced remodeling th at occurs prior to menopause. This postmenopausal remodeling favors bone resorption (Doran & Khosla, 2000). Without estrogen suppressing osteoclast formation and activation, as well as inducing osteoclast apoptosis, more osteoclasts are active on the bon e surface. The suggested osteoblast formation that is promoted by estrogen is also lost (R iggs, Khosla, & Melton). Bone resorption exceeds formation in the hypoestrogenic state of menopaus e. In addition, the suppressive effect of estrogen on potent cytokines involved in bone resorption such as IL-1, IL-6, and TNFis lost and bone resorption is further increas ed (Vaananen & Harkonen, 1996). The role of RANK, RANKL and OPG has rece ntly been shown to play a role in postmenopausal osteoporosis. RANKL is pr oduced by osteoblasts and is a membrane bound factor of the tumor necrosis factor family show n to stimulate osteoclast differentiation (Troen, 2003). RANK is a membrane bound receptor and also a member of the tumor necrosis factor family. OPG is a decoy receptor which can bi nd to RANKL in the place of RANK and inhibit osteoclastogenesis (Troen). Aubin and Bonnelye (2000) found that estrogen inhibits the production of RANKL and subsequently RANKL-induced osteoclastogenesis. Hofbauer and 40

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colleagues (1999) found that estrogen increase d production of the decoy receptor OPG in cell cultures of human osteoblast cel ls. Eghbali-Fatourechi and co lleagues (2003) found that an estrogen deficit state induced the expression of RANKL promoting increased bone resorption, however they were unable to determine if it was a direct effect of the estrogen deficiency, or the increased expression of cytoki nes such as IL-1 and TNFin menopause which are also known to increase expression of RANKL. The decreased estrogenic state of menopause al so increases the sensitivity of the bone to parathyroid hormone (PTH), which is responsi ble for calcium homeostasis in the body. The increased sensitivity to PTH causes even higher bone resorption which leads to an increasing level of serum calcium. The body compensates by increasing the excret ion of calcium through the urine and decreasing calcium absorption in the in testinal system preventing hypercalcemia (Barrett & Barrett, 2005; Riggs, Khosla, & Melton, 1998). As women age a second cause of bone loss is identified. Approximately 10-15 years after the menopause serum levels of PTH begin to rise Research has shown that aging impairs the intestinal absorption of calcium and the renal reabsorption of filtered calcium, causing a decrease in circulating calcium. PTH secretion increases and calcium is mobilized from the bones, where 99% of calcium is stored (Riggs, Khosla, & Melton, 1998, 2002). This increased resorption of calcium from the bones causes bone loss which resu lts in fragile bones and increased risk of osteoporotic fractures. The diagnosis of osteoporosis is currently done with either the presen ce of an osteoporotic fracture or the use of bone mineral density m easurements. Biochemical markers of bone turnover are also being used to assess bone remo deling, but have not prove n useful in predicting the fracture risks associated with osteoporosis, and are currently being used more in research, 41

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specifically for testing antiresorptive drugs (Kenny & Prestwood, 2000). Bone mineral density (BMD) measurement is the current acceptable form of diagnosing osteoporosis and osteopenia. BMD can be measured in a variety of ways including single and dual energy X-ray absorptiometry (DEXA) scan, ultrasound meas urement, computed tomography and radiography (Kanis, 2002). Bone mineral density has been pr oven to correlate well with the load bearing ability of bone and the potent ial risks of fracture (NIH C onsensus Development Panel on Osteoporosis Prevention, Di agnosis, and Therapy, 2001). Dual energy X-ray absorptiometry (DEXA) scans are used to measure the mineral content of the skeleton, specifically the si tes prone to fractures such as the vertebrae and the hip. The results are given in standard deviation (SD) measurements as compared to a young healthy population and the measurement obtained is called the T-score (Kanis, 2002). Osteoporosis is diagnosed when the T-score of the hip is at or below 2.5 SD of the young adult female. Osteopenia, or low bone mass, is diagnosed when the T score of the hip is between 1 SD and 2.5 SD below the young adult female. Severe osteoporos is is diagnosed when the T score of the hip is more than 2.5 SD below the young adult female or the diagnosis of at least one fracture due to bone fragility (Kanis). The major use of DEXA s cans is the assessment of the mineral content of the bones and the risks of fracture. The info rmation obtained from DEXA scans can aid the health care practitioner in treating osteoporosis prior to a fracture with antireasorptive drugs such as the commercially available bisphosphonates, hormone replacement therapy, or calcitonin. Biochemical Markers of Bone Remodeling Biochemical markers of bone remodeling are divided into two groups, markers measuring bone resorption and markers measuring bone form ation. Markers commonly used to measure bone resorption are urine pyridinolines and deoxypyridolines, commonly referred to as crosslinks. In addition, newer type I collagen Ctelopeptides (CTx) and Ntelopeptides (NTx) are 42

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markers of resorption measured by both serum and urine sampling. Markers commonly used to measure bone formation are bone specific al kaline phosphatase (BSA P), osteocalcin, and propeptides of type I collagen, all serum markers (NIH Consensus Development Panel on Osteoporosis Prevention, Dia gnosis, and Therapy, 2001). The collagen of mature bone is held in place by non-breakable cross-links formed primarily by amino acids. The primary crosslinks in bone are hydroxylysyl-pyridinoline and lysyl-pyridinoline commonly known as pyridino line (Pyr) and deoxypyridi noline (D-Pyr, DPD) (Ziambaras & Civitelli, 1998). When bone is broken down during the process of resorption, the collagen cross-links are liberated, released into th e systemic circulation and then excreted in the urine intact, as they are not metabolized or reused in the body again (Eyre, 1994). D-Pyr is present in high concentrations in the collagen of bone and the dentin of the teeth, and since bone is the primary source of collagen in the body, it is an excellent ma rker for the collagen breakdown that occurs during bone resorption (Z iambaras & Civitelli). Immunoassays are available which measure the amount of free Pyr or D-Pyr concentrations in the urine and are the basis for the biochemical markers of bone resorption. In addition, imm unoassays have been developed to identify the amount of the terminal fragments of the type I collagen peptide chain known as the N-terminal telope ptide (NTx) and the C-terminal telopeptide (CTx). The Nterminal of the type I collagen molecule is t hought to be responsible for approximately 60% of the D-Pyr in human bone collagen and the Cterminal responsible for the remaining 40% (Ziambaras & Civitelli) making them a useful source for measuring bone resorption. These markers are not affected by a pe rsons diet and are specific for type I collagen; however since bone remodeling follows a diurnal pattern, they are best collected consistently and in the early morning hours (Hanley, 2000). 43

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Studies using the C-terminal telopeptide (C TX) have shown that CTX is an excellent marker of bone resorption in osteoporosis. Chailurkit and colleagues (2001) compared numerous bone biochemical markers in evaluating the effect of hormone replacement and calcium supplementation alone and in combination and concluded that serum CTX is an excellent indicator for determining response to treatment. The effect of estrogen withdrawal has been well documented. Sornay-Rendu and colleagues (2003) used serum CTX to evaluate the effect on bone remodeling in women who were on estrogen replacement and then discontinued treatment. A rapid increase in bone remodeling was documen ted using biochemical markers of both bone resorption and formation, specifically serum CT X. Serum CTX was significantly lower in a study which compared the response of several bone biochemical markers to alendronate therapy, and serum CTX was declared to be the most effective bone biochemical marker used in the study (Fink et al., 2000). Recently se rum CTX was utilized in evaluating the effectiveness of a once a month dose of Ibandronate (Boniva ). Serum CTX was significantly decreased after three months of treatment with once-monthly Boniva (Reid, 2006). Serum CTX is unaffected by diet, but fasting has been shown to reduce the effect of the diurnal pattern of high levels in the early morning hours and low levels in the afternoon (Del mas et al., 2000). It is recommended that to reduce the effect of the diurnal pattern of serum CTX that al l specimens be collected after the subject has fasted and the timing be tightly cont rolled, collecting specimens at the same time of the day both before and after treatment. Mature active osteoblasts secr ete several non-collagenous prot eins such as osteocalcin, bone specific alkaline phosphatase and type I pro-collagen peptides such as N-terminal PINP and C-terminal PICP, during the formation phase of bone remodeling (Srivastava et al., 2005). Osteoblasts secrete these non-collag en proteins, in addition to type I collagen, and then the newly 44

PAGE 45

formed bone is mineralized in two phases when hydroxyapatite is deposited. Since formation involves the laying of both type I collagen and the non-collagenous proteins, it is possible to measure the rate of bone formation by measurin g the amount of these non-collagenous proteins in the blood. Alkaline phosphatase is an enzyme that origin ates from a variety of tissues in the human body, specifically the liver, intestines, spleen, bone, kidney, an d placenta (Delmas et al., 2000). In the healthy adult the alkaline phosphatase in serum comes in equal parts from the liver and bone. Total alkaline phosphatase is not specific in determining the tissue of origin, but the bone specific alkaline phosphatase (BSAP) is specific to the bone tissue, and is considered a highly specific test (Delmas et al.). Bone specific alkaline phosphatase us es an antibody that recognizes and binds to a glycoprotein on the surface of the osteoblast cell known as an immunoassay. There is some cross reactivity between bone an d liver alkaline phosphatase, approximately 1020%, but it is still considered hi ghly specific (Delmas et al.). In the osteoblast, prior to secr etion, the type I collage n is held in a triple helix formation by disulfide bonds between the C-terminal propeptid es and stored in the golgi complex until it is time for secretion. After the type I collagen is secreted by the os teoblast, the N-terminal and the C-terminal peptides are removed and released into the systemic circulation, allowing them to be measured through serum assays (Hanley, 2000). Th ese peptides are specific for newly formed type I collagen and are termed PINP for the amino N-terminus and PICP for the carboxy Cterminus of the peptide exte nsion (Delmas et al., 2000). Osteocalcin is a non-collagenous protein with an affinity for binding to hydroxyapatite secreted by the osteoblast. Osteocalcin is ofte n called Gla-protein as it contains three gammacarboxyglutamic acid residues which are believed to be responsible for the calcium binding 45

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properties of the osteocalcin protein (Hanley, 2000) Though the exact function of osteocalcin is unclear, it is considered to be a reliable mark er of osteoblast function (Delmas et al., 2000). Osteocalcin, like deoxypyrid inoline, has a diurnal pattern and peaks in the early morning and is dependent on adequate kidney function for excreti on (Hanley). Therefore it should not be relied on in someone with kidney diseas e or inadequate kidney function. Biochemical markers are used extensively in research involved with osteoporosis. The markers used vary widely, and sometimes combinations of markers are us ed in the same study. Biochemical markers are used to study bone lo ss, risk of fracture, and the efficacy of medications such as estrogen, bisphosphonates, calcium, calcitonin, parathyroid hormone, and the phytoestrogens. Currently, in addition to dua l energy X-ray absorptiometry (DEXA) scans, biochemical bone markers are a staple in osteoporo sis research, and are beginning to be used in clinical practice. It is known that bone mineral density (BMD) is different in men and women as well as different ethnic groups. African American women have been shown to have higher BMD at every site tested in DXA scans and a slower rate of bone loss from the femur and spine than Caucasian women (Aloia, Vaswani, Yeh, & Flaste r, 1996). In addition to African Americans having a larger bone mass, Asians have a lower bone mass than Caucasian women, with Hispanic women similar to Caucasian women. Barrett-Connor and coll eagues (2005) found that African American women had the highest BMD followed by Hispanic women, Native American women, Caucasian women, and fi nally Asian women. Though Asian women have the lowest BMD they have lower fracture risks then Hispanic and Caucasian women (Barrett-Conner et al.). There is also an ethnic difference noted in biochemical bone markers. Aloia and colleagues (1996) found differences in biochemi cal bone markers between whites and blacks, 46

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with black women having lower levels of bone turnover markers, and higher levels of parathyroid hormone and serum calcitriol, a meta bolite of vitamin D. Han and colleagues (1997) found that postmenopausal African-American women had statistically signifi cant lower levels of serum osteocalcin than Caucasian women, but ther e was not a statistically significant difference in bone specific alkalin e phosphatase. Aloia and colleagues ( 1998) found that several indices of bone turnover were lower in postmenopausal African-American women than postmenopausal Caucasian women such as bone specific alkaline phosphatase, osteocalcin, urine hydroxyproline, urine pyridinoline cross-links, and urine N-terminal telopeptide. Gundberg and associates (2002) found that osteocalcin was lower in African-A merican women compared to Caucasian and Hispanic women, and bone specific alkaline phosphatase was lo wer in Caucasian women than African-American and Hispanic women. These studies show differen ces in individual biochemical bone markers dependent on race/eth nicity, which would make comparing groups with different ethnic origins more difficult. It would be more bene ficial to look at an intervention tested on one specifi c racial/ethnic group, and then compare each racial/ethnic group to another to see how each group indivi dually responded to the intervention. Biochemical markers of bone remodeling have l ong been used in drug intervention trials. Estrogen replacement in the postmenopausal year s has been shown to have statistically significant changes in biochemical markers of bo th bone resorption and fo rmation. Studies have shown that with estrogen therapy both markers, serum osteocalcin and urine deoxypyridinoline, have significantly decreased, evidence that bone remodeling has decreased, a goal for the prevention of the bone loss that accompanies menopause (Castracane, 2005; Lindsey, 2002; Riggs, 2003; Watts, 2000; Zhan, 1999). Resear ch studies on antiresorptive agents, the bisphosphonates alendronate and risedronate, have used biochemical bone markers. Serum 47

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osteocalcin, bone specific alkaline phosphatase (BSAP), urine D-Pyr, and urine NTx all decreased in women taking alendronate (Delmas, 2000; Greenspan, 2005). Studies involving many phytoestrogens have also used bone biochemical markers for assessing the effect of their product on bone remodeling. Yamori and colleagues (2002) found a statistically significan t decrease in urine deoxypyridi noline after ten weeks of soy supplementation in postmenopausal women compared with placebo. Nikander and colleagues (2004) reported a statistically significant decrease in urine D-Py r in the phytoestrogen soy group compared with placebo after three months of th erapy, but no change in bone specific alkaline phosphatase. Changes were noted in the N-termin al and C-terminal telopeptides, markers of bone resorption, in the soy group, but they we re not statistically significant. Black cohosh, Cimicifuga racemosa, has long been grouped in the phytoestrogen category due to the fact that its exact mechanism of acti on was unclear. It remains to be proven if black cohosh works on estrogen receptors, dopamine receptors, or a combination of both receptor types (Viereck, Emons, & Wuttke, 2005). In ovariectomized rats, Niblein and Freudenstein (2003) found that black cohosh had significantly reduced urine Pyr and D-Pyr after nine weeks of therapy. In Germany, Wuttke and colleagues (2003) found that black cohosh has a positive influence on the bone turnover index (BSAP/Crosslaps ratio) of 62 postmenopausal women and may have implications in the prevention of osteoporosis. Effect of Calcium and Vita min D Supplementation on Bioc hemical Bone Markers in Postmenopausal Women It is currently accepted as a standard of care to recommend both calcium and vitamin D supplementation to postmenopausal elderly wome n. Current recommendations for women not taking estrogen replacement are calcium 1500mg /day and vitamin D 400 IU/day (Goldfeder, 48

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2005). Biochemical bone markers have been used in the past to evaluate th e effect of calcium on bone turnover, both resorption and formation. In 1994 Blumsohn and colleagues noted that supp lementation with calcium citrate in the evening hours showed a decrease in morning measurements of urine DPD and NTX, whereas supplementation of calcium in the morning hour s had no effect on either urine DPD or NTX excretion the following morning. Kenny and co lleagues (2004) found calcium supplementation with calcium citrate for 12 weeks decreased urinary DPD, CTX, and NTX while supplementation with calcium carbonate showed no significant change in the same biochemical markers. Ulrich and colleagues (2004) found that calcium carbonate supplementation for 18 weeks in early postmenopausal women showed no change in serum OC, BSAP, and CTX or urinary NTX. Supplementation with an effe rvescent Sandocal 400 mg tablets one daily in healthy young adults showed a decrease in serum CT X, demonstrating in th is study that calcium can decrease bone remodeling in young adults (Sidideen & Swaminathan, 2004). Role of Black Cohosh ( Cimicifuga Racemosa ) Black cohosh is a native plant in North America and Canada. Black cohosh was originally used by the Native Americans for a variety of female complaints including: dysmenorrhea, childbirth, snakebite and pain relief (Hardy, 2000). Black cohosh has been known by many names including snakeroot, squawroot, rattle root, rattle weed, rattle to p, and bug-bane. The names involving the word rattle were believed to be due to the distinctive rattle appearance to the flowering part of the black cohosh plant (Skidmor e-Roth, 2004). It is im portant not to confuse black cohosh with blue or yellow cohosh. Black cohosh is best known by the name Cimicifuga racemosa and is still often refe rred to by that name. It has since been renamed Actaea racemosa due to a reclassification into a different genus; however many researchers interchange the names, and use both Cimicifuga and Actaea (Upton, 2002). The word cohosh is an Algonquin Indian 49

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word meaning it is rough. Black cohosh was used by many Native American tribes including the Cherokee, Delaware, Iroquois, Mikmaq, and the Penobscot (Upton). The tribes used the plant as a treatment for a variety of complaints including: hives, rheuma tic conditions, sedative and gynecological. The first published reference to black cohos h occurred in Carl von Linnes work in 1749 and its uses were listed as treatments for swelling and female problems (Upton, 2002). In 1831 the use of black cohosh was in troduced in the United States through a paper written by Young and published in the American Journal of Medi cal Science. Black cohosh was used as a treatment for smallpox in 1832, a belief that was supported for forty years. In 1820 black cohosh was listed in the United States Pharmacopoeia as an anti-inflammatory and antispasmodic. Black cohosh appeared in the United States Di spensatory in 1833 and remained there for 122 years as a stimulant for the kidneys, skin and pulmonary mucous membranes. In 2001 it was proposed to include black cohosh in the United States PharmacopoeiaNational Formulary, and it is currently approved in Germany for reli ef of dysmenorrhea and menopausal symptoms. Black cohosh is an herbaceous perennial. It consists of a stem, leav es, flowers, fruit and root system. The stem of the plant is approxima tely eight feet tall with leaves approximately 615 cm long and 6-16 cm wide and green in colo r (Upton, 2002). The flowers are cream colored and the fruit is a 5-10cm oval brown pod with multip le seeds. The rhizome is 2-15 cm long with many tightly packed curving branches with a brownish-black exterior. The roots are 3-16 cm long and dark brown in color. The plant is found only in the United States and Canada. Currently the entire supply of black cohosh is from the Unite d States, specifically Kentucky, Tennessee, Georgia, Ohio, North Carolina, Michig an, South Carolina, Virginia, West Virginia and Wisconsin. The plant is found in forests, meadows, creek margins and mountains. The 50

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rhizome and root are harvested when the plants are at least two years in age and the plant is in the dormant cycle (Upton, 2002). The rhizome portion of the black cohosh plan t is used in commerc ial preparations of black cohosh extract. The exact chemical compos ition of any black cohosh supplement is varied according to the manufacturer, and may contai n many different compounds. The main active ingredients are the trite rpene glycosides, and a black cohosh supplement may contain as many as 20 different types of triterpene glycosides (U pton, 2002). Commercial preparations of black cohosh contain approximately 2.5% triterpene glycosides and include the following: actein, 23epi-26-deoxyactein, and cimiracemoside A. In addition, commercial preparations contain varying amounts of aromatic acids, flavonoids, ta nnins, resins, fatty acids starch, and sugars (Upton). The flavonoids are phytoestrogenic in nature but th eir presence in black cohosh preparations are currently under debate. Three fl avonoids have been report ed to be present in black cohosh preparations: biochanin A, formonon ectin, and kaempferol (Upton). The presence of formononectin was identified in early studies, but re cent studies have failed to prove its presence in current commercial preparations of black cohosh (Kennelly et al., 2002). The presence or absence of biocha nin A and Kaemferol has been debated and cannot be proven; further studies are recommended (Upton). The exact composition of black cohosh can be measured by mass spectroscopy and is recommended for ensuring the consistency of the product. The exact metabolism and excretion (Johnson & vanBreemen, 2003), and the exact mechanism of action of black cohosh in humans remains unknown (Skidmore-Roth, 2004). It has long been proposed that black cohosh contained substances were estrogenic in nature, or were similar to the selective estr ogen receptor modulators (SERMs). Due to the multitude of chemical components in a black cohosh preparation, it has been difficult to 51

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determine the exact mechanism of action. Black cohosh has been classified as a phytoestrogen because it was believed to behave similarly to estrogen and was plant derived. However, all phytoestrogens have different mechanisms of acti on, and most are believed to exert their effects through the estrogen receptors, ER and ER (Wuttke, Jarry, Westphalen, Christoffel, & Seidlova-Wuttke, 2003). Studies on black cohosh have produced conflicting results because, although believed to work by exerting an influence on target organs vi a estrogen receptors, several studies have shown that that the com pounds in black cohosh do not directly bind with estrogen receptors. Init ially, black cohosh was thought to be es trogenic in nature due to the fact that preparations of black c ohosh, specifically Remifemin, decreased circulating levels of luteinizing hormone (LH), but not follicle stimulating hormone (FSH) in postmenopausal women (Duker, Kopanski, Jarry, & Wuttke, 1991). Howeve r, several studies have shown that black cohosh does not have estrogenic e ffects, a fact that has become important to women seeking options for relief of postmenopausal vasomotor symptoms who have been treated for breast cancer. Liu and colleagues (2001) found that usi ng four different assays to measure estrogen receptor binding in vitro, black cohosh did not bind to estrogen receptors and and did not exert an estrogenic activity through the estrogen receptors. Lupu and colleagues (2003) found that black cohosh did not demonstrate any estrogen ic activity in assays measuring the activation of the estrogen-response-element (ERE) require d for estrogen receptor function. Black cohosh has also demonstrated no effect on the MC F-7 cell line, obtained from human breast adenocarcinoma which contains estrogen receptors, and is therefore a model for estrogenresponsive cells (Amato et al., 2002; Einbond et al., 2004; St romeier et al., 2005). The possibility that black cohosh is a new SERM has been proposed. A SERM is a substance which has estrogen-like effects in some target organs and anti-e strogenic effects or no 52

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effect at all on other target organs. Seidlova-Wuttke and a ssociates (2003) found that black cohosh exerted estrogenic activity by significantly reducing serum levels of LH and decreasing the amount of bone loss in ovariectomized rats co mpared to placebo. It was proposed that black cohosh contained an unidentified component whic h exerted selective estrogenic activity. Jarry and colleagues (2003) also suggest the presence of an unidentified component in black cohosh preparations which they termed ER They found that black cohos h competed with radioactivelabeled estradiol in a cytosolic ER preparation from pork uteri, demonstr ating phytoestrogen-like activity in vitro. Viereck and colleagues (2005) also suggest that black cohosh is not a classic phytoestrogen, and due to the fact that it has ER binding and an estrogen agonistic effect on bone tissue, and no estrogenic activity on breast or endometrial tissue, it should be classified as a SERM. In addition, it is postulated that the mechanism of action of black cohosh may be through serotonin receptors or dopamine receptors. The possibility that black cohosh may have an effect on cytokines, which themselves have an effect bone remodeling, has been proposed. It is known that an in creased expression of cytokines such as: tumor necrosis factor(TNF), Interleukins (IL) 1, 4, 6 and 7, receptor activator of NF-KB ligand (RANKL), and osteoprotegerin (O PG) can either stimulate or inhibit bone resorption (Khosla, 2001; Reddy & Roodma n, 2004; Turner, Riggs, & Spelsberg, 1994; Turner, Rickard, Spelsbery, & Sibonga, 2003). Black cohosh was shown to inhibit the production of IL-4, and TNFin human mast cells (Kim et al., 2004) and increase the expression of OPG mRNA in cu ltured human osteoblas t cells (Viereck et al., 2005). Qiu and colleagues (2007) found that one of the trite rpene glycosides found in black cohosh (25acetylcimigenol xylopyranoside [ACCX]) blocked in vitro osteoclastogenesis induced by RANKL or TNF alpha. 53

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The exact mechanism of action of black cohos h remains unclear, but it is accepted that black cohosh does not act through the common estrogen receptors ER and ER It has been suggested that black cohosh might act through an unknown receptor, or possibly the serotonin receptors. It has also been suggested that black cohosh may have an effect on the regulation of cytokines which regulate bone remodeling. Ho wever, the exact mechanism of action in postmenopausal women remains unknown. The use of black cohosh by postmenopausal wome n has increased in the last few years, possibly from fear of synthetic es trogens, from a desire to use something more natural, and/or a desire to be more in control of the decision for taking medication or supplement. Recently studies have shown that black cohosh is effectiv e in relieving the vasomotor symptoms of the postmenopausal woman. The most common and disrupting vasomotor symptom in the postmenopausal woman is the hot flash. Black cohosh has shown promising results in reducing the number of hot flashes in postmenopausal wo men (Nappi et al., 2005; Osmers et al, 2005; Pockaj et al., 2004; Uebelhack et al., 2006; Verhoeven et al., 2005). Recently it has been suggested that in addi tion to relief of vasomotor symptoms, black cohosh may have a proventive effect on the bone lo ss that occurs in the postmenopausal period. Studies done in ovariectomized rats, a rat m odel of osteoporosis, show changes in bone biochemical markers. Niblein and Freudenstein (2003) found that biochemical markers of bone resorption (PYR, DPY) decrea sed, and BMD loss was significantly less, in ovariectomized Sprague-Dawley rats when supplemented with black cohosh. Seidlova -Wuttke and colleagues (2003) found a significant bone sparring effect in the tibia of ovariectomized rats with black cohosh supplementation over the control group, but less than the estradiol group. Serum osteocalcin was lower in both the estradiol a nd the black cohosh groups, but serum crosslaps 54

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were reduced only in the estradiol group. A study done in Germany by Wuttke and colleagues (2003) in postmenopausal women found an improvement in climacteric complaints, a decrease in the biochemical marker of bone resorption Crosslaps, and an incr ease in the biochemical marker of bone formation, bone-specific alkali ne phosphatase. They concluded that black cohosh had a positive effect on osteoblast activit y and decreased osteoclast activity. A repeat study done by Wuttke and colleagues (2006) in postmenopausal women found a statistically significant increase in osteoblast ac tivity, but no significant change in osteoclast activity after 12 weeks of black cohosh. These st udies show that there is pote ntial for black cohosh as an acceptable supplement to decrease the bone loss that accompanies menopause. Further studies are needed to understand the mechanism of acti on of black cohosh and its effect on bone and the bone remodeling that accompanies menopause. Adverse Effects of Black Cohosh Few adverse effects of standardized bl ack cohosh at the recommended dosage are mentioned. Skidmore-Roth (2004) lists nausea vomiting, hypotension and slow heart rate. Upton (2002) reported headache, vertigo, weight gain, nausea, vomiting, and a stimulant effect. The National Center for Complementary and A lternative Medicine (NCCAM) list in their official material on black cohosh the following si de effects: headaches, stomach discomfort, heaviness in the legs and weight problems (2005). Though adverse events are rare in standardized doses many preparations of black cohosh are not standardized and prepared from different parts of the plant. Recently several cas es of liver failure have been linked to black cohosh and several have been re ported in the United States. A 54 year old postmenopausal woman taking 1,000 mg of black cohosh daily presente d to the clinic with symptoms of fatigue, forgetfulness, and a 10 pound weight loss. Liver failure was diagnosed and a liver transplant was required (Lynch, Folkers, & Hutson, 2006). In addition, a 50 year old postmenopausal woman 55

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taking 500 mg of black cohosh daily presented to a hospital with jaundice, dark urine and lightcolored stools. Liver failure was diagnosed a nd a successful liver transplant was performed (Levitsky, Alli, Wisecarver, & Sorrell, 2005). Several cases of hepatitis and liver failure requiring transplant have been documented outside the United States. L de and colleagues (2007) found that the liver in rats fed varying doses of black cohosh s howed liver cell death by apoptosis both in vitro and in vivo, suggesting that liver damage is a possible adverse event with black cohosh administration and should be looked for in all subjects who take the supplement. Standardization of Black Cohosh The use of herbal medicine dates back thousa nds of years originating in China and India and is still used quite extensively in Asia. In the United States herbal medi cine began in colonial times and was greatly influenced by the Native American culture. Many herbal medications were folk remedies passed on through the gene rations (Bedi & Shenef elt, 2002). Currently herbal products are sold in the United States as dietary supplements and are therefore not subject to stringent regulations with regard to standardization, safe ty, or quality. In or der to be classified as a dietary supplement, a herb may not claim to prevent, treat, or cure a disease (Bent & Ko, 2004). Many herbs are sold as extracts of the orig inal plant that have been obtained by boiling or percolating the herb in water or alcohol, which removes the active ingredient (Bent & Ko). Many herbal products contain a number of active i ngredients, some as yet unidentified, as well as contaminants such as pesticides, prescrip tion drugs, and heavy metals (Barnes, 2003). In 1994 congress passed the Dietary Supplement Health and Education Act which set forth the definition for a dietary supplement as well as how they may be promoted, guidelines for labeling the product, and placed the burden of proving the product unsafe on the Food and Drug Administration (U.S. Food and Drug Administra tion, 1994). This law does not require the manufacturers of dietary supplements to c onform to current good manufacturing practices 56

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(CGMP). On March 13, 2003 the Food and Drug Administration (FDA) published a proposed rule which would require that manufacturers ensure that their dietary supp lement did not contain contaminants or impurities, and that the supplem ent be labeled accurately with regard to the active ingredients, as well as other ingredients contained in the supplement (USFDA, 2004). Under this new proposal dietary supplement manu facturers would be requ ired to evaluate the purity, quality, strength and compositi on of their products as well as report all adverse effects to the FDA (USFDA, 2004). Currently there are se veral manufacturers that produce standardized products that contain a specific quantity of the active herbal ingredient and have removed the harmful or toxic ingredients (Barnes, 2003). However, there are no current legally imposed regulations for standardization of dietary herbal supplement, and the term standardization may vary from manufacturer to ma nufacturer (National Institute of Health Office of Dietary Supplements, 2004). Standardization of black cohosh refers to the amount of the active i ngredient obtained from the root of the plant known as triterpene glyc osides, or sometimes referred to as triterpene saponins, which is expressed as 26-deoxyactein (Na tional Institutes of Health Office of Dietary Supplements, 2005). Each standardized tablet of 20 mg or 40 mg (dependent on manufacturer) of black cohosh extract contains 1 mg of tr iterpene glycosides. Chen and colleagues (2002) isolated a triterpene glycoside known as 26-de oxyactein from the root of the cimicifuga racemosa plant as well as 23-epi-26 deoxyactei n formerly identified as 27-deoxyactein, a triterpene glycoside used for calculating the total triterpene content of black cohosh preparations using chromatography on silica gel. As the triterp ene glycosides are the active ingredient in the black cohosh plant, Panossian and colleagues (2 004) recommends standardizing dosages to the triterpene glycosides and the one most commonly used is 23-epi-deoxyactein, occasionally still 57

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known as 27-deoxyactein. Most commercial black cohosh root products standardize their product to contain 2.5% triterpene glycosides, which is equal to 1m g of 26-deoxyactein per tablet (National Institute of Health Offi ce of Dietary Supplements, 2005). Future Research on Black Cohosh The use of dietary supplements has continued to rise in the past decade due to a number of reasons. Many Americans believe th at dietary supplements are more natural, and therefore better for them. In addition, dietary supplements are ava ilable without a costly vi sit to the health care provider, and are often less e xpensive than prescription medication. However, dietary supplements have not undergone th e rigorous research that is requi red in the United States by the Food and Drug Administration and therefore lack in formation on quality, efficacy, and safety. In addition, dietary supplements are not required by law to report adverse effects and drug interactions, further compromising patient safety. Black cohosh has gained popularity recently as a dietary supplement for the relief of menopausal vasomotor symptoms specifically, the hot flash. Recently more studies are being done utilizing randomized, double-blind, placebo-contro lled clinical trials evaluating the effect of black cohosh on vasomotor symptoms and th e quality of life of pos tmenopausal women. Literature review revealed one study conduc ted by Wuttke and colleagues (2006) that investigated the effect of black cohosh on bone metabolism. Wuttke found that black cohosh stimulated osteoblast activity, but had no statistica lly significant effect on osteoclast activity. The sample size was small (N=62 randomized into 3 study groups) and further studies replicating these findings have not been located. This study utilized a randomized, double-bli nd, placebo controlled trial investigating the effect of a daily dose of black cohosh, standardized to 1 mg of triterpene glycosides in the form of a 40mg tablet once daily for 12 weeks. In addition, subjects in the c ontrol and experimental 58

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group were given calcium carbonate and vitamin D supplementation. Bone biochemical markers of resorption and formation were evaluated at the onset of the study and after 12 weeks of therapy. To minimize the effects of confoundi ng variables such as exercise, smoking, and excessive alcohol intake subjects with those behaviors were excluded from the study. To minimize the effects of ethnicity on bone mine ral density and bone biochemical markers only Caucasian women were included in this initial study. As bone remodeling occurs at different rates depending on age and the amount of postm enopausal years only women greater than one year post menopause and less than 6 years were in cluded. This study attempted to evaluate the effect of black cohosh on the most homogenous sample possible. Results from this study will be used to educate postmenopausal women on the possi bility of a viable alternative to estrogen replacement to prevent postmenopausal bone loss. 59

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Figure 2-1. Human femur Figure 2-2. Haversian syst em (Barrett & Barrett, 2005). 60

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Figure 2-3. Osteoclast (Barrett & Barrett, 2005). 61

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Declining levels of circulating estradiol Loss of inhibition of cytokine production production of RANKL production of osteoprotegerin Increased production of cytokines TNF IL-1, !L-6 osteoclast formation And activation osteoclast apoptosis Loss of regulatory Effect on bone remodeling ? Role in Osteoblast formation Less activation of ER and ER in bone cells gene transcription bone resorption bone formation Imbalance in bone remodeling Results in BMD loss Postmenopausal Osteoporosis Figure 2-4. Postmenopausal oste oporosis theoretical model. 62

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Black cohosh Su pp lementation Possible stimulation of Serotonin receptors Effect on Unknown estrogen rece p tors Possible effect on Cytokines OPG, RANKL, TNF Interleukins Effect on Bone remodeling Effect on bone Biochemical markers Figure 2-5. Black cohosh theoretic al model on mechanism of acti on. Broken arrows indicate proposed, but unproven theories of the m echanism of action of black cohosh. 63

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Table 2-1. Role of cytokines in bone re sorption and the influence of estrogen Cytokines Mechanism Estrogenic Effects Resorption RANK Receptor for Rankl RANKL Interacts with the RANK R eceptor Inhibits other cytokines Known to upregulate RANKL Stimulates OPG Decoy receptor for RANKL, interferes with RANKL signaling Stimulates production of OPG Inhibits TNF Increases RANKL expression Inhibits production of TNF Stimulates IL-1 Increases RANKL expression Mediates effects Stimulates IL-4 Inhibits IL-6 Inhibits effects Stimulates IL-7 Mediates effects Stimulates TGF Increases OPG expression and stimulates RANK Increases production of TGF Inhibits 64

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CHAPTER 3 METHODS This study was designed to determin e the effect of black cohosh ( Cimicifuga Racemosa) on the biochemical bone markers of resorption and formation in the postmenopausal woman. In the postmenopausal woman it is known that du e to estrogen deficiency, bone remodeling increases resulting in fragile brittle bones, often leading to bone fracture. The dual process of bone remodeling, resorption and formation can be assessed with the use of commercially available bone biochemical markers. This ch apter includes the following sections: research design, subjects, measures, procedure, statistic al analysis, and ethica l considerations. Research Design This study was a randomized, placebo-controll ed, double-blind clinical trial. After obtaining informed consent, qualified subjects were randomized into two separate groups, an experimental group and a control group. Both groups had an initial set of bone biochemical markers measured. Serum C-terminal telopeptid e (CTX) was utilized as the marker for bone resorption. Serum osteocalcin (OC) was utilized as the marker of bone formation. After the biochemical markers of bone remodeling were measured each group received the study medication. All participants received calcium carbonate and vitamin D (OsCal +D) supplementation in tablet form. The experime ntal group took a standardized dose of black cohosh 40 mg (triterpene glycosides 2.5%) once da ily for 12 weeks. The placebo group took an identical appearing placebo capsule containing l actose once daily for 12 w eeks. After 12 weeks, measurements of the two biochemical markers for bone resorption and forma tion were repeated. Sample Subjects were recruited using fl yers and advertisement in local hospitals, doctors offices, womens clubs, and word of mouth in North Central Florida. In an effort to encourage subject 65

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participation a $20.00 honorarium was provided to all subjects who completed the study. A prorated amount was given to subjects who complete d at least 3-6 weeks of the study. Inclusion criteria were: Women who were naturally or surgically menopausal for at least one year but not more than six years. Women able to give voluntary consent Women not taking any hormone replacement th erapy or selective estrogen receptor modulators (SERMs) for the past three months Women who had not been diagnosed with osteoporosis or an osteoporosis-related bone fracture Women who were sedentary and no t involved in a regular exerci se program, defined as at least 30 minutes at a time three times a week Women who were Caucasian female s between the ages of 35 and 60 Women who had not taken black cohos h for the past three months Women were excluded from the study if they: Were current smokers Failed to take the medication as directed per study protocol Had a patient-reported history of kidney or li ver disease, diabetes, parathyroid disease or documented osteoporosis with DEXA scan. Were lactose intolerant Became ill or were diagnosed with osteoporosis or fracture during the study time period Changed their mind about being included in the study Had a history of taking bisphosphona tes at any time in their life The above inclusion and exclusion criteria were altered slightly during the trial due to low subject recruitment and an impending expiration da te on all of the study medication. All changes in the inclusion and exclusion criteria were approved by the Do ctoral Supervisory Committee, the University of Florida Institutional Review Board, and the State of Florida Department of Health Institutional Review Board. The follo wing changes were made. Women who were surgically menopausal and wome n with thyroid disease under control with medication were included in the study. The age range was change d to reflect the incl usion of surgically menopausal women and was changed from 45-60 to 35-60. The length of time from surgical and naturally-occurring menopause remained unchanged. Subjects were provided with free calcium 66

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carbonate (Os-cal +D) and vitamin D supplementa tion during the time period of the study, and given education on postmenopausal osteoporosis. To achieve a power of 80% and an alpha level of 0.05 using a one -tailed test and a medium effect size 0.6 the study required 23 su bjects per group. To allow for the accepted attrition rate of 20%, the goal for recruitment was 25 subjects per group. The effect size was computed by averaging the effect size of th e two bone biochemical markers. Fifty subjects consented to the study. Forty eigh t subjects met the inclusion crit eria and were randomized into the study. Forty six subjects completed the st udy, 23 per study group. Two subjects withdrew from the study prior to the second blood sa mpling. One subject (study drug Bplacebo) withdrew due to a subject-report ed weight gain. The subject d eclined to be reweighed by the principal investigator. The second subject (st udy drug A-black cohosh) to withdraw from the study did so because the principal investigator was unable to obtain the second blood sample after two attempts on two separate occasions. Measures Biochemical markers of bone remodeling have been used for over 20 years in bone and osteoporosis research, and are more specific to bone tissue (Delmas et al., 2000). As bone remodeling is divided into two phases, resorp tion and formation, two bone biochemical markers were used. Serum CTX was used to measure the C-terminal peptide fragments that are released when type I collagen is broken down during the resorp tion process. Serum CTX testing was done on serum samples using a Metra serum crosslaps CTX Elisa kit (Quidel Corporation, San Diego, CA). The serum crosslaps CTX Elisa is an enzy me immunological test for the products of the Cterminal telopeptides releas ed during bone resorption. 67

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Serum osteocalcin (OC) was used to meas ure the activity of osteoblasts during bone formation. Serum osteocalcin testing was done on a serum sample using a Metra Osteocalcin EIA kit (Quidel Corporation, San Diego, CA). The Metra osteocalcin assay is a competitive immunoassay which uses osteocalcin coated stri ps, a mouse anti-osteocal cin antibody, an antimouse IgG-alkaline phosphatase conjugate, and a pNPP substrat e to detect and measure the osteocalcin in the sample. Demographic variables were assessed using a questionnaire which included age, weight, height, body mass index (BMI), last menstrual period (LMP), postmenopausal or surgical menopause, and current medications. The demographic questionnaire (Appendix A) was administered at the beginning of the study after the participant ha d signed the informed consent and prior to the serum sample co llection. The demographic questionn aire is a one page fill in the blank questionnaire. The participant was assi gned a number at the onse t of the study and the questionnaire was coded with the participant s number. The medical history questionnaire (Appendix B) is a two page form that the partic ipant filled out after co mpleting the demographic questionnaire. The medical history questionnaire provided the researcher with important facts on the participants health history and allowed the researcher to determine if the participant was eligible for the study. Operationalization of the Variables The independent variable is the administrati on of a standardized dose of black cohosh, 40mg once daily and is a dichot omous variable with the partic ipant either taking the black cohosh or not. Each standardized capsule of black cohosh contained 40 mg of black cohosh root, or the equivalent of 1mg of tr iterpene glycosides. The placebo medication was packaged in an identical appearing capsule and was taken once daily. The identical-appearing placebo was 68

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compounded and packaged in clear gelatin cap sules by Francks Compounding Pharmacy in Ocala. The dependent variables are the two bioche mical markers of bone remodeling and are continuous variables. Serum C-terminal telopeptide (CTX) was used to assess bone resorption. The CTX results are expressed as ng/ml. Serum osteocalcin (OC) is the marker that was used to assess bone formation. Serum osteocalcin is measured in ng/ml and does not need to be corrected unless the sample was diluted for any reason, but in this study, the samples did not need to be diluted. The monoclonal antiosteocalcin antibody has a high specificity show ing 100% reactivity when compared to bovine osteocalcin, which shares a signi ficant homology with human oste ocalcin. The antibody has a high specificity for reco gnizing intact osteocalcin and not fr agments of bone resorption such as the Nand Cterminal propeptides. The Metr a Osteocalcin kit requires 25L of sample for analysis. Demographic variables such as age, weight, height, BMI, and years postmenopause are continuous variables. Demographic stat istics are discussed in Chapter four. Procedure Participants that met the incl usion criteria and agreed to participate in the study were advised of the benefits and risks of the study, an d then asked to sign an informed consent which had been approved by the Institutio nal Review Board (IRB) of both the University of Florida and the State of Florida Department of Health. Afte r entry into the study the participant was given a participant number, and then randomized into eith er the experimental or the control group using a computer generated randomization table. 69

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Laboratory Tests Protocol After randomization, the subject was asked to fill out the de mographic questionnaire and the medical history questionnaire. If the inclusion criteria were met and none of the exclusion criteria were met, the subject was included in the study. All serum samples were obtained during the morning hours and the subject di d not need to be fasting. Blood was drawn by venipuncture using aseptic technique from an antecubital or dorsal hand vein using a 21-gauge needle at the beginn ing of the study, and ag ain after 12 weeks on the study medication. Two 10ml red top serum separator tubes were collected at each blood draw. The tubes were centrifuged at 5000g for 10 minutes to separate the serum component. After the serum was separated, it was pipetted into three or four 1.5 ml aliquo ts and then packed in dry ice for transport according to OSHA standards. The principal investigator completed a four hour training program required by the Department of Transportation (DOT) titled Shipping and Transport of Biological Materials. This training is required to transport blood in a vehicle from one site to another. The frozen serum was trans ported to the University of Florida for storage in the College of Nursing Office for Research S upport (ORS) physiology wet lab. It was frozen and stored at -80 in the College of Nursing wet laboratory freezer until all specimens were collected. The longest time period a specime n was stored in the freezer was 350 days. At the completion of the study both the samp les from the beginning and the end of the study for each participant were analyzed in th e wet lab by the Principa l Investigator under the supervision of the laboratory supe rvisor and the guidance of the Do ctoral Supervisory Chair. All the samples were analyzed in the College of Nu rsing wet lab by the princi pal researcher over the course of several days. Samples were thawed immediately prior to use. All samples used in the bone assays were freshly thawed samples, refrozen samples were not utilized for this st udy. After the samples 70

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were thawed they were thoroughly mixed using a vortex spinner. After mixing the serum was pipetted into the assay plates provided in the assay kits. The assays were performed by the principal investigator over the course of two weeks in the Coll ege of Nursing physiology wet lab. Each assay kit tested 40 samples in duplicate pl us the standards and controls. Each assay kit required 4-6 hours to complete. All assays we re done in duplicate, and the controls and standards of both assays were w ithin the range provided by the manu facturer of the assay kits. Assays were performed following specific di rections provided by the manufacturer. Medication Protocol After completing the initial laboratory tests, the medica tion, which was prepared by Francks pharmacy, was dispensed to the participant. The subject received either the black cohosh or an identical appear ing placebo. The black cohosh was obtained from a local GNC (General Nutrition Corporation) retail store in Ocala. All the bottles of black cohosh bore the same lot number. The black cohosh was manufactured for GNC by the Nutra Manufacturing Company. A certificate of analysis from the Nutra Manufacturing Company is included in this dissertation (Appendix C). Th e bottles of black cohosh were then taken to the compounding laboratory of Francks Pharmacy to be repackaged as study medication. The study medications were packaged by the pharmacy and were in iden tical appearing plastic brown containers. The bottles were now labeled either study drug A or study drug B. Th e medication was then given to the participant by the blinded researcher. The participant was given 28 capsules of either study medication A or study medication B depending on group assignment, and instructed to take a capsule once daily for four weeks. After f our weeks if there were no adverse events, the participant received 28 more capsules. After eight weeks, if there were no adverse events, the participant received the final 28 capsules. At th e end of twelve weeks, the second serum sample was drawn. In addition to receiving the black co hosh or placebo, all participants were provided 71

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with calcium carbonate and vitamin D supplementa tion in the form of Oscal caplets + D which they were instructed to take by mouth twi ce daily. During the study th e participants were contacted by phone weekly to address any concer ns or questions and to detect any adverse events. In addition, the particip ants were given the cell phone numbe r of the principal researcher to call in the event of an emergency. Statistical Analysis Data was analyzed using SPSS (SPSS Inc., Chic ago, IL). Descriptiv e statistics including means, standard deviation, and frequency distributions were analyzed. Leve nes test for equality of variances and the students ttest for equality of means we re conducted to analyze the group differences. Repeated measures Analysis of Covariates (ANCOVA) was used to determine the difference in the bone biochemical markers w ithin groups, between groups, and within-bybetween group interaction after controlling for age and body mass index (BMI). A p value of 0.05 was required for statistical significance. Ethical Considerations All participants who met the in clusion criteria for the study were included. The research proposal was reviewed and approved by the Inst itutional Review Board (IRB) at the Health Sciences Center at the University of Florida and the State of Flor ida Department of Health. In addition, an investigational ne w drug number (IND) was obt ained from the Food and Drug Administration (FDA). The IND letter from the FDA is included as Appendix D. Participants were asked to sign an informed consent which outlin ed the risks, benefits, and goals of the study. All participants were assured that they were fr ee to leave the study at any time for any reason if they were uncomfortable with the study protocol. All participants were provided with calcium and vitamin D supplementation which is currently a standard of care for postmenopausal women. Subjects were randomized using a computer generated randomization table to ensure that all 72

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subjects had an equal opportunity to receive the study medication. Confidentiality was protected by using assigned subject numbers and no names, a nd current HIPAA laws were adhered to. All material related to the study subj ect was kept in a numbered file. All confidential subject files were kept inside the principa l investigators secure home in a locked filing cabinet. 73

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Table 3-1. Research Design for a two group st udy including an experimental group and a placebo group. R= randomization, O1= First laboratory values of CTX and OC, X= introduction of the black cohosh intervention, O2= Second laboratory values of CTX and OC. Duration of the study from O1 to O2 was 12 weeks. RGroup 1 O1 X O2 RGroup 2 O1 O2 Table 3-2. Timeline for study participants. Week 1 Week 2-4 Week 5-8 Week 9-12 Week 12 Visit 1: met the subject, inform subject about study, complete informed consent, complete demographic questionnaire and medical history form and answer questions. Visit 2: draw morning blood. Provide subject with 28 days worth of study medication and give subject instructions for taking the medication correctly, answer questions Each week subjects were called by phone to determine if there were any problems or questions. Visit 3: met with the subject, determine if there are any adverse events, answer any questions, and provide the subject with the next 28 days worth of study medication. Weekly calls to subject to determine if there were any problems or questions. Visit 4: met with the subject, determine if there are any adverse events, answer any questions, and provide the subject the final 28 days of study medication. Weekly calls to subject to determine if there were any problems or questions. Subject completed the final 28 days of medication. Visit 5: met with the subject to draw morning blood samples. Answer any questions the subject had and make a final determination if any adverse events occurred. 74

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Baseline Variables Serum CTX Serum o steoc alcin Sample 46 women between 1 and 6 years Postmenopausal Control Group Identical appearing Placebo by mouth daily Calcium carbonate + Vitamin D daily N=23 Experimental Group Black Cohosh 40 mg by mouth Daily Calcium carbonate + Vitamin D daily N=23 Outcome Variables Serum CTX Serum osteocalcin R Demographic Variables Age Years since Menopause BMI Education Figure 3-1. Conceptual framework and relationship of the research variable 75

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CHAPTER 4 ANALYSIS AND RESULTS Data analysis for this study was conducted using SPSS statistical so ftware version 11.5 (SPSS Inc., Chicago, IL). Descriptive statistics were obtained to provide summary measures for the data. Bivariate statistics were analyzed to compare the two groups, the black cohosh and the placebo group. Analysis of covariates (ANC OVA) was performed to test the research hypotheses and to answer the research questions. Forty-eight subjects we re recruited into the study to de termine the effect of the dietary supplement black cohosh ( Cimicifuga racemosa) on bone biochemical markers of resorption and formation in postmenopausal women. All subjects were recruited from the North Central Florida area. All subjects were females between the ages of 35 and 60 that had been estrogen depleted for at least one year and less than six years. A total of 48 subjects were recruited into the study, 46 subjects completed the study. Two subjects voluntarily dropped from the study, one due to weight gain and the other due to the inability to obtain the second blood sample by the principal investigator. All subjects were recruited through word of mout h, flyers, or discussion of the study in a public forum. Demographic Statistics All of the subjects who participated in the study were female and between the ages of 35 and 60. The overall mean age of the women was 53.44 (SD=4.70) with the minimum age of 36 and a maximum age of 60. The mean age of the subjects taking drug A was 54.08 (SD= 4.95) and the mean age of the subjects taking dr ug B was 52.79 (SD= 4.44). Age difference was not significant (p=.552) for Levenes test and the difference was not sta tistically signif icant (t-test= .951, p= .346). The largest portion of women in the study were in their fifties (83%), followed by women in their forties (6.3%), sixty year old women -(6.3%), and wo men in their thirties 76

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(4.2%). The overall mean body mass index (BMI) was 29.43 (SD= 5.71) with the minimum BMI of 20.14 and a maximum of 44.48. The mean BMI of subjects taking drug A was 29.02 (SD=5.38) and the mean BMI of those taking drug B was 29.84 (SD=6.09). Body Mass Index (BMI) was not significant (p= .502) for Levenes test and the differe nce between treatment groups was not significant (t-test= -.494, p= .623). The educational status of the subjects is as follows: 15 had a high school diploma (31.3%), 10 had an Associate degree (20.8%), 10 had a Masters Degree (20.8%), 8 had a Bachelors degrees (16.7%), 3 ha d completed a GED certificate (6.3%), and 2 had completed a doctoral degree (4 .2%). The years postmenopausal were five years (35%), four years (13%), three years (17 %), two years (10%) and one year (25%). The overall mean for years postmenopausal was 3.23 (SD= 1.63). The mean for years postmenopausal in the black cohosh group wa s 3.71 (SD=1.46) and the placebo group was 2.75 (SD=1.67). Years postmenopausal was not signifi cant (p=.225) for Levenes test, however the difference in mean years postmenopausal betwee n groups was significant (t-test= 2.114, p=0.04). Thirty (62.5) of the subjects were married, 13 (27.1%) were divorced, and 5 (10.4%) of the subjects were single. Thirty-f ive (72.9%) of the subjects were employed full time, 7 (14.6%) of the subjects were employed part time, 4 (8.3%) of the subjects were unemployed, and 2 (4.2%) of the subjects were retired. Blood pressure was assessed two times in the st udy, at the onset and at the conclusion. The mean group onset systolic blood pressure was 122.17mm/Hg (SD=16.01) and the mean group onset diastolic was 78mm/Hg (SD=8.31). The mean group conclusion systolic blood pressure was 123.09mm/Hg (SD=16.37) and the mean group conclusion diasto lic blood pressure was 77.28mm/Hg (SD=8.43). The mean onset systol ic blood pressure of subjects taking drug A was 121.21mm/Hg (SD=16.17) and subjects taking drug B was 122.13mm/Hg (SD= 16.14). The 77

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mean conclusion systolic blood pressure of subjects taking drug A was 123.67mm/Hg (SD= 18.50) and those taking drug B was 122.48mm/Hg (SD= 14.20). Systolic blood pressure at the onset (p= .680) and conclusion (p= .300) of the study were not significant for Levenes test and the difference between treatment groups was not si gnificant at the onset (t -test= -.411, p= .683) or at the conclusion (t-t est= .246, p= .807). The mean group onset diastolic blood pressure was 78.0mm/Hg (SD=8.31) and the mean group conclusion diastolic blood pressure was 77.28mm/Hg (SD=8.43). The mean onset diastolic blood pressure for those subject s taking drug A was 75.54mm/Hg (SD= 7.70) and subjects taking drug B was 80.46mm/Hg (SD= 8.31) The mean conclusion diastolic blood pressure for subjects taking drug A was 75.88mm/Hg (SD=10.06) and those taking drug B was 78.74mm/Hg (SD= 6.21). Diastolic blood pressure at the onset (p =.798) and at the conclusion (p=.099) of the study were not significant for Levenes test and there wa s no significant (p=.249) difference in means in the conc lusion diastolic blood pressure between the two groups. The onset mean diastolic blood pressure was signi ficantly (t-test=-2.125, p= 0.039) different between the two treatment groups. The means and standa rd deviations are summarized in Table 4-1. Frequency and percentages for categorical data are summarized in Table 4-2. Bivariate statistics were performed betw een the experimental and placebo group to evaluate any differences between the two groups. Age and body mass index (BMI) were analyzed using the independent samples t-test. Le venes test for equality of variances was not significant for either age (p=.552) or BMI (p=.502) demonstrating that both the black cohosh and the control group did not violate the assumption of homogeneity of variances. The t-test for equality of means was not significant for age (p=.346) or BMI (p=.623). 78

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Bivariate statistics were performed between the experimental and the placebo groups to evaluate differences in blood pressure. Levenes test for equality of variances was not significant for onset systolic blood pressure (p=.680), conclusion sy stolic blood pressure (p=.300), onset diastolic blood pressure (p=.798) and conclu sion blood pressure (p=.099) demonstrating that both the black cohosh and th e placebo group did not vi olate the assumption of homogeneity of variances. The ttest for e quality of means was significant for differences in onset diastolic blood pressure (t-test= -2.125, p=.039) but was not significant for conclusion diastolic blood pressure (t-tes t= -1.169, p=.249), onset systolic blood pressure (t-test= -.411, p= .683), and conclusion systolic blood pressure (t-test=.246, p=.807). Cross tabs analysis was conducted on educat ion and marital status as these were not continuous variables. The cross tabs analysis using Pearson Ch i-Square between the two groups (black cohosh or placebo) for education (p= .216) and marital status (p=.158) were not statistically significant. Hypotension is a documented side effect of black cohosh. Two separate analyses of covariates (ANCOVA) controlling for age and BMI were conducted to evaluate the effect of black cohosh on blood pressure, one on systolic blood pressure and one on diastolic blood pressure. The assumption for homogeneity of variance and normality were met in both analyzes. The results of the ANCOVA revealed no significant difference on systolic blood pressure levels within administration (pretreatment vs. posttreatment), F (1,43) = 3.655, p = .063; no significant group, (drug A vs. drug B) by administration (pre treatment vs. posttreatment) interaction on systolic blood pressure levels, F (1,43) = .314, p = .578; and no significant difference on systolic blood pressure levels between group (experiment al vs. control), F (1,43) = .032, p = .859. There 79

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was also no significant differen ce in BMI between the two groups Results are summarized in table 4-3. A second ANCOVA was conducted on diastolic blood pressure. Th e results of the ANCOVA revealed no significant di fference on diastolic blood pressure levels with groups (pretreatment vs. posttreatment), F (1,43) = 2.306, p = .136; no signifi cant group (drug A vs. drug B) by administration (pretreatment vs. pos ttreatment) interaction on diastolic pressure levels, F (1,43) = .469, p = .497; and no signifi cant difference on diastolic blood pressure between groups (experimental vs. control) F (1,43) = 3.909, p = .054. The results are summarized in Table 4-3. Research Question The purpose of this study was to determine if a standardized (2.5% tr iterpene glycoside) commercial preparation of the dietary supplemen t black cohosh would alter bone remodeling in the postmenopausal female. Bone remodeling is a coupled process including both resorption and formation. Usually in postmenopausal women both resorption and formation increase, but not in equal amounts, which results in a net bone loss. It was hypothesized that both resorption and formation would decrease with the administra tion of black cohosh. Bone resorption and formation were measured with two commercially available bone biochemical assays. Serum Cterminal telopeptide (C TX) was used to measure bone resorption and serum osteocalcin (OC) was used to measure bone formation. In order to determine if there was a statistically significant effect on C-terminal telopeptid e (CTX) from pretreatment to posttreatment, and between groups (black cohosh versus placebo), an analysis of covariates ( ANCOVA), controlling for age and BMI, was conducted. The assumptions for homoge neity of variance and normality were met. The results of the ANCOVA revealed no significan t difference in CTX levels within groups upon administration of black cohosh (pretreatment vs. posttreatment), F (1,42) = .332, P = .568; 80

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no significant group (drug A vs. drug B) by admini stration of black cohosh (pretreatment vs. posttreatment) interaction on CTX levels, F (1, 42) = .071, p = .791; and no significant difference on CTX levels between groups (e xperimental vs. control), F ( 1,42) = .095, p = .759. The results are summarized in Table 4-4. To determine if there was a statistically significant effect on serum osteocalcin (OC) levels from pretreatment to posttreatment a nd between groups who took a standardized dose of black cohosh and those that did not, while c ontrolling for both age and BMI, an ANCOVA was conducted. The assumptions of homogeneity of variance and normality were met. The results of the ANCOVA revealed no significant difference on OC levels within groups administration of black cohosh (pretreatment vs. posttreatment), F (1,42) = .184, p = .670; no significant group (drug A vs. drug B) by admini stration of black cohosh (pre treatment vs. posttreatment) interaction, F (1,42) = .255, p = .616; and no significant difference on OC levels between groups (experimental vs. control), F (1,42) = .172, p = .680. The results are summarized in table 4-4. 81

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Table 4-1. Means and Standard Deviations for Age, BMI, Years Postmenopause and Blood Pressure. Overall N= 46 Overall Minimum Overall Maximum Black Cohosh n= 23 Placebo n=23 Significance Age (years) 53.44 (SD=4.70) 36 60 54.08 (SD=4.95) 52.79 (SD=4.44) p= 0.346 Body Mass Index (BMI) 29.43 (SD=5.71) 20.14 44.48 29.02 (SD=5.38) 29.84 (SD=6.09) P=0.623 Years Postmenopausal 3.23 (SD=1.63) 1 5 3.71 (SD= 1.46) 2.75 (SD=1.67) P=0.040 Systolic Blood Pressure (mm/Hg) Onset Conclusion 122.17 (SD=16.01) 123.09 (SD=16.37) 92 95 148 165 121.21 (SD=16.17) 123.67 (SD=18.50) 123.13 (SD=16.14) 122.48 (SD=14.20) p=0.683 p=0.807 Diastolic Blood Pressure (mm/Hg) Onset Conclusion 78.00 (SD=8.31) 77.28 (SD=8.43) 60 57 96 101 75.54 (SD=7.70) 75.88 (SD=10.06) 80.46 (SD=8.31) 78.74 (SD=6.21) p=0.039 p=0.249 82

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Table 4-2. Frequency and Percentage on Education Education Frequency Percentage Associate Degree 10 20.8 Bachelors Degree 8 16.7 Doctoral Degree 2 4.2 GED certificate 3 6.3 High School Diploma 15 31.3 Masters Degree 10 20.8 83

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Table 4-3. Analysis of covariates on Systolic and Diastolic Blood Pressure after Controlling for Age and BMI. Source df F Sig. Power Within Groups Systolic Blood Pressure Levels Error Systolic Blood Pressure Levels*Drug Error 1 43 1 43 3.655 111.839 .314 111.839 .063 .578 .078 .007 .464 .085 Between Groups Systolic Blood Pressure Levels Drug Error 1 43 .032 389.259 .859 .001 .054 Within Groups Diastolic Blood Pressure Levels Error Diastolic Blood Pressure Levels*Drug Error 1 43 1 43 2.306 33.358 .469 33.358 .136 .497 .051 .011 .318 .103 Between Groups Diastolic Blood Pressure Levels Drug Error 1 43 3.909 100.359 .054 .083 .489 84

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Table 4-4. Analysis of covariates on CTX and OC Levels after Controlling for Age and BMI. Source df F Sig. Power Within Groups CTX Levels Error CTX Levels*Drug Error 1 42 1 42 .332 .025 .071 .025 .568 .791 .008 .002 .087 .058 Between Groups CTX Levels Drug Error 1 42 .095 .165 .759 .002 .060 Within Groups OC Levels Error OC Levels *Drug Error 1 42 1 42 .184 1.313 .255 1.313 .670 .616 .004 .006 .070 .078 Between Groups OC Levels Drug Error 1 42 .172 20.022 .680 .004 .069 85

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Table 4-5. Means and Standard Deviation of Serum Osteocalcin and C-terminal Telopeptide at the Onset and Conclusion of the Study Black Cohosh Placebo Significance Osteocalcin (ng/mL) Onset N=24 10.652 SD=2.78 11.187 SD=.4.16 p=0.603 Conclusion N=23 11.359 SD=2.40 11.510 SD=3.65 p=0.861 C-terminal Telopeptide CTX (ng/mL) Onset N=24 0.484 SD=.253 0.564 SD=0.322 P=0.346 Conclusion N=23 0.556 SD=0.330 0.575 SD=0.344 P=0.848 86

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CHAPTER 5 CONCLUSIONS AND DISCUSSION Menopause is a natural midlife transition that all women will experience as they age. Menopause is broadly defined as the final mens trual period and the termination of ovarian function resulting in a significant decrease in circul ating estrogen. This d ecrease in circulating estrogen produces a skewed form of bone remodeling, resulting in a net bone loss. This condition is termed postmenopausal osteoporosis. Postmenopausal osteopor osis currently affects more than eight million American women, and that number is expected to increase significantly in the next ten years. This net bone loss resu lts in a more fragile brittle bone, predisposing women to fractures. Every year more than 1.5 million fractures occur due to osteoporosis, resulting in an increased morbid ity and mortality. Estrogen replacement therapy has been shown to prevent bone loss and theref ore decrease the incidence of po stmenopausal osteoporosis. The publication of the Womens Health Initiative (WHI) study in 2002 produced a fear of estrogen therapy in both women and their healthcare pr oviders. Many health care providers will not prescribe estrogen therapy to healthy women, let alone women with chronic diseases such as diabetes, hyperlipidemia and hypertension. To find some relief for the vasomotor symp toms and to exert some autonomy over their lives, many postmenopausal women have turned to dietary supplements, resulting in a multibillion dollar industry. Dietary supplements such as black cohosh, evening primrose oil, chastebery tree extract, and many soy products are marketed to the postmenopausal woman looking for symptom relief. Many of these suppl ements are poorly regula ted and have little to no research documenting their effects. This study was done in an effort to determin e if the dietary supplement black cohosh had an effect on bone remodeling, and may therefore be an alternative option for retarding the bone 87

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loss that occurs during the menopausal year s. This study was a randomized double-blind, placebo-controlled clinical trial to evaluate the effect of a standardized dose of black cohosh on the two processes of bone remodeling, resorp tion and formation. Healthy postmenopausal women who currently chose not to take estrogen supplements were invited to participate. Discussion of the Findings Forty-eight healthy postmenopausal Caucasia n women were recruited into the study. Fortysix women completed the study. After cons ent the women were randomized into either an experimental group or a control group. After randomization serum blood samples were obtained and the women were provided with either study drug A or study drug B and instructed to take one capsule orally every day for 12 weeks. All women were provided with a bottle of Oscal + D and instructed to take one calcium tablet by mouth twice daily. Serum samples were obtained after 12 weeks of drug therapy. Bone bioc hemical assays were performed on both the pretreatment and posttreatment serum samples and the results were analyz ed using Analysis of Covariates (ANCOVA) after c ontrolling for age and BMI. Descriptive and bivariate statis tics revealed both groups had e qual number of subjects at 23 per group. All women were Caucasian females between the ages of 36 and 60. There was no statistically significant difference in ages be tween the black cohosh (52.79) and the placebo group (52.79). All of the subjects had been wit hout a menstrual period for at least one year but less than six years. Women with hysterectomy were included in the study if they had also had removal of the ovaries as well as the uterus. This was done in an effort to increase recruitment. Of the 48 subjects who entered the study 39 (81.3 %) were naturally menopausal and 9 (18.7%) were surgically menopausal. Thirty-one (64.6% ) subjects reported experiencing hot flashes entering the study and 17 (35.4%) reported no hot flashes prior to entering the study. Women entering the study were generally healthy and were included in the study if they had a health 88

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problem unrelated to bone metabolism, or their health problem was under control with medication. Women were excluded from the study if they had any medical problem which could interfere with bone metabolism such as diabetes parathyroid disease or osteoporosis. Women were also excluded if they had a health probl em which would prevent them from taking black cohosh such as liver or kidney disease or breast or endometrial cancer. Smokers were also excluded from the study. Women who were taking any form of estrogen supplement (pill, patch, injection, or ring) or were on a SERM (Evista) were excluded from the study. Women with medical conditions under control, unaffected by black cohosh, or with no effect on bone remodeling were recruited into the study. Women with thyroid disease were recruited into the study if they were controlled with medication and were currently euthyroid. The thyroid status was self repor ted and no thyroid labs were draw n and the patient medical files were not assessed. Forty (83.8%) subjects repo rted no thyroid disease and 8 (16.7) reported controlled thyroid disease. Ten (21%) of the women had hypertension controlled with medication. Four (8%) of the s ubjects reported having mitral valve prolapse (MVP) with no current symptoms. Bone biochemical assays were performed us ing two commercially available kits. Bone resorption was assessed utilizing an assay which measured the C-terminal telopeptide (CTX). There was no difference in mean values fo r CTX between the black cohosh group (.484ng/mL) and the placebo group (.563ng/mL) on th e first serum sample done prio r to instituting treatment. After 12 weeks of treatment serum samples were collected again. There was no difference in the mean values for CTX between the black cohosh group (.556ng/mL) and the placebo group (.575ng/mL). After 12 weeks of treatment w ith black cohosh there was no statistically significant difference in the mean values for serum CTX within each treatment group 89

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(pretreatment vs. posttreatment) or between treat ment groups (black cohosh vs. placebo). The results of this study reveal that a standardized dose (2.5% triterpene glyc osides) of black cohosh 40mg taken once daily by mouth has no effect on bone resorption. Bone formation was assessed utilizing an assay which measured serum osteocalcin (OC). There was no difference in mean values fo r OC between the blac k cohosh group (10.65ng/mL) and the placebo group (11.19ng/mL ) on the first serum sample done prior to instituting treatment. After 12 weeks of treatment seru m samples were performed again. There was no statistically significant difference in the mean values for OC between the black cohosh group (11.35ng/mL) and the placebo group (11.51ng/mL). After 12 weeks of treatment with black cohosh there was no difference in the mean valu es for serum OC within each treatment group (pretreatment vs. posttreatment) or between treat ment groups (black cohosh vs. placebo). The results of this study reveal that a standardized dose (2.5% triterpene glyc osides) of black cohosh 40mg taken once daily by mouth has no effect on bone formation. Blood pressure was assessed at th e beginning and at the end of the study in all subjects. All blood pressure measurements were obtained by th e principal investigator and were performed using an electronic blood pressure cuff. Seve ral subjects (21%) had e ssential hypertension prior to entering the study. Hypotension is a documente d side effect of black cohosh. There was no difference in the mean systolic blood pressu re between the black cohosh group (121.20mm/Hg) and the placebo group (123.12mm/Hg) at the onset of the study. There was no difference in the mean systolic blood pressure between the bl ack cohosh group (123.67mm/Hg) and the placebo group (122.48) at the conclusion of the study. Ther e was a statistically significant difference in the mean diastolic blood pressure between the black cohosh group (75.54mm/Hg) and the placebo group (80.46mm/Hg) at the onset of the st udy. However, at the conclusion of the study 90

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there was no difference in mean diastolic blood pressure between the black cohosh group (75.88mm/Hg) and the placebo group (78.74). The resu lts of this study show that black cohosh had no effect on blood pressure measurement. This study shows that even though black c ohosh may help women with the vasomotor symptoms of menopause it does not have an effect, either positive or negative on bone metabolism. This is an important fact for h ealth care providers to understand. As women age during menopause, bone loss is occurring. Women who choose not to take estrogen replacement have few options available to them to pr otect or rebuild bone. Many women are choosing supplements because advertising has led them to believe that supplements are better for them, and they are easily accessible without a pres cription. This study demonstrates that a standardized 40mg dose of black cohosh once daily has no effect on bone metabolism in women who are recently postmenopausal. Research Hypotheses The research hypotheses presented in this study were not supported. Two hypotheses were postulated and each will be discussed individually. Hypothesis 1: Postmenopausal women taking a standardized 40mg (2.5% triterpene glycoside) dose of an oral black cohosh supplement daily for twelve weeks will show a decrease from baseline in the level of serum C-terminal telopeptide, a biochemical marker of bone resorption. This hypothesis was not supported by the results of this study. The serum C-terminal telopeptide drawn at the conclu sion of the study did not significan tly change from the baseline levels drawn at the onset of the study in either the black cohosh group or the placebo group. However, it is possible that a low sample size coul d have led to a Type II error. This will be discussed later in this chapter. 91

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Hypothesis 2: Postmenopausal women taking a standardized 40 mg (2.5% triterpene glycoside) dose of an oral black cohosh supplement daily for twelve weeks will show a decrease from baseline in serum osteocalcin, a biochemical marker of bone formation. This hypothesis was not supported by the result s of this study. The serum osteocalcin drawn at the conclusion of the st udy did not significantly change fr om the baseline levels drawn at the onset of the study in either the black cohosh group or the placebo group. Again, the possibility of a Type II error is pr esent due to a low sample size. Strengths of the Study One of the strengths of the study is the study design. There are few randomized placebocontrolled, double-blind c linical trials on the dietary suppl ements used by postmenopausal women. Many of the studies done on black cohos h have been done wit hout a placebo, without blinding or randomization, and many were measuri ng only severity and frequency of hot flashes. Several studies have been conducted in Germa ny measuring bone metabolism, but those have study flaws as well. The strength of this st udy design is that it wa s randomized, double-blinded, and placebo-controlled. The study drug used in this study was obtained from a reputable source and was accompanied by a certificate of analysis which stated that there were no contaminants present. The entire supply of study drug came from the same lot number, which helped to assure that subjects were getting the same consistency and strength of study medication. Efforts to encourage treatment fidelity to the study protocol were as follows: 1) subjects were called weekly to remind them to take the study drug, as well as determine if any adverse events had occurred; 2) subjects met with the principal invest igator every four weeks and were told to return the previous bottle of st udy medication, which was assessed for pill count; 3) subjects were given 92

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the phone number of the principal investigator in case there was any problems with adhering to the study protocol. A strength of the study is that all of the bone biochemical assays were performed by the same person, the principal researcher. The prin cipal researcher perfor med all of the assays required for the study on freshly thawed serum samples. No refrozen serum samples were utilized. The researcher was a novice lab technician, but all samp les were assayed in exactly the same manner increasing intrarater reliability. All serum samples were collected, centrifuged, and stored in the same manner by the same research er guaranteeing that the samples were handled with consistency. Limitations of the Study Design Limitations Every study no matter how well planned will have limitations. This study had several study design limitations. One limitation dealt with the subjects medical history. The medical history questionnaire was filled out by the subject prior to beginning the study. The researcher counted on the subject honestly filling out the fo rm. No medical records or lab reports were obtained by the researcher prior to or during the study. If the subject had lab work documenting menopause or a stable thyroid c ondition the researcher did no t obtain any medical records, relying on the subject for the correct value and interpretati on. One question on the medical history questionnaire asked the que stion Do you currently partic ipate in a regular exercise program? Subjects had a difficult time determining what exercise constituted a regular exercise program. If they walked 20 minut es once a week they considered this regular exercise. This question is often on health histories and subjects will often exaggerate how much they exercise. Some subjects considered exercise caring for their grandchildren for the day. It was left up to the researcher to determine what constituted regular exercise and the definition of regular exercise 93

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was at least 30 minutes of exercise at least three times a week. In addition, subjects were advised not to start a regular exercise program as regular exercise can have an effect on bone metabolism. This encouragement not to exercise for the duration of the twelve week study could constitute a break in the standard of care for postmenopausal women. As health care providers we encourage all women to participate in a regular exercise program for bone, cardiac, and generalized good health. As with all studies, subject recruitment pr oved to be more difficult than expected. Inclusion and exclusion criteria were strict and many wome n who were interested in the study were excluded for any number of reasons. The incentive for women to take black cohosh is that they consider it a natural alte rnative to estrogen and it is av ailable without a prescription. Therefore women who had contraindications to estrogen replacement therapy such as breast cancer, older age, heart attack, or stroke felt that they should be able to take black cohosh. They however were excluded from the study. Two subj ects with a history of breast cancer told me their physician said they should take black cohosh and did not unders tand why they were excluded from the study. Many women who ha d completed menopause many years before, but had been taken off estrogen therapy were also interested in the st udy. It was very frustrating for the researcher to be turning down more people than were accepted into the study. In an effort to boost subject recruitment several changes in the inclusion/exclusion criteria were made halfway through data collec tion. It was decided to accept women who had a hysterectomy as long as they had their ovaries removed as well. The number of years postmenopause remained at less than six years, but the age range was changed from 45-60 to 3560. This enabled the researcher to accept nine more subjects. Women with thyroid disease 94

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under control with medication were also accepted into the study. This change allowed 8 more subjects into the study. A recalculation of sample size was done approximately halfway through the study due to low recruitment. The power remained at 80% a nd the alpha level remained at 0.05 but the effect size was altered. Initially, the effects size wa s based on the lower effect size of the bone biochemical marker serum osteo calcin. The serum C-terminal te lopeptide had a higher effect size. It was decided by the researcher to lower the effect size by averagin g the effect size of the two bone markers. This new effect size re quired only 23 subjects per group instead of 49 subjects per group. This smaller sample size increases the risk for a Type II error. To increase recruitment in to the study the researcher also used a convenience, nonrandom sampling technique. The researcher recruited heavily at her place of employment, a local hospital in Marion County, by visiting many of the departments and talking with the female employees. All departments of the hospital were visited and the resear cher discussed the study with the staff for approximately 5-10 minutes. Ni neteen (40%) subjects we re recruited from the hospital environment. The other subjects were recruited by word of mouth (37%) and flyers (23%) posted at health food stores, groceries stor es, the local health department, churches, and the University of Florida parking garage. Using this form of sampling can pose a threat to the external validity of the study. Another limitation of the study was that th e investigator did no t devise a method for determining if the subjects took th eir calcium supplement correctly or at all. The investigator called the subjects weekly to inqui re if the subject had experienced any adverse effects, and to remind them to take the study medication. The investigator did not follow up on compliance to the calcium regimen unless the subject provided that information without prompt. Calcium can 95

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have an effect on bone remodeling and this may have had an effect on the possibility of a Type II error. Another limitation of the study is that the bone biochemical assays were performed by the principal investigator. The principal investigat or is a novice lab technician and mistakes in performing the assays might have occurred. One subject in the study had very high levels of serum osteocalcin, markedly different from the other subjects in the study. The assay was performed again on a fresh sample of the subjects serum and the results were not significantly different. This resulted in the subject being cla ssified as an outlier, a nd therefore dropped from the statistical analysis. The inve stigator performed the assays af ter the minimal training required by the laboratory. The assays were performed by the researcher, but overseen by the laboratory manager at the College of Nursing physiology wet lab and under the guidan ce of the Doctoral Supervisory Chair. Statistical Analysis Limitations All studies are at risk for Type II errors. Type II errors can occur when the researcher incorrectly fails to reject th e null hypothesis. The data ma y not support rejecting the null hypothesis when in truth the null hypothesis is not true. The data from this study do not reject the null hypothesis, and it was dete rmined that there was no change in bone biochemical markers from baseline after 12 weeks of black cohosh th erapy. If in the population black cohosh does significantly lower bone remodeling, then a Type II error occurred. A T ype II error may occur with small sample sizes and an incorrect sample size, both of which are possible in this study Conclusions The conclusion drawn from this study is th at the black cohosh did not demonstrate any effect on bone remodeling in the postmenopausal female subjects. The study revealed no statistically significant differences in either the bone resorption or bone formation marker after 96

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taking a standardized 40mg dose of black cohosh for 12 weeks. However, it continues to be difficult to rule out the possibili ty that black cohosh has an effect on bone remodeling. It is possible for this researcher to conclude that the results of the study occurred due to a small sample size and the utilization of the larger effect size. Another conclusion drawn from this study is that black cohosh ha d no effect on either systolic or diastolic blood pr essure. Blood pressures were m easured at the onset and the conclusion of the study period and no significant change in blood pr essure was demonstrated. It can be concluded that black cohosh is a safe alternative for postmenopausal women who are normotensive or have controlled hypertension. This study revealed different findings from those documented in the study by Wuttke and colleagues (2006) who described an increase in os teoblast activity with a standardized dose of black cohosh. Wuttkes study had three groups: bl ack cohosh, conjugated estrogens, and placebo with a total N= 62 (approximate ly 20 subjects per group). The dose of black cohosh and the duration of treatment were the same as the cu rrent study. The inclusi on criteria for the Wuttke study were not as strict. All women between th e ages of 40 and 60 were included in the study. The number of years postmenopause was not addr essed and women who had been amenorrheic for 6 months were included. Wo men with a Body Mass Index (BMI) of greater than or equal to 30 were excluded from the study. The difference in the results of the cu rrent study and the study done by Wuttke et al. could be the more stringent inclusion and exclusion criteria in the current study. Most importantly, the study by Wuttke et al. included both preand postmenopausal women with highly variable estr ogen status whereas the current study was restricted to women who were estrogen deplete for 1-6 years. 97

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Recommendations for Future Research Although this study did not find any statistically significant effect on bone remodeling with black cohosh, it is possible for future studies to observe a positive effect. Prior studies conducted in Germany measuring the effect of black cohosh on bone metabolism have shown positive results. Currently there are other studies in progress evaluating the effect of black cohosh on bone in both Germany and the United States. In addition, studies measuring the effect of black cohosh on hot flashes continue, as the studies previously conducted have demonstrated conflicting results. The sale of black cohosh is a multimillion dollar industry and numerous brands and variations of it exist in the retail market. It is important that research continue on black cohash as women will continue to take this supplement as it is easy to obtain, and has a good word of mouth reputation. The future direction in bl ack cohosh research is to: 1) conduct more randomized, double-blind, placebo-controlled clinical trials on the effect of black cohosh on bone remodeling; 2) conduct stud ies to identify the mechanism of action of black cohosh; 3) continue research on its effect on the severity and frequency of hot flashes; 4) conduct studies to identify the mechanism of action for liver damage and failure in women taking black cohosh. Implications for Clinical Practice It is imperative for the womens healthcar e provider to be knowledgeable about the common dietary supplements postmenopausal women take. It is often not acknowledged that women take herbal supplements in the healthcar e providers office. It is important for the healthcare provider to understand th at most dietary supplements have little to no clinical research done on them, and what is done is often poorly designed and sponsored by the supplement company. The current goal of the nursing healthcare provider is to document the use of dietary 98

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supplements and to educate the client on the cu rrent research, contrai ndications, and adverse effects of the most commonly used dietary supplements. It is also in the realm of nursing research to continue with well desi gned clinical trials on dietary supplements. Although this study did not demonstrate that black cohosh had any effect on bone remodeling, future nursing studies may. It is vitally important th at nurse researchers perform well-designed clinical tr ials on dietary supplements. Th e American public is spending billions of dollars on dietary supplements that have little to no clinical research (Bent & Ko, 2002). Postmenopausal women aged 45-60 are th e top spenders on dietary supplements, many purchasing products to alleviate the vasomotor sy mptoms of menopause such as hot flashes and insomnia (Kang, Ansbacher, & Hammoud, 2002). The future direction of dietary research should include studies to evalua te effectiveness, safety, a nd the interaction of dietary supplements with other drugs. Consumers and hea lth care providers need to be educated about dietary supplements. Nurse researchers are in a great position to participate fully in supplement research and then educate the consumer w ith the most up to date information. 99

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APPENDIX A DEMOGRAPHIC INFORMAT ION QUESTIONNAIRE Name ________________________________________ Last first m.i. Address _____________________________________ ______________________________________________ Telephone home ( ) work ( )_ ________________ Age ___________ Date of Birth _______________________ Marital Status ______________________ Highest educational degree ____ Middle School ____ GED Certificate ____ High School Diploma ____ Associate Degree ____ Bachelors degree ____ Masters degree ____ Doctoral degree ____ Other degrees Please list ________________________ Present work status ____ Not employed ____ Disabled ____ Working Part time ____ Working Full time ____ Retired Last menstrual period ________________________ Date form completed __________________ 100

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APPENDIX B MEDICAL HISTORY QUESTIONNAIRE Name _____________________________ Id number __________________ General Health History Yes No ___ ___ 1. Do you consider yourself to be generally healthy? ___ ___ 2. Have you ever been diagnosed with a heart condition? ___ ___ 3. Have you ever been diagnosed with high blood pressure? ___ ___ 4. Have you ever had a heart attack? ___ ___ 5. Have you ever had a stroke? ___ ___ 6. Have you ever been diagnosed with diabetes? ___ ___ 7. Have you ever been diagnosed with kidney disease? ___ ___ 8. Have you ever been diagnosed with a thyroid condition? ___ ___ 9. Have you ever been diagnosed with a parathyroid problem? ___ ___ 10. Have you ever been diagnosed with osteoporosis? ___ ___ 11. Have you ever been diagnosed with breast cancer? ___ ___ 12. Have you ever been diagnosed with a blood disorder? Medication History Yes No ___ ___ 1. Have you ever taken bi sphosphonates (Actonel, Fosamax, Boniva)? ___ ___ 2. Have you ever taken parathyroid hormone (Forteo, Teriparatide)? ___ ___ 3. Are you currently taking estrogen replacement? ___ ___ 4. Are you taking any over the counter dietary supplements? List _________ ____________________________________________________________ ___ ___ 5. Are you taking any medication for osteoporosis? ___ ___ 6. Are you currently taking Evista? ___ ___ 7. Are you currently taking calcium supplements? ___ ___ 8. Are you currently taking oral contraceptives (birth control pills)? 9. Please list all medications and supplements you are taking below: _______________________________ _________________________ _______________________________ _________________________ _______________________________ _________________________ Social History Yes No ___ ___ 1. Do you currently smoke or use tobacco products? ___ ___ 2. Do you currently drink alc ohol? If yes how much and how often?___ _______________________________________________________ ___ ___ 4. Do you currently particip ate in a regular exercise program? If yes please list __________________________________________ 101

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Reproductive History Yes No ___ ___ 1. Are you currently menopausal (having no menstrual period)? Date of last menstrual period _______________________ ___ ___ 2. Do you still have your uterus? ___ ___ 3. Do you still have your ovaries? ___ ___ 4. Are you currently experiencing hot flashes? ___ ___ 5. Have you ever experienced hot flashes? ___ ___ 6. Have you been told by a h ealth care provider that you are menopausal or postmenopausal? ___ ___ 7. Have you had lab work done by a health care provider th at confirms you are menopausal? Signature of Participant __________________________________ Signature of Investigator __________________________________ Date Form Completed ____________________________________ ______ Recommended for study ______ Not recommended for study 102

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APPENDIX C CERTIFICATE OF ANALYSIS 103

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APPENDIX D INVESTIGATIONAL NEW DRUG NUMBER (IND) 104

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LIST OF REFERENCES Bedi, M. K., & Shenefelt, P. D. (2002). Herbal therapy in dermatology. Achives of Dermatology, 138, 232-242. Akesson, K. (2003). New approaches to pharmacological treatment of osteoporosis. Bulletin of the World Health Organization, 81, 657-664. Aloia, J. F., Mikhail, M., Paga n, C. D., Arunachalam, A., Yeh, J. K., & Flaster, E. (1998). Biochemical and hormonal variables in bl ack and white women matched for age and weight. Journal Lab Clinical Medicine, 132, 383-389. Aloia, J. F., Vaswani, A., Yeh, J. K., & Flaster, E. (1996). Risk of oste oporosis in black women. Calcified Tissue International, 59, 415-423. Amato, P., Christophe, S., & Mellon, P. L. (2002) Estrogenic activity of herbs commonly used as remedies for menopausal symptoms. Menopause, 9, 145-150. Athanasiou, K. A., Zhu, C. F., Lanctot, D. R., Agrawal, C. M., & Wang, X. (2000). Fundamentals of biomechanics in tissue engineering of bone. Tissue Engineering, 6, 361381. Aubin, J. E., & Bonnelye, E. (2000). Osteoprotegerin and its ligand: A new paradigm for regulation of osteoclastog enesis and bone resorption. Osteoporosis International, 11, 905-913. Barnes, J. (2003). Quality, efficacy and safety of complementary medicines: fashions, facts and the future. Part I. Regulation and quality. Journal of Clinical Pharmacology, 55, 226233. Barrett, E. J. (2005). Organizati on of endocrine control. In W. F. Boron, & E. L. Boulpaep (Eds.), Medical Physiology (pp. 1005-1021). Philadelphia: Elsevier Saunders. Barrett, E. J., & Barrett, P. (2005) The parathyroid gland and vitamin D. In W. F. Boron, & E. L. Boulpaep (Eds.), Medical Physiology (pp. ). Philadelphia: Elsevier Saunders. Barrett-Connor, E., Siris, E. S., Wehren, L. E., Mill er P. D., Abbott, T. A., & Berger, M. L. et al. (2005). Osteoporosis and fracture risk in women of different ethnic groups. Journal of Bone and Mineral Research, 20, 185-194. Blair, H. C., Robinson, L. J., & Zaidi, M. (2005). Osteoclast signaling pathways. Biochemical and Biophysical Research Communications, 328, 728-738. Blumsohn, A., Herrington, K., Hannon, R. A., Shao, P., Eyre, D. R., & Eastell, R. (1994). The effect of calcium supplementation on th e circadian rhythm of bone resorption. Journal of Clinical Endocrinolog y and Metabolism, 79, 730-735. 106

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Boivin, G., & Meunier, P. J. (2002). The degree of mineralization of bone tissue measured by computerized quantitative contact microradiography. Calcified Tissue International, 70, 503-511. Bord, S., Horner, A., Beavan, S., & Compston, J. (2001). Estrogen receptors and are differentially expressed in developing human bone. The Journal of Clinical Endocrinology & Medicine, 86, 2309-2314. Castracane, V. D., Kraemer, G. R., Oge n, B. W., & Kraemer, R. R. (2005, May 20). Interrelationships of serum estradiol, estrone, and estrone sulfate, adiposity, biochemical bone markers, and leptin in postmenopausal women. Maturitas, Epub(ahead of print), 19. Chailurkit, L., Ongphiphadhanakul, B., Piase u, N., Saetung, S., & Rajatanavin, R. (2001). Biochemical markers of bone turnover and response of bone mineral density to intervention in early postmenopausal women: An experience in a clinical laboratory. Clinical Chemistry, 47, 1083-1088. Chen, S., Li, W., Fabricant, D. S ., Santarsiero, B. D., Mesecar, A., & Fitzloff, J. F. et al. (2002). Isolation, structure elucidation, and absolu te configuration of 26-deoxyactein from Cimicifuga racemosa and clarification of nomenclature associated with 27-deoxyactein. Journal of Natural Products, 65, 601-605. Dawood, M. Y. (2000). Menopause. In L. J. Copeland, & J. F. Jarrell (Eds.), Textbook of gynecology (2 ed., pp. 603-629). Philadelph ia: W.B. Saunders Company. Dawson-Hughes, B. (2000). Calcium, vitamin D, and bone metabolism. In L. V. Avioli (Ed.), The osteoporotic syndrome (4th ed., pp.). San Diego: Academic Press. Dawson-Hughes, B., Dallal, G. E., Krall, E. A., Sadowski, L., Sahyoun, N., & Tannenbaum, S. (1990). A controlled trial of the effect of calcium supplementation on bone density in postmenopausal women. The New England Journal of Medicine, 323, 878-883. Dawson-Hughes, B., Harris, S. S., Krall, E. A., & Dallal, G. E. (1997). Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older. The New England Journal of Medicine, 337, 670-676. Delmas, P. D. (2000). Markers of bone turnover for monitoring treatment of osteoporosis with antiresorptive drugs. Osteoporosis International, Supp 6, S66-76. Delmas, P. D., Eastell, R., Garnero, P., Seibel, M. J., & Stepan, J. (2000). The use of biochemical markers of bone turnover in osteoporosis. Osteoporosis International, Supp 6, S2-17. Doran, P. M., & Khosla, S. (2000). Senile osteoporosis. In J. E. Henderson, & D. Goltzman (Eds.), The osteoporosis primer (pp. 225-235). Cambridge, United Kingdom: Cambridge University Press. 107

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Gomez, B., Ardakani, S., Evans, B. J., Merrell, L. D., Jenkins, D. K., & Kung, V. T. (1996). Monoclonal antibody assay for free ur inary pyridinium cross-links. Clinical Chemistry, 42, 1168-1175. Greendale, G. A., Lee, N. P., & Arriola, E. R. (1999). The menopause. Lancet, 353, 571-579. Greenspan, S. L., Resnick, N. M., & Parker, R. A. (2005). Early changes in biochemical markers of bone turnover are associated with long-term changes in bone mineral density in elderly women on alendronate, hormone replacement th erapy, or combination therapy: A threeyear, double-blind, placebo-controlle d, randomized clinical trial. The Journal of Clinical Endocrinology & Metabolism, 90, 2762-2767. Gundberg, C. M., Looker, A. C., Nieman, S. D., & Calvo, M. S. (2002). Patterns of osteocalcin and bone specific alkaline phosphatase by age, gender, and race or ethnicity. Bone, 31, 703-708. Han, Z. H., Palnitkar, S., Rao, D. S., Nelson, D., & Parfitt, A. M. (1997). Effects of ethnicity and age or menopause on the remodeling and turnover of iliac bone: Implications for mechanisms of bone loss. Journal of Bone and Mineral Research, 12, 498-508. Hanley, D. A. (2000). Biochemical markers of bone turnover. In J. E. Henderson, & D. Goltzman (Eds.), The osteoporosis Primer (pp. 239-252). Cambridge, United Kingdom: Cambridge University Press. Hardy, M. L. (2000). Herbs of special interest to women. Journal of the American Pharmaceutical Association, 40, 234-241. Hofbauer, L. C., Khosla, S., Dunstan, C. R., Lacey, D. L., Spelsberg, T. C., & Riggs, B. L. (1999). Estrogen stimulates gene expression an d protein production of osteoprotegerin in human osteoblastic cells. Endocrinology, 140, 4367-4370. Igarashi, P. (2005). Regulation of gene expression In W. F. Boron, & E. L. Boulpaep (Eds.), Medical Physiology (pp. 115-144). Philadelphia: Elsevier Saunders. Jarry, H., Metten, M., Spengler, B ., Christoffel, V., & Wuttke, W. (2003). Invitro effects of the cimicifuga racemosa extract BNO 1055 Maturitas, 44 (Supp 1), S31-38. Jee, W. S. (1988). The skeletal tissues. In L. Weiss (Ed.), Cell & tissue biology (6 ed., pp. 213254). Baltimore, MD: Urban & Schwarzenberg. Jee, W. S. (1999). Structure and function of bone tissue. In F. Bronner, & R. V. Worrell (Eds.), Orthopaedics: Principles of basic and clinical science (pp. 3-27). Boca Raton, FL: CRC Press. 109

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BIOGRAPHICAL SKETCH Alice Peters Carlisle graduated from Palm B each Community College with an Associate of Science degree in nursing in 1979. She began he r nursing career working as a staff nurse on a medical-surgical floor at Good Samaritan Hospital in West Palm Beach Florida. In 1981 she went to work in the labor and delivery unit, and that was the beginni ng of a lifelong love of womens health care and the care of pregnant women. She worked as a labor and delivery nurse in West Palm Beach, Vero Beach, and Gainesville Florida until 1995, when she began her career as a nurse midwife. Alice completed a Bachelor of Science degree in nursing in 1992 and a Master of Nursing with a certificate in nur se midwifery in 1995 at th e University of Florida. She began working as a nurse midwife in private practice in Ocala Fl orida with Rasik Nagda MD in January 1996 and stayed at that practice until June 2004. In 2003 Alice began her doctoral studies at the Un iversity of Florida wi th an interest in postmenopausal womens health. In 2004 she left private practice and began working part time for Midwives of Ocala, a hospital owned midwifer y service. She continues to work for them providing prenatal care and hospita l birth to the women of Marion County. Since 2004 Alice has also worked part time as a family planning nur se practitioner at the Marion County Health Department Belleview clinic. Alice was also an American Society of Ps ychoprophylaxis in Obstet rics (ASPO) certified childbirth educator and taught childbirth clas ses in Palm Beach County from 1981 until 1989. She continues to use that knowledge to educate pregnant women in her current position. Alice was also a part time adjunct clin ical professor at the University of Florida College Of Nursing from 2005 to 2006. 117

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Alice is an active member of the American College of Nurse Midwives (ACNM), and served as the secretary of the Gain esville-Ocala chapter fo r four years. Alice is also a member of the American Nurses Association (ANA), Flor ida Nurses Association, and Sigma Theta Tau (Alpha Theta chapter). 118