Oxidative Stress and the Risk of Osteoporosis: The Role of Dietary Polyphenols and

Nutritional Supplements in Postmenopausal Women

by

Nancy N. Kang

A thesis submitted in conformity with the requirements for the degree of Masters of Science

Graduate Department of Nutritional Sciences

University of Toronto

© Copyright by Nancy N Kang (2012)

ii

Oxidative Stress and the Risk of Osteoporosis: the Role of Dietary Polyphenols and

Nutritional Supplements in Postmenopausal Women

Nancy N Kang

Masters of Science

Department of Nutritional Sciences

University of Toronto

2012

Abstract

Previous findings have indicated that oxidative stress plays a role in the development of

osteoporosis and that individual polyphenols, by virtue of their antioxidant properties, may

mitigate these damaging effects. Nutritional supplements, greens+ bone builderTM, containing

polyphenols and other micronutrients beneficial for bone health are of recent interest as

complementary strategies in the management of osteoporosis. A randomized controlled study

was conducted to explore the combined effects of the nutrients found within the supplement

on bone health for 8 weeks. Total polyphenol content and antioxidant capacity increased

whereas oxidative stress parameters and the bone resorption marker, crosslinked C-

telopeptide of type I collagen decreased after supplementation. There was no significant

change in the bone formation marker, procollagen type I N-terminal propeptide. This thesis

shows an association of polyphenols with other micronutrients acts through their antioxidant

capacity to decrease oxidative stress parameters and bone resorption, thus potentially

reducing the risk for osteoporosis.

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Acknowledgments

It is with immense gratitude that I acknowledge the support and help of my

supervisor, Dr. A. Venketshewer Rao (aka “Venket”), who had to bear with me throughout

my thesis with his patience and ingenious knowledge. You continually conveyed a spirit of

adventure in regard to research and scholarship, as well as an excitement in regard to

teaching. Without your commitment and guidance, this thesis would not have been possible.

I am honoured to have you as a supervisor.

I would like to further express my sincerest gratitude to my co-supervisor, Dr. Leticia

G. Rao (aka “Letty”), who has the attitude and the substance of a genius. With her

enthusiasm, inspiration, and efforts to explain things clearly and simply, she helped to make

my thesis gratifying. I could not have wished for a better or friendlier co-supervisor. As well,

this thesis, too, would not have been completed or written.

I would like to thank all the members of my committee, Drs. R. Bazinet, H.

Tenenbaum and R.G. Josse, for offering their time and individual expertise to support and

guide me though this exciting and daunting journey.

I am indebted to many of my colleagues for helping me allocate my resources within

their labs. Bala, thank you for allowing me to store my samples in your freezer at the

FitzGerald Building. Dr. Semple’s technicians: Michael Kim and Edward Speck for their

guidance and knowledge when needed as well as storing my urine samples in their freezer.

Thank you to Christopher Spring and Pamela Plant at the core lab of St. Michael's Hospital

LKSKI for the use of the freezers and the full spectrophotometer. A very special thanks to

Dr. P. Connelly and Graham Maguire, not only do I thank you both for generously allowing

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me to use their lab space in the new Li Ka Shing Knowledge Institute (LKSKI), but for your

valued friendship, advice and guidance you have granted me throughout this journey.

In addition, I thank many of my student colleagues; Loren Chan, Katrine De Asis and

Dawn Snyder, for their help with some of the assays and for providing a stimulating and fun

environment to laugh, learn and grow in. It was a pleasure to work with each and every one

of you.

Funding is shared by the Canadian Institutes of Health Research (CIHR) and the

industrial partner, Genuine Health Inc, Toronto, Canada. We also thank Genuine Health Inc.

for providing the supplement and placebo products used in this study.

I wish to thank all of my great friends for helping me get through the difficult times;

smiles, giggles, emotional support, camaraderie, entertainment, and caring each of you

blessed me with. Noteworthy is Michael, my foodie boy friend, thank you for taking such

great care of me- knowing when I should relax, exercise and eat.

I am grateful to the secretaries of the department of Nutritional Sciences, Louisa and

Emeliana, for helping the departments to run smoothly and for assisting me in many different

ways to get this thesis completed on time.

I wish to thank my entire extended Kang and Simmonds family for providing a loving

environment for me. In tribute to Uncle David Simmonds, Rest in Peace.

Lastly, and most importantly, I wish to thank my family; sister, Jane; brother, Leo;

parents, Kyung-nim “Kay” and Dae-soo “David”. You have been very supportive and such

great role models for always believing in me. You taught me how hard work and

perseverance pays off in the end. I thank you for always comforting me and offering endless

encouragement. To them I dedicate this thesis.

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Table of Contents

ABSTRACT II ACKNOWLEDGEMENTS III LIST OF TABLES VII LIST OF FIGURES VIII LIST OF APPENDICES X LIST OF ABBREVIATIONS XI CHAPTER 1. INTRODUCTION 1 2. LITERATURE REVIEW 2

I. Postmenopausal Osteoporosis A. Overview 3 B. Bone biology: Remodelling and pathophysiology 4

of osteoporosis C. Risk factors for osteoporosis 10 D. Bone biology: Biomarkers of bone turnover as 13

predictors of bone loss and individual risk of fracture in participants with postmenopausal osteoporosis

II. Oxidative Stress A. Overview 18 B. Oxidative damage to cell components 18 C. Defense mechanisms 19 D. Oxidative stress and the risk for the 20

development of osteoporosis III. Antioxidant Phytochemicals

A. Overview 24 B. Structure and food sources 24 C. Dietary intake 29

3. RATIONALE, HYPOTHESIS AND OBJECTIVES 30

I. Rationale 31 II. Hypothesis 32 III. Objectives 32 IV. Subjects and Methods

A. Participant recruitment and sample collection 33 B. Serum analysis 34 C. Urine analysis 35

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4. ANTIOXIDANT EFFECTS OF A NUTRITIONAL SUPPLEMENT 37

CONTAINING POLYPHENOLS IN POSTMENOPAUSAL WOMEN: A RANDOMIZED CONTROLLED STUDY

I. Abstract 38 II. Introduction 39 III. Methods 41 IV. Results 42 V. Discussion 48

5. DIETARY POLYPHENOLS AND NUTRITIONAL SUPPLEMENTS 51

SIGNIFICANTLY DECREASED OXIDATIVE STRESS PARAMETERS AND THE BONE RESORPTION MARKER COLLAGEN TYPE 1CROSS-LINKED C-TELOPEPTIDE IN POSTMENOPAUSAL WOMEN

I. Abstract 52 II. Introduction 54 III. Methods 56 IV. Results 57 V. Discussion 65

6. GENERAL DISCUSSION AND CONCLUSIONS 71

I. Discussion of results 72 II. Implication of findings 79 III. Limitations of study 80 IV. Future area of research 81 V. Conclusions 82

7. LIST OF REFERENCES 83 8. APPENDICES 95

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LIST OF TABLES TABLES PAGE CHAPTER 2

Table 2.1: Biochemical markers of bone turnover 15 including bone formation and bone resorption markers. Table 2.2: Classification of flavonoids, general chemical 26 structure and food sources. Table 2.3: Classification of non-flavonoids, general chemical 27 structure and food sources.

CHAPTER 4

Table 4.1: Participant characteristics and baseline values 44 for total antioxidant capacity and oxidative stress parameters for each treatment group.

CHAPTER 5

Table 5.1: Participant characteristics and baseline values 59 for total polyphenol content, total antioxidant capacity, oxidative stress parameters, and bone turnover markers for each treatment group.

viii

LIST OF FIGURES

FIGURES PAGE CHAPTER 2

Figure 2.1: Model for bone remodelling. 7 Figure 2.2: Signalling pathway of ROS affecting osteoblasts and osteoclasts. 10 Figure 2.3: The role of oxidative stress in osteoporosis 23 and how/where antioxidants play a role in mitigating ROS. Figure 2.4: Classification of polyphenols. 25

CHAPTER 3

Figure 3.1: Illustration of hypothesis 32 Figure 3.2: Study design. 34

CHAPTER 4 Figure 4.1a: Significant time by treatment effect on total 45 antioxidant capacity after 8 weeks of supplementation. Figure 4.1b: The change in Trolox observed in the Treatment 45

group was significantly different from the change seen in the Placebo group at each time point.

Figure 3.2a: Significant time by treatment effect on lipid 46 peroxidation after 8 weeks of supplementation. Figure 3.2b: The change in lipid peroxidation observed in 46

the Treatment group was significantly different from the change seen in the Placebo group at each time point.

Figure 3.3a: Significant time by treatment effect on protein 47 thiols after 8 weeks of supplementation. Figure 3.3b: The change in protein thiols observed in the 47

Treatment group was significantly different from the change seen in the Placebo group at each time point.

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CHAPTER 5 Figure 5.1a: The Supplement group had significantly 60 decreased in CTX after 8 weeks.

Figure 5.1b: After 8 weeks of supplementation, the 60 change in CTX seen in the Supplement group was significantly

different from the change seen in the Placebo group.

Figure 5.1c: The Supplement group had no significant 60 change in PINP after 8 weeks.

Figure 5.1d: After 8 weeks of supplementation, the 60 change in PINP seen in the Supplement group was not significantly

different from the change seen in the Placebo group. Figure 5.2a: The Supplement group had significantly 61 increased total polyphenol content after 8 weeks. Figure 5.2b: After 8 weeks of supplementation, the 61 change in total polyphenol content observed in the Supplement group was significantly different from the change seen in the Placebo group. Figure 5.3a: The Supplement group had significantly increased 62 total antioxidant capacity after 8 weeks. Figure 5.3b: After 8 weeks of supplementation, the change in 62 total antioxidant capacity observed in the Supplement group was significantly different from the change seen in the Placebo group. Figure 5.4a: The Supplement group had significantly decreased 63

lipid peroxidation after 8 weeks of supplementation. Figure 5.4b: After 8 weeks of supplementation, the change in 63 lipid peroxidation observed in the Supplement group was significantly different from the change seen in the Placebo group. Figure 5.5a: The Supplement group had significantly increased 64

protein thiols after 8 weeks of supplementation. Figure 5.5b: After 8 weeks of supplementation, the change 64 in protein thiols observed in the Supplement group was significantly different from the change seen in the Placebo group.

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LIST OF APPENDICES APPENDIX I PAGE greens+ bone builderTM ingredients 95 APPENDIX II Informed consent package for clinical study 97 APPENDIX III Food record 109

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LIST OF ABBREVIATIONS

1O2 singlet oxygen 8-iso-PGF2α 8-iso-Prostaglandin F2α 8-OH-dG 8-Hydroxy-2’-deoxyguanosine A, B, C ABTS 2, 2, azinobis (3-ethylbenzothiazoline-6-sulfonic acid) ABTS•+ 2, 2, azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation ALP alkaline phosphatase ANOVA analysis of variance AOPP advanced oxidation protein product BAP bone alkaline phosphatase BMD bone mineral density BMI body mass index BMU(s) basic multicellular unit(s) BTM(s) bone turnover marker(s) CAT catalase CO2 carbon monoxide CRF(s) clinical risk factor(s) CTX collagen type I cross linked C-telopeptide D, E, F, G DPD deoxypyridinoline DXA dual X ray absorptiometry ELISA enzyme-linked immunosorbent assay FDA Food and Drug Administration GPx glutathione peroxidase Grx-5 glutaredoxin 5 H, I, J, K H2O water H2O2 hydrogen peroxide HClO hypoclhorous acid HRT hormone replacement therapy ICTP type I collagen C-telopeptide IL-1/6 interleukin-1/6 INRA Institut National de la Recherche Agronomique L, M, N M-CSF monocyte/macrophage colony–stimulating factor Mn-SOD manganese superoxide dismutase

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MDA malondialdehyde MSC mesenchymal stem cells NFκΒ nuclear factor-κΒ NO•

nitric oxide radical NTX crosslinked N-telopeptides of type I collagen O, P O2• superoxide radical O3 ozone OCN osteocalcin OH•

hydroxyl radical OPG osteoprotegerin OSI oxidative stress index PICP procollagen type I C-terminal propeptide PINP procollagen type I N-terminal propeptide PTH parathyroid hormone PYD pyridoline Q, R, S RANκ receptor activator of nuclear factor κΒ RANκL receptor activator of nuclear factor κΒ ligand REB research ethics board RO•

alkoxyl radical ROO• peroxyl ROS reactive oxygen species Runx2 runt-related transcription factor 2 SOD superoxide dismutase T TAC total antioxidant capacity TAS total antioxidant status TBARS thiobarbituric acid reactive substances TEAC Trolox equivalent antioxidant capacity TNF tumor necrosis factor TOS total oxidative status TRAP5b type 5 tartrate resistant acid phosphatase U, V, W, X, Y, Z USDA United States Department of Agriculture WHO World Health Organization

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CHAPTER 1

INTRODUCTION

The research work undertaken in this thesis focuses on the role of the additive effects of

polyphenol antioxidants with other micronutrients (Appendix I), which have been shown to

be important for bone health, in lowering the risk of osteoporosis due to oxidative stress. For

better understanding of these relationships, the following sections include a discussion of

postmenopausal osteoporosis, oxidative stress and polyphenol antioxidants.

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CHAPTER 2

LITERATURE REVIEW

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I. Postmenopausal Osteoporosis

IA. Overview

Osteoporosis is a skeletal disease characterized by low bone mass and structural deterioration

of bone tissue and structure, leading to an increase in bone fragility and susceptibility to

fractures, most frequently in the hip, wrist and spine [1]. Osteoporosis is commonly referred

to as the “silent thief” as there are no noticeable symptoms associated with its progression

until it manifests in the form of a fragility fracture [2]. It has been estimated that

approximately 1 in 3 women over the age of 50 will suffer a fragility fracture at some point

in their life-time [3].

There are two types of osteoporosis, primary and secondary types. Primary osteoporosis is

classified into two subtypes: (a) type I osteoporosis (also known as postmenopausal

osteoporosis), which is a common bone disorder in postmenopausal women and is mainly

due to estrogen deficiency resulting from menopause, and (b) type II osteoporosis (also

referred to as age-related osteoporosis or senile osteoporosis), which is related mainly with

aging in both women and men [4]. Alternatively, secondary osteoporosis refers to bone

disorders that are the consequence of various other medical conditions, or of changes in

physical activity, or are adverse outcomes of therapeutic interventions, generally

pharmological, for certain disorders [4, 5]. The gold standard for diagnosis of osteoporosis is

dual energy X ray absorption (DXA) scan, which measures the bone mineral density (BMD).

Using DXA measurements, BMD is expressed as the Z-score, which measures the variance

between the patient and the expected value for patient’s age and sex, and T-score, which

measures the variance between the patient the young adult of the same sex and race [6]. The

World Health Organization (WHO) developed guidelines for the use of clinically diagnosing

osteoporosis based on BMD T-scores: +1 to -1 (normal BMD), -1 to -2.5 (osteopenia/low

BMD), -2.5 or lower (osteoporosis), and -2.5 or lower with fragility fractures (severe

osteoporosis) [7].

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Postmenopausal osteoporosis in particular, is becoming more prevalent as the population

ages and therefore the impact of this condition on public health is becoming increasingly

critical [8]. Approximately 1.6 million hip fractures occur worldwide each year and by 2050,

this prevalence could reach between 4.5 million and 6.3 million [3]. Postmenopausal women

experience an increased rate of bone turnover, which in turn has a detrimental effect on bone

microarchitecture and is associated with an increased risk of osteoporotic fractures [9].

Specifically, early postmenopausal women experience an accelerated bone loss; an average

bone mineral loss at a rate of 0.5-2% per year, which then slows down later with age [10]. In

2005, Johnell and Kanis showed that an average 50 year old woman has approximately 40–

50% lifetime risk of an osteoporotic fracture leading to significant personal and societal

burden [11].

IB. Bone biology: Remodelling and pathophysiology of osteoporosis Bone is a dynamic tissue that is constantly being remodelled to preserve a healthy, functional

skeleton. Remodelling is essential to maintain bone mass, to repair microdamage of the

skeleton, to avoid accumulation of too much old bone, and for mineral homeostasis [5].

There are two major types of bone: (a) cortical, which is protective and provides a

mechanical function, and (b) trabecular, which provides strength and is more metabolically

active (25% per year) than cortical bone (3% per year) [4, 12]. The higher turnover may be

due to the large bone surface area, which is in close contact with the bone marrow in which

regulatory factors are present (discussed below) [12-15]. Given its higher metabolic activity,

trabecular bone is the primary site of bone remodelling; hence, it is the main site for various

bone diseases to occur, particularly metabolic bone diseases including osteoporosis [4].

However, it is also noteworthy that metabolic bone diseases affect both trabecular and

cortical bone.

Bone remodelling is carried out by a functional and anatomic structure known as the basic

multicellular unit (BMU) and requires the coordinated action of three major types of bone

cells: osteoclasts, osteoblasts and osteocytes [4].

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Cells of Bone

Osteoclasts are large, multinucleated cells that differentiate from mononuclear cells of the

monocyte/macrophage lineage upon stimulation by two integral factors: the

monocyte/macrophage colony–stimulating factor (M-CSF) and the receptor activator of

nuclear factor κΒ ligand (RANκL) [4]. M-CSF enables proliferation, survival and

differentiation of osteoclast precursors, whereas RANκL is the most important cytokine that

prepares the precursor cells for osteoclast differentiation. RANκL binds to the receptor

RANκ on the surface of osteoclast precursors and osteoclasts, and is the primary activator of

osteoclast formation and action [16]. Osteoclasts have a catabolic role and break down

components of bone through the action of lysosomal enzymes at specific sites [17]. In

contrast, osteoblasts are anabolic, and thus, promote the formation of new bone by producing

the organic constituents of bone (unmineralized bone matrix), and subsequently take part in

its two stage mineralization process [16]. Mesenchymal stem cells (MSCs) yield

osteoprogenitors, which grow and differentiate into pre-osteoblasts and then mature into

osteoblasts via the Wnt signaling pathway [4, 17]. Even though the synthesis of

unmineralized bone matrix is relatively fast and can take place within 6–12 hours from

initiation, the secondary mineralization takes much longer (1–2 months) [17]. When bone

formation is complete, the osteoblasts become surrounded by the new bone matrix and

differentiate into osteocytes [17]. Osteoblasts also secrete osteoprotogerin (OPG), a

circulating inhibitor of RANKL [17]. OPG binds to and sequesters RANKL and so, prevents

its binding to RANK; thus, OPG acts as a potent anti-resorptive cytokine by inhibiting

osteoclast formation and thus, activity [17, 18]. Osteocytes are the most abundant cells in

bone and may play an important part in transmitting local strain information to allow BMUs

to regulate the bone content in response to the local need. They might function as

‘mechanostats’ and under conditions of strain would prompt the remodelling of bone [4, 17].

Bone remodelling or turnover in an individual BMU is a physiological process that follows a

time sequence lasting approximately four months wherein old or damaged bone is eliminated

by osteoclasts then replaced by new bone formed by osteoblasts [4, 16]. There are four

overlapping phases [4, 16]: (1) Osteoclast precursors are require factors produced by marrow

stromal cells, osteoblasts or T-lymphocytes to differentiate into multinuclear osteoclasts.

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Two essential factors are the M-CSF and RANκL [19]. (2) Osteoclasts resorb bone,

producing a resorption cavity – a process that takes approximately 3 weeks: once the

multinucleated osteoclasts have matured, they attach to a bone surface to seal off a resorption

zone. Osteoclasts then secrete numerous enzymes (e.g. the protease Cathepsin K) and acid

into this zone causing bone resorption. (3) Osteoblasts produce collagenous bone matrices

and complete its mineralization, resulting in the synthesis of such bone matrix proteins such

as collagen type 1, osteopontin, osteocalcin, bone specific alkaline phosphatase and bone

sialoprotein. Additionally, the osteoblasts produce different factors that are stored within the

newly synthesized bone for future use and released during subsequent remodelling cycles.

Following behind the osteoclasts during bone resorption are the newly recruited osteoblasts

to fill the site of resorption. Thus, osteoclasts consist of cells that are of different ages and the

successive set of osteoblasts are of the same age [20]. (4) As bone formation persists,

osteoblasts (i) become entrenched more deeply into the bone, eventually becoming

surrounded by bone and are henceforth defined as osteocytes; (ii) enter apoptosis; or (iii) stay

on the bone surface to become bone-lining cells.

A model illustrating the 4 phases of bone remodelling is show in Figure 2.1.

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Figure 2.1: Model for bone remodelling (Adapted from [4])

It is important to note that the organization of the BMU found in trabecular bone is different

from cortical bone. In cortical bone, the front (cutting cone) of the BMU forms a cylindrical

canal through the bone, which is filled by osteoblasts (closing cone) [21]. Unlike cortical

bone, the BMU travels across the trabecular surface by creating a trench rather than a canal,

and osteoblasts fill the resorption cavity. The lifespan of a BMU is approximately 2-8 months

[13, 22]. The whole remodelling process renews about 10% of bone per year, thus renewing

the entire skeleton in approximately 7-10 years [20, 21].

Postmenopausal Homeostasis of Bone and its Dysregulation

In mature, healthy bone, the balance between bone resorption and bone formation is tightly

regulated and maintained to ensure that there are no significant alterations in bone mass or

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mechanical strength after each remodelling cycle [4, 16]. However, an imbalance between

bone resorption and formation may arise under certain pathological conditions. This

dysregulation of the homeostatic relationship between bone forming and bone resorbing cells

leads to abnormal bone remodelling and the development of bone disorders including

osteoporosis [4]. The greatest change in bone remodelling occurs at menopause, where there

are more resorption cavities due to an increase in bone resorption. Moreover, bone formation

does not increase proportionately with the increases seen in resorption and so resorption

cavities are not completely refilled with new bone. This deficit in bone replacement during

menopause causes increasing loss of bone mass, which if left untreated, is permanent [5, 16].

This might be related to the fact that at menopause, estrogen, which normally exerts an

inhibitory effect on osteoclast function, becomes deficient [17]. A more detailed discussion

regarding the pathophysiology of osteoporosis is below.

Pathophysiology of Osteoporosis: a brief overview

The exact pathogenesis of osteoporosis is not known. It is recognized that both bone

resorption and bone formation are amplified in postmenopausal osteoporosis; nonetheless,

there is an imbalance between bone resorption and bone formation in favour of bone

resorption [4]. Lerner and colleagues suggest that it is the increased frequency of resorption

cavities and the reduced ability of individual osteoblasts to synthesize new bone that cause

the decrease in bone mass as well as in bone strength [5]. Alternatively, Raisz et al.

presumed that impairment of osteoblast renewal, rather than a defect in osteoblast function,

was involved [23]. Consequently, the number of new osteoblasts that differentiate and lay

down successive lamellae of new bone is reduced.

Indeed, an increase in bone resorption seems to be the key stimulus for bone loss in the

setting of acute estrogen deficiency. It is evident that osteoblasts, osteocytes, and osteoclasts

express functional estrogen receptors [18]. These receptors are also expressed in MSCs, the

precursors of osteoblasts, which physically support future osteoclasts, T-cells, ß-cells, and

most other cells in the human bone marrow [18]. It is well established that activation of

estrogen receptors in osteoblasts by estrogen induces their anabolic activities and reduces the

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pathway by which osteoblasts can activate osteoclasts activity [5]. In contrast, stimulation of

estrogen receptors in osteoclast progenitor cells diminishes osteoclast formation, and

activation of estrogen receptors in mature osteoclasts prevents their bone-resorbing activity.

Furthermore, via a complex interaction between bone cells and the immune system, lack of

estrogen stimulates T-cells to release various inflammatory cytokines, which estrogen

normally inhibits [18]. Some (e.g., interleukin [IL]-1, IL-6, and tumor necrosis factor [TNF]-

α) promote osteoclast recruitment, differentiation, and prolonged survival, while others (e.g.,

IL-7) inhibit osteoblast differentiation and activity, and cause apoptosis of osteoblasts [18].

The ultimate outcomes of estrogen deficiency are therefore increased bone resorption and

impaired bone formation and hence, bone loss.

Thus, the pathogenesis of osteoporosis in women likely involves augmented bone resorption

by osteoclasts, related to changes in estrogen levels at menopause. Another factor that is

involved in the development of osteoporosis is age. Age is centered on bone formation by

osteoblasts, and involves various distinct factors linked to the aging process in both men and

women such as the formation and accumulation of reactive oxygen species (ROS) [4]

inducing oxidative stress. ROS, the radical forms of oxygen, are by-products of respiration

and oxidase enzyme activity in the mitochondria, and of cellular responses to numerous

external stimuli ranging from inflammatory cytokines to ionizing radiation [4]. There is an

age-related rise in ROS levels that stems from age-related increases in ROS production

and/or a reduction in antioxidants, which counteract these adverse effects [4, 24]. Age-

dependent increases in ROS have been shown to reduce osteoblastogenesis and hence, bone

formation [4, 24]. A proposed signaling pathway of ROS affecting osteoblasts and

osteoclasts is shown in figure 2.2. The role of oxidative stress and antioxidants in

osteoporosis will be discussed further in the following sections.

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Figure 2.2: ROS-activated signalling pathways affecting mesenchymal stem cells (MSC),

osteoblasts (OB) and osteoclasts (OC).

IC. Risk Factors for Osteoporosis Osteoporosis is a multi-factorial disease. Risk factors can be categorized into two main

groups: non-modifiable risk factors and modifiable risk factors. The non-modifiable risk

factors are those that cannot be changed, including age, sex, race, family history, early

menopause, as well as medical comorbidities. The modifiable risk factors include

geographical region and lifestyle (alcohol, smoking, exercise, and diet).

Non-modifiable Risk Factors:

Age

Approximately 90% of hip fractures occur in people who are over the age of 50 [25]. For

any BMD, the fracture risk is much higher in the elderly than in the young [26-28]. This is

partially because the bone remodelling balance tips toward bone mineral loss, leading to an

increased risk of fracture.

Sex Differences

Women generally have a lower peak bone mass compared to men [29]. Furthermore, women

are more susceptible to bone loss than men, especially postmenopausal women due to their

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deficiency of estrogen. Estrogen plays a vital role in bone remodelling in that it promotes an

increased osteoblast activity, favouring bone formation [24, 30]. These factors, among others

contribute to the fact that postmenopausal women are at greater risk for the development of

fracture than men of the same age. For instance, it has been shown that all else being the

same, females have a 40-50% chance of developing fractures due to osteoporosis whilst

males only have a 13-22% chance [11].

Race

Studies have shown that variations in the incidence of osteoporotic fractures incidence can

also be explained by exist depending on race. Osteoporosis is considered to be more common

amongst the Caucasian and Asian populations [31, 32] and one study also showed that

vertebral fracture was more prevalent in Japanese women than in American Caucasian

women [33]. As well, the incidence of osteoporosis and fractures of the hip and spine is

lower in Black than in Caucasian subjects [34-36]. Furthermore, the lifetime risk of a hip

fracture amongst the Black population is approximately 50% of the rate observed in the

Caucasian population [37]. Interestingly, despite lower BMD measurements in Asians than

Black women, the National Osteoporosis Risk Factor Assessment study noted that they

shared a relatively low risk for osteoporotic fractures of the forearm when compared with

American Caucasian, Hispanic and Native American women [38].

Family and Medical History

A family history of fractures also appears to be a risk factor for future fractures [39]. A

family history of hip fracture has been shown to correlate to increased risk for fracture

independent of BMD [40]. Furthermore, it has been shown that the risk of hip or wrist

fracture was increased in women with a family history of wrist or hip fracture [41, 42]. As

well, women with a family history of osteoporosis were more likely to develop osteoporosis

in comparison to those who did not have such a family history [43].

Risk factors for fracture also include a previous fracture related to (presumably osteoporosis

related) fragility [44], body mass index [41, 45, 46], resting pulse rate over 80 beats per

minute [47], certain therapeutics in addition to diseases that directly or indirectly affect bone

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remodelling (asthma, rheumatoid arthritis, Crohn’s disease, etc) and conditions that affect

mobility and balance (e.g. poor vision) that contribute to increased risk of falling [37, 39, 48,

49].

Modifiable Risk Factors:

Geographical Region

Variation in the prevalence of osteoporosis has also been shown to differ on the basis of

geographic location [50, 51]. For example, it has been reported that there is an increased

incidence of osteoporosis in subjects living in northern countries as compared to those living

in southern countries [50, 52, 53].

Lifestyle: Diet and Exercise

It is accepted that a healthful diet of minerals including, but not limited to manganese,

copper, selenium, and zinc is important insofar as the prevention of many diseases and

disorders are concerned. A diet low in calcium might lead to increases in the synthesis and

secretion of parathyroid hormone, which activates osteoclast activity. This could conceivably

lead to increased bone resorption so as to normalize serum calcium that might otherwise be

reduced due to the dietary deficit [54]. As well, vitamin D is also essential because it

mediates calcium absorption from the intestines into the blood. It is recommended in Canada

for women over the age of 50 years to consume to that 800 to 2000 IU of vitamin D and

1,200 mg of calcium is consumed daily to prevent the development of osteoporosis [55, 56].

Therefore, a diet low in calcium and vitamin D may increase the risk of osteoporosis.

Exercising has also been shown to affect skeletal growth [57-59]. It has been documented

that people with a more sedentary lifestyle are more likely to have a hip fracture than

individuals who are active. For instance, women who sit for more than nine hours a day are

twice more likely to have a hip fracture than those who sit for less than six hours a day [60].

In contrast, studies have reported that exercise had no effect on preventing or treating

osteoporosis [61].

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Cigarette smoking and excessive alcohol consumption are two other contributing lifestyle

choices that are modifiable risk factors for osteoporosis. It has been documented that people

who used to smoke or still currently smoke have a greater risk for any type of fracture,

including one related to osteoporosis when compared to those who are never smoked [62].

When compared to individuals who consumed a moderate or no intake of alcohol, those who

consumed an excessive amount of alcohol (> 2 units/day) were at an increased risk of

sustaining any osteoporotic fracture [27, 63]. This may be caused by the effects of alcohol on

osteoblasts, and could also affect the hormonal cascades involving calcium metabolism and

vitamin D deficiency [64].

ID. Bone biology: Biomarkers of bone turnover as predictors of bone loss and individual risk of fracture in participants with postmenopausal osteoporosis

Bone turnover markers (BTM) reflect whole body rates of bone resorption and bone

formation, and provide a dynamic analysis of ongoing changes within the skeleton. This is

advantageous over BMD measurements using DXA for various reasons that include: (a)

DXA offers a static measurement of bone mineral content and density [65, 66], even though

bones are physiologically active and undergo a continuous process of remodelling in each

BMU; (b) BMD changes occur more slowly (e.g. could take a couple of years) than changes

in the levels of the biomarkers which can be seen in weeks in response to changes in activity

of disease (with or without treatment) [67]; (c) from a clinical use perspective, half of the

patients with incident fractures have baseline BMD measurements above -2.5 SD or below

the average value of young healthy women, thus limiting the sensitivity of this particular

outcome measure. In fact, as a consequence of this relatively poor sensitivity, other

approaches have been developed to determine the risk for osteoporotic bone fractures to

develop. In this regard, recently, the WHO introduced a prognostic tool to evaluate fracture

risk of patients called the FRAX® [68]. It assesses the 10-year risk of osteoporotic-related

fracture based on individual patient models that combines clinical risk factors (CRF) as well

as BMD at the femoral neck.

An in depth review showed several studies that examined the association between baseline

levels of biochemical markers and the rate of subsequent bone loss estimated by changes in

14

BMD by DXA over long periods of up to 10 years among elderly and younger

postmenopausal women [69]. Many of these studies showed a significant association

between high bone turnover with faster subsequent bone loss, although the association was

inconsistent between the skeletal sites. Thus, a single measurement of bone markers cannot

accurately identify individuals with rapid bone loss. However, this was acknowledged in one

study where BMD and BTM were assessed, showing that by averaging serial measurements

over time, it may be possible to improve the predictive value relative to bone loss in elderly

women compared to a single baseline assessment [70].

Bone remodelling can be assessed accurately by the measurement of BTM in blood or urine.

These have been useful in reflecting the effects of therapeutic agents on the cell’s activities

related to either or both bone formation or resorption. Assessment of BTM alone cannot be

used to diagnose osteoporosis because the values for osteoporotic and normal patients

overlap considerably; hence DXA is a much stronger tool in this case, but as noted above,

even in the case of DXA, there is considerable overlap [71]. However, measurement of BTM

can give a good indication about the future risk for bone loss and fractures and is highly

effective in monitoring the efficacy of antiresorptive therapy in patients with osteoporosis[67,

72, 73]. They can be categorized as bone formation markers and bone resorption markers

(Table 2.1).

15

Table 2.1: Biochemical markers of bone turnover including bone formation and bone

resorption

Bone formation Markers Bone Resorption Markers

Serum Serum Urinary

Serum osteocalcin (OC) collagen type I cross

linked C-telopeptide

(s-CTX)

Total pyridoline

(PYD)

Serum Total Alkaline Phosphatase (ALP) Carboxyterminal

telopeptide of type I

collagen (ICTP)

Free-pyridoline

(f-PYD)

Serum Bone-Specific Alkaline

phosphatase (BALP)

Tartrate resistant

acid phosphatase

(TRACP)

Total

deoxypyridoline

(DPD)

Serum procollagen type I C-terminal

propeptide (PICP)

Tartrate resistant

acid phosphatase 5b

(s-TRACP 5b)

Free-

deoxypyridoline

(f-DPD)

Serum procollagen type N-terminal

propeptide (PINP)

Collagen type I

cross linked N-

telopeptide (NTX)

Collagen type I

cross linked C-

telopeptide (u-CTX)

Bone formation markers reflect osteoblast activity. These include bone specific alkaline

phosphatase (BAP), osteocalcin (OC) and procollagen type I C- and N-terminal propeptide

(PICP and PINP, respectively). Bone resorption markers are indicative of either enzymes of

the osteoclastic cells such as the 5 b isoenzyme of tartrate resistant acid phosphatase

(TRAP5b) or the proteolytic fragments of bone collagen matrix, including the type I collagen

16

crosslinks, pyridinoline (PYD) and deoxypyridinoline (DPD), and their telopeptides, carboxy

terminal telopeptide (CTX) and amino terminal telopeptide (NTX) [65]. For the purpose of

this thesis, PINP and CTX will be discussed in further detail as they were the markers used

for the studies conducted.

Type I collagen is an important and most abundant component in the bone matrix where

osteoblasts secrete its precursor proteins, procollagen and tropocollagen, during bone

formation. Type I collagen is a triple helical structure consisting of two identical a1 chains

and one a2 chain with a non-helical region where the amino (N)-telopeptide and carboxy (C)-

telopeptide attach to the crosslinks [65, 67, 74]. As collagen is being synthesized, the N- and

C-terminal extensions of the procollagen type 1 are cleaved by enzymes and are referred to

as PINP and PICP respectively [75-77]. The PINP concentration has been shown to be

proportional to the amount of new collagen laid down during bone formation. To note, type I

collagen propeptides sources may vary, however, most of the non-skeletal tissues have

illustrated a slower rate of turnover than bone, thus the propeptides seen in circulation are

mainly derived from bone [74]. PINP is a highly specific and sensitive bone turnover marker

for monitoring anabolic treatment [78]. One of the advantages of using this assay is that the

only source of variation is dependent on the small circadian rhythm.

During bone resorption, an osteoclast enzyme such as cathepsin K hydrolyzes the crosslinks

and releases the N (NTX)- or C (CTX)-telopeptide into circulation as markers of bone

resorption [79, 80]. The CTX-telopeptide consists of two isomeric forms, the native (α CTX)

and the age-related (ß CTX) [74, 81]. The CTX-telopeptide is highly vulnerable to

isomerization or racemization, resulting in αL, ßL, αD, and ßD [65, 74]. The degree to which

collagen is isomerized may possibly provide information on the age-dependent changes of

collagen in health and disease [82, 83]. As there are various assays to determine CTX, a

sandwich enzyme-linked immunosorbent assay (ELISA) for the measurement of β-CTX in

serum is one of the most commonly used measures. CTX is detected by using two

monoclonal antibodies that recognize the β-isomerized octapeptide on the non-helical (C)-

telopeptide [65]. To date, CTX is considered to be the most sensitive marker of bone

resorption. However, CTX has some disadvantages in that the samples must be collected at a

17

specific period of the day and in a fasting state. For instance, CTX levels are much lower in

the afternoon than in the morning and are related to the ingestion of food [75, 84].

18

II. Oxidative Stress

IIA. Overview Oxidative stress is characterized by an increased level of ROS that disrupts the intracellular

reduction–oxidation (redox) balance [85]. This balance heavily depends on the equilibrium

between the concentrations of ROS and antioxidants, reacting with ROS, to protect other

intracellular molecules from oxidative damage. ROS are oxygen-derived pro-oxidants that

can damage cell structures that are comprised of lipids, proteins or DNA, as well as some

enzymes that defend the cell [86]. Damaging any of these structures can lead to the damage

of bone cells. The two common groups of ROS are radicals and nonradicals. The radicals

have one or more unpaired electrons, making them highly reactive as they donate or obtain

another electron from another free radical to become stable [87]. These include nitric oxide

(NO•), superoxide ion (O2•-), hydroxyl (OH•), peroxyl (ROO•), and alkoxyl radicals (RO•) and

one form of singlet oxygen (1O2) [88]. Nonradicals, also highly reactive, include

hypochlorous acid (HClO), hydrogen peroxide (H2O2), organic peroxides, aldehydes, ozone

(O3) and O2 [88]. All of these oxygen radicals and nonradicals are created as by-products of

aerobic metabolism [89]. Other factors which can generate ROS include cigarette smoke,

physical stress, ultraviolet radiation, chemicals, fried foods, environmental pollutants and the

aging process [77, 90-93].

IIB. Oxidative damage to cell components Oxidative Damage to Lipids

All cellular membranes contain high concentrations of unsaturated fatty acids, therefore they

are highly vulnerable to oxidation [94]. This damage is commonly referred to as lipid

peroxidation, which occurs in three majors steps: initiation, propagation and termination. In

general, initiation involves the attack of a ROS and removing a hydrogen atom from the

lipid, thus creating a fatty acid radical [87]. When oxygen is present in the surroundings, the

fatty acid radical will react with it creating a peroxy fatty acid radical, leading to the

19

production of other fatty acid radicals undergoing the same reaction. This initiates the

propagation step and allows the process to continue [87, 95]. Finally, termination follows

when two radicals interact or when a radical interacts with an antioxidant, producing a non-

radical. This can occur when there is ample concentration of the radical species [87, 95]. The

lipid peroxidation marker, malondialdehyde (MDA) has been used as a measure of osteoclast

function [96] because osteoclasts generate O2•-, which can result in the oxidation of lipids. This

can be manifested as increased serum concentrations of MDA (or other lipid peroxidation

markers) [1].

Oxidative Damage to Protein

Proteins can undergo oxidation by interacting with ROS; involving peroxidation, damage to

specific side-chains, change in their tertiary structure and degradation [87, 97-100]. Several

lines of research have indicated that OH• and RO• and nitrogen-reactive radicals are the main

ROS that cause protein damage [97]. Oxidizing proteins can lead to the loss of enzymatic and

structural activity, alteration in cellular functions (energy production), improper modulation

of cell signaling, and induce apoptosis [97, 98, 100, 101].Other common protein oxidation

products are aldehydes, keto compounds and carbonyls [102].

Oxidative Damage to DNA

ROS can interact with DNA and cause alterations by modifying DNA bases, increasing the

susceptibility to lose purines, and damaging the deoxyribose sugar as well as the DNA repair

system. Notably, not all ROS can damage DNA. For instance, O2•- and H2O2 have been

shown not to react with DNA [87]. In contrast, OH• can react and affect all DNA bases,

whereas 1O2 selectively attacks only guanine [87, 103]. Damage to DNA can cause mutations

and lead to various chronic diseases including osteoporosis [104-106].

IIC. Defense mechanisms There are three main lines of defense against the development of ROS and their actions (i.e.

oxidative stress). These include mechanisms of prevention, repair and defense against

antioxidant effects. The preventative mechanism involves the use of metal chelating proteins

20

and the endogenous antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and

superoxide dismutase (SOD). Repair mechanisms include DNA repair enzymes, lipase,

protease and transferase. These are responsible for repairing damage and reconstituting

tissues [107]. The antioxidant defenses include the dietary antioxidants. These can function

as singlet oxygen quenchers, radical scavengers, reducing agents, or radical chain-

suppressors, breakers or terminators [107, 108]. Free radical scavengers in the human diet are

derived mainly from plants and include carotenoids, flavonoids, vitamins C and E and certain

plant polyphenols [109-111]. These antioxidants break the chain of oxidation during the

propagation step by interacting with the free radical at the site of attack in that they become

the targets for oxidation rather than important biomolecules or tissues. Therefore, these

antioxidants prevent oxidation of nearby cellular components such as proteins, lipids and

DNA [107].

IID. Oxidative stress and the risk for the development of osteoporosis Oxidative stress occurs when the production of free radicals exceeds the ability of

antioxidant defense system to eliminate these oxidants [104, 112]. When they are not

counteracted or prevented from forming in the first place, free radicals can change the

integrity of and thus, damage several biomolecules, such as DNA, proteins and lipids [104].

There is increasing evidence implying that oxidative stress is responsible, at least in part for

the pathophysiological processes of the aging and may be involved in the pathogenesis of

atherosclerosis, neurodegenerative diseases, cancer, and diabetes. Recently, it was proposed

that ROS might be responsible for the development of osteoporosis. Several in vitro and

animal studies have shown that oxidative stress diminishes bone formation by reducing the

differentiation and survival of osteoblasts [104], while ROS also activate osteoclasts and

thus, enhance bone resorption [104]. Clinical studies have also suggested that the

involvement of ROS and/or antioxidant systems may play a role in the pathogenesis of bone

loss [113-115]. Mackinnon et al. showed that supplementation with lycopene significantly

reduced oxidative stress parameters and bone resorption among early postmenopausal

women, suggesting that oxidative stress, induced by ROS, may be associated with the

pathogenesis of bone loss [114].

21

Baek et al. found a significant negative association between the levels of 8-Hydroxy-2’-

deoxyguanosine (8-OH-dG), an oxidative DNA adduct, and BMD at four bone sites (lumbar

spine, total hip, femoral neck, and trochanter) [104]. The level of 8-OH-dG in urine, tissue,

and serum is commonly used as a marker of oxidative stress in vivo. Furthermore, a positive

correlation has been demonstrated between serum 8-OH-dG levels and levels of the bone

resorption marker, type I collagen C-telopeptide (ICTP). This result and the aforementioned

negative relationship between 8-OH-dG levels and bone mass imply that subjects with higher

oxidant levels lose more bone through increased osteoclast-mediated bone resorption. In vitro

studies conducted by Baek and colleagues demonstrated that ROS promoted production of

RANκL and M-CSF, thus stimulating osteoclastogenesis and osteoclast activity in a primary

human bone marrow cell culture. These results indicate that osteoporosis, at least in part,

could be caused by oxidative damage.

Zhang et al. reported a negative correlation between BMD in the femur and the levels of the

advanced oxidation protein product (AOPP) in the plasma as well as between femur BMD

and plasma malondialdehyde (MDA) levels in rats [112]. It was also shown that AOPP

prevents osteoblast cell differentiation. AOPP and MDA are markers of oxidative damage to

proteins and lipids, respectively. Sontakke et al. showed that MDA has an effect on

osteoclasts [96]. The contribution of oxidative stress in age-related bone loss was further

established by the positive correlation between SOD activity and BMD in the femur [112]. In

the broader sense, several studies have reported a negative correlation between lipid

oxidation and BMD [1, 116]. Thus, these data suggest that the buildup of proteins and lipids

that have been modified by oxidation are related to age-related bone loss, whereas the

presence of antioxidant enzymes can prevent bone loss.

Experimental data have demonstrated that a rise in superoxide radical generation is related to

induced osteoclast-mediated bone resorption, and vice versa [117, 118]. Garrett et al.

reported that superoxide radicals are generated by osteoclasts and is associated with their

ability to resorb bone, and in turn promotes the production of osteoclasts [119]. Superoxide

radicals generated by osteoclasts in an extracellular compartment, even in the absence of

22

other enzymes can degrade bone matrix proteins [120]. In contrast, the superoxide scavenger

desferal-manganese complex reportedly reduces the amount of osteoclastic superoxide

radicals and decreases bone resorption in a dose-dependent manner [117].

Yang et al. and Dreher et al. provided further evidence that ROS participate in bone

resorption, with direct involvement of osteoclast-generated superoxide and bone

degradation[121, 122]. It has been documented that osteoblasts generate antioxidants such as

GPx to defend against H2O2, as well as transforming growth factor-β (TGF-β), which plays a

role in depressing bone resorption [122, 123]. GPx has also been reported to prevent

RANκL-induced osteoclastogenesis [85]. Interestingly, recent studies suggested that ROS

may be needed as a signaling intermediates for osteoclast differentiation, and that

antioxidants could limit bone resorption in vivo [124, 125].

Attindag and others was able to determine the levels of oxidative stress found in osteoporotic

patients. They reported lower levels of total antioxidative status (TAS), and higher levels of

total oxidative status (TOS) as well as oxidative stress index (OSI) in osteoporotic patients as

compared to healthy controls [126]. As well, there was a significant negative correlation

between BMD and OSI in the lumbar and femoral neck region. Studies have also

demonstrated that there is an association between oxygen-derived free radicals with

osteoclastic bone resorption stimulated by parathyroid hormone (PTH), IL-1β, and TNF-α

[127, 128]. It has also been demonstrated that TNF-α, directly or indirectly might activate

RANκL, OPG and NFκB, leading to the up-modulation of osteoclastogenesis. Increased

plasma TNF-α levels have been related to induce oxidative stress observed in

postmenopausal women, which in turn, may lead to augmented bone resorption and changes

cytokine production. It has been proposed that NFκB is essential for osteoclast formation as

an oxidative stress-responsive transcription factor. Thus, free radicals may elevate bone

resorption through activation of NFκB, which regulates the production of cytokines that

induce the formation of osteoclasts [129].

23

Utilizing osteoclast precursors from bone marrow and osteoblast/pre-osteoclast co-culture,

Chen et al. demonstrated that ethanol-induced RANκL expression by osteoblasts depended

on elevated intracellular levels of superoxide anion and hydrogen peroxide through enhanced

NADPH oxidase activity, implying that ROS promote osteoclast differentiation and

subsequent bone resorption [130]. In addition to influencing the differentiation process, ROS

may also affect the lifespan of osteoblasts. Glutaredoxin 5 (Grx5) is a highly expressed

protein in bone that might be involved in the protection against ROS. Linares et al. showed

that when over-expressed; Grx5 inhibits ROS generation and protects osteoblastic cells from

ROS-induced apoptosis through a mechanism that relies on manganese superoxide dismutase

activity (MnSOD) [131]. Altogether, these data illustrate that ROS not only directly promote

RANκL-induced osteoclastogenesis, but also stimulate bone loss by promoting apoptosis and

decreasing the differentiation and activities of osteoblasts. Therefore, by affecting both cell

types as well as the bone cell communication, oxidative stress seems to play a significant role

in bone cell function and the development of such bone disorders such as osteoporosis.

A general illustration demonstrating the role of oxidative stress and antioxidants in

osteoporosis and other chronic diseases is shown in figure 2.3.

Figure 2.3: The role of oxidative stress in osteoporosis and how/where antioxidants play a role in mitigating ROS

24

III. Phytochemicals Antioxidants

IIIA. Overview Although several micronutrients such as vitamins C, D, and B and calcium, magnesium, zinc,

copper, manganese, and boron are known to have antioxidant properties, there is increased

interest in other phytochemical antioxidants that are naturally present in plant derived foods.

These phytochemical antioxidants belong to one of the two groups: 1) carotenoids, which are

lipid soluble and 2) polyphenols, which are water soluble. greens+ bone builderTM, the test

material used in the thesis, is the major antioxidant phytochemicals present in this

supplement.

IIIB. Structure and Food Sources Polyphenols

Polyphenols are a class of water-soluble molecules naturally found in plants. They are

defined, as compounds having molecular masses ranging from 500 to 3000–4000 Da and

possessing 12-16 phenolic hydroxy groups on five to seven aromatic rings per 1000 Da of

relative molecular mass [132]. To date, over 8000 polyphenols have been identified [133].

Polyphenols can be divided into 2 main groups: flavonoids and non-flavonoids [134-136].

An organizational scheme of classifying polyphenols is shown in Figure 2.4.

25

Figure 2.4: Classification of polyphenols

Flavonoids

Flavonoids represent the most common group of polyphenols [137]. These consist of 2

phenyl rings connected by a chain of 3 carbon atoms forming a heterocyclic 6-membered

ring with oxygen and 2 carbon atoms from an adjacent phenyl ring [138]. They can be further

subclassified into anthocyanidins (e.g., cyanidin, delphinidin, malvidin), flavanols (e.g.,

catechin, epicatechin), flavonols (e.g., quercetin, fisetin), and flavones (e.g., luteolin),

isoflavones, and anthrocyanidins. Many flavonoids are found as glycosides in nature, which

makes it difficult to characterize [133]. The different flavonoids, their chemical structures

and sources are shown in Table 2.2.

Polyphenols

Flavonoids Non-flavonoids

Anthoxanthins Anthocyanins

Flavanols

Flavonols

Flavanones

Isoflavones

Flavones

Phenolic acids

Stilbenes

Coumarins

Lignans

26

Table 2.2 Classification of flavonoids, general chemical structure and food sources

Class Subclass Structure Common flavonoid Food examples

Anthocyanidins

Cyanidin

berries, purple

cabbage, beets,

grape seed

extract, red wine

Anthoxantins

Flavanols

Catechins

white, green and

black teas

Proanthocyanidins

chocolate, fruits

and vegetables,

red wine, onion,

apple skin

Theaflavins black teas

Flavonols

Quercetin

red and yellow

onions, tea, wine,

apples,

cranberries,

buckwheat, beans

Flavanones

Narigenin citrus fruits

Hesperidin citrus fruits

Silybin blessed milk

thistle

Isoflavones

Genistein

Diadzein

soy, alfalfa

sprouts, red

clover, chickpeas,

peanuts, other

legumes.

Flavones

Tangeritin

tangerine and

other citrus peels

Luteolin celery, thyme,

green peppers,

Apigenin chamomile,

celery, parsley

27

Non-Flavonoids

The non-flavonoid group account for approximately one-third of the polyphenols and

consists of phenolic acids, stilbenes (e.g. reservatrol), coumarin, and lignans [132, 139].

Phenolic acids can be subclassified into 2 groups: hydroxybenzoic acids (e.g. gallic, ellagic,

salicylic and vanillic acid) and hydroxycinnamic acids (e.g. caffeic and ferulic acid) [140,

141]. The different non-flavonoids, their chemical structures and sources are shown in Table

2.3.

Table 2.3 Classification of non-flavonoids, general chemical structure and food sources

Class Subclass Structure Common

flavonoid

Food

examples

Phenolic

Acids

Hydroxycinnamic

acids

Caffeic acid coffee beans

Hydroxybenzoic

acids

Gallic acid

gallnuts,

sumac, witch

hazel, tea

leaves, oak

bark,

Stilbenes

Resveratrol gapes skins,

red wine

Courmarin

aesculetin and

scopoletin

citrus fruits,

strawberries,

apricots,

cherries and

cinnamon

Lignans

Secoisolaiciresinol flaxseeds

28

The polyphenol content in plant foods and beverages has differed among many scientific

publications. There are many factors that influence the value of the polyphenol content and

profiles in plants, including: plant variety or cultivar, growth conditions (climate and soil),

crop management (irrigation, fertilization, pest management), state of maturity at harvest,

postharvest handling, storage, and processing [138, 142, 143]. With this in mind, it is

understandable as to why it has been difficult to determine the exact value of the content of a

given phenolic compound in a given food. Furthermore, specific polyphenols may only be

present in some varieties and absent in others. For instance, blue potatoes or black rice

contain anthocyanins, but are not present in their non-colored varieties (white potatoes or

white rice).

Recently, the United States Department of Agriculture (USDA) created a detailed food

database based on a compilation of literature sources [144]. This table contains values for

individual flavonoid compounds for 500 foods. In addition to the USDA database, Institut

National de la Recherche Agronomique in France (INRA) has developed a new Web-based

database called Phenol-Explorer, a compilation of the scientific literature of 502 polyphenols

found in foods [145]. This seems to be a more user-friendly database as it categorizes into

phenolic acids, lignans and stilbenes in addition to flavonoids.

Extracted from the either database, there are foods that contain very high amounts of

polyphenols [145, 146]. However, since certain foods and beverages that are rich in

polyphenols such as sage (flavones), capers (flavonols), oregano (hydroxycinnamic acids),

and wine (resveratrol) are consumed in very low amounts; they contribute very little to the

total intake [138]. On the contrary, some foods and beverages that contain small amounts of

polyphenols such as potatoes (chlorogenic acid) or tea (quercetin) are consumed in large

amounts, which can contribute significantly to polyphenol intake. Other polyphenols such as

stilbenes do not appear in the database because there are no known food sources that have 20

mg/100 g or greater [138].

29

IIIC. Dietary Intake The daily intake of polyphenols has been estimated to be approximately 1g/day [147]. This is

more than that of all other known dietary antioxidants, which is 10 times greater than that of

vitamin C and 100 times higher than those of vitamin E and carotenoids [148]. However,

several factors have been addressed that explains the challenge in estimating polyphenol

intake: [138] 1) Polyphenols have a vast amount of chemical structures. 2) Polyphenols are

present in a large variety of foods. For instance, quercetin can be present in several foods,

whereas flavanones are predominately present only in citrus foods. 3) Their content in a

given food can vary to a wide extent, as mentioned earlier, and 4) There have been no

standardized methods to estimate the amount of polyphenols in foods and methods of

analysis can vary between publications.

To date, there are no detailed intake values for all polyphenols. Having said that, catechins

and proanthocyanidins make up for approximately 75% of the total flavonoids, the largest

group of polyphenols, ingested. However, phenolic acids represent a majority of the

polyphenols ingested among coffee consumers. There are numerous factors that may

influence the polyphenol intake in a population. The major factors that influence the type and

quantity of polyphenols consumed include cultural habits (soy consumption in Asian

countries) and food preferences (wine, berries). Furthermore, polyphenol intake has been

shown to vary with age, sex and ethnicity, all which have been known to affect food choices

[149].

In addition to the dietary sources of polyphenols, nutritional supplements also contribute

towards their intake. Several nutritional supplements including greens+ bone builderTM are

now being marketed as sources of polyphenols.

30

CHAPTER 3

RATIONALE, HYPOTHESIS AND OBJECTIVES

31

I. Rationale

Evidence suggests that in postmenopausal women, the delicate balance between bone

resorption by osteoclasts and bone formation by osteoblasts is tipped towards increased

resorption [150], thus leading to net loss of bone over time. Although the mechanisms

underlying osteoporosis are not completely known, there is evidence suggesting that

oxidative stress caused by reactive oxygen species (ROS) is associated with its pathogenesis

[113, 116, 124, 151]. As other mechanisms exist in the development of osteoporosis, ROS

has shown to mediate several signalling pathways affecting bone remodelling. There is

renewed interest in the use of natural antioxidant components of foods and nutritional

supplements as complimentary strategies due to the adverse effects of pharmaceutical drugs

for the prevention and treatment of osteoporosis.

Nutritional supplements such as greens+TM (Genuine Health Inc., Toronto), a blend of several

botanical products, have been shown recently to contain a combination of water-soluble

antioxidant polyphenols naturally present in food. Previous research in our laboratory has

shown that polyphenols from greens+TM stimulated the formation of mineralized bone

nodules in human osteoblasts, raising the possibility that it may be effective in the

management of osteoporosis [152]. Another nutritional supplement, the bone builderTM

(Genuine Health Inc., Toronto), containing antioxidants, vitamins, minerals, trace minerals,

and amino acids, all of which have been shown individually to be beneficial to bone health

have also been shown in our laboratory to stimulate bone formation [153] The third

nutritional supplement, greens+ bone builder ™ (Genuine Health Inc., Toronto) has been

shown to be more effective in stimulating bone formation than either greens+TM or bone

builderTM alone [154]. As greens+ bone builderTM has been shown to be the more effective

stimulator of bone formation in vitro [154], we rationalized that this supplement will reduce

the risk for development of osteoporosis in postmenopausal women.

32

II. Hypothesis

The overall hypothesis is that intervention with greens+ bone builderTM, a good source of

polyphenols and micronutrients will increase the serum antioxidant capacity and urinary total

polyphenol content and decrease serum markers for oxidative stress in postmenopausal

females. Supplementation with greens+ bone builderTM will also decrease the bone resorption

marker, CTX and increase bone formation marker, PINP, thus reducing the risk of

osteoporosis (fig. 3.1).

Figure 3.1: Illustration of hypothesis

III. Objectives

The objectives are to investigate whether greens+ bone builderTM is effective in decreasing

the risk of osteoporosis in postmenopausal women (50-60 years of age) by:

1. Increasing serum antioxidant capacity and decreasing lipid peroxidation and

protein oxidation, and

2. Increasing urinary polyphenol content and if there is a negative correlation with

serum bone turnover markers, CTX and PINP.

33

IV. Subjects and Methods

IVA. Participant Recruitment and Sample Collection Participant recruitment was conducted from the years 2008-2011. Female participants

between the ages of 50-60 years, who were at least one year postmenopausal, were recruited

by telephone and advertisements and were asked to sign an informed consent in

conformation with the guidelines of the St Michael’s Hospital Research Ethics Board (REB)

(Appendix II). Exclusion criteria included participants who smoked cigarettes or were on

medications affecting bone metabolism, including those for heart disease, high blood

pressure, diabetes and/or osteoporosis. Based on these criteria, a total of 48 subjects were

selected to participate in the study. All participants were required to submit 7-day dietary

records (Appendix III) outlining foods, beverages and nutritional supplements they

consumed as well as baseline 12-hour fasting blood and 2nd void urine samples at the first

visit. Another set of dietary records as well as fasting blood and urine samples were collected

following a one-week washout period during which patients were asked to refrain from

drinking or eating foods rich in polyphenols and herbal/vitamin-supplements. The

participants were then assigned randomly to two groups: 1) greens+ bone builderTM (N=24),

or 2) the placebo diet (rice flour), (N=24), for a period of 8 weeks. Fasting blood and urine

samples were collected after 4 and 8 weeks following their respective treatment periods (Fig.

3.2). Blood samples were processed to obtain serum. Both the serum and urine samples were

labeled, kept frozen and stored at -80˚C until the time of analysis.

34

Figure 3.2: Study design

IVB. Serum Analyses Processing of Blood Samples

Unless otherwise specified, all materials were obtained from Sigma Aldrich Canada,

Oakville, ON, Canada. Fasting blood samples collected from participants were centrifuged at

2,500 RPM within 1 hour of collection. The serum was then collected, aliquoted equally into

4 eppendorf tubes (minimum of 250 µl each) and keep frozen at -80oC. The frozen samples

were thawed for analysis of total antioxidant capacity (TAC) [155], protein oxidation (thiols)

[156] and lipid peroxidation (TBARS) [157].

Total Antioxidant Capacity

Total antioxidant capacity (TAC) was measured, using the Trolox-equivalent antioxidant

capacity assay (TEAC) [155], to determine the overall capacity of antioxidants in the serum

using 2,2, azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), a blue-green

chromophore. The absorbance was read at 734 nm to determine decolourization (Milton Roy

Spectronic 1001 Plus, PA, USA), and compared to the decolourization produced by Trolox, a

vitamin E analogue as a standard.

Oxidative Stress Parameters

35

Protein oxidation was determined by estimating the content of protein-sulfhydryl groups

(thiols) in serum [156]. The optical density was read at 412 nm (Milton Roy Spectronic 1001

Plus, PA, USA) and protein thiol concentrations were calculated using an optical dentistry

reading at a wavelength of 13600 cm-1 M-1. A high concentration of protein thiols

corresponds to reduced levels of protein oxidation.

Lipid peroxidation in the serum was measured using the thiobarbituric acid-malondialdehyde

(TBA-MDA) assay and was reported as thiobarbituric acid reactive substances (TBARS).

The optical density was read at 535 nm (Milton Roy Spectronic 1001 Plus, PA, USA) and the

concentration of TBARS was calculated using an absorptivity of 156 mM-1cm-1.

Bone Turnover Markers

Both serum crosslinks (CTX), a marker of bone resorption, and procollagen type I N-

terminal propeptide (PINP), a marker of bone formation, were measured using an

electrochemiluminescent immunoassay (ECLIA) technique, which were performed

automatically by the Cobas® e601 (Roche Diagnostics, Germany).

IVC. Urine Analysis Total Polyphenol Content

Total urinary phenolic compounds were measured in 2nd void urine samples by using the

methodology described by Medina-Remón et al.[158]. This method was improved from the

Roura et al. protocol to measure a broader spectrum ofphenolic compounds contained in

different foods [159]. Oasis® MAX96-well plate SPE cartridges were selected because they

provided the best recovery. Absorbance was measured at 765 nm in the SpectraMax M5e

Microplate Reader (Molecular Devices, LLC., US). This spectrophotometer allowed the

absorbance in solutions contained in all 96 wells of this type of plate in to be determined in

approximately in 10 s. To measure the creatinine levels in the urine samples, the modified

Jaffé alkaline picrate method was used in, also with microtiter 96-well plates. Total

polyphenols were expressed as mg gallic acid equivalent (GAE) per g creatinine.

36

Ethics

This protocol was approved by the Research Ethics Board at St. Michael's Hospital, Toronto,

Ontario, Canada (approved June 11, 2008) (Appendix II).

37

CHAPTER 4

Antioxidant effects of a nutritional supplement containing

polyphenols in postmenopausal women: a randomized controlled

study

This chapter has been accepted for publication to the Journal of Aging: Research and

Clinical Practice as a research paper entitled: “Antioxidant effects of a nutritional

supplement containing polyphenols in postmenopausal women: a randomized controlled

study” by N.N Kang, A.V. Rao, K. De Asis, L.A. Chan, L.G. Rao

38

I. Abstract Background: Oxidative stress is an important factor in the development of osteoporosis.

Antioxidants counteract the damaging effect of oxidative stress, which may reduce the risk of

osteoporosis. Nutritional supplements, such as greens+TM and greens+ bone builderTM that

contain water-soluble polyphenols and other micronutrients beneficial for bone health, are of

recent interest as complementary strategies in the management of osteoporosis.

Objective: Clinically evaluate the antioxidant properties of greens+ bone builderTM in

postmenopausal women.

Design: Forty-seven postmenopausal women, 50-60 years old were recruited for a

ten-week clinical study. During week 1, participants recorded their baseline food intake.

During week 2, the participants refrained from consuming polyphenol-rich foods, beverages

and supplements. The participants were then randomized to either Treatment group

consuming 1 scoop (equivalent to ¼ cup) daily of greens+ bone builderTM (N=23) or Placebo

(N=24) group for a period of 8 weeks. Blood samples were collected at 0, 4 and 8 weeks of

supplementation, processed and assayed for serum total antioxidant capacity (TAC), lipid

peroxidation and protein oxidation as markers of oxidative stress.

Results: Statistical analysis showed that after 4 and 8 weeks, the Treatment group

significantly increased their serum total antioxidant capacity and decreased in lipid

peroxidation and protein oxidation while the Placebo group showed no significant changes.

These were also significantly different from those of the Placebo group.

Conclusions: Results suggest that a daily supplementation with greens+ bone builderTM may

be important in reducing oxidative damage, thus reducing the risk of osteoporosis in

postmenopausal women.

39

II. Introduction

Osteoporosis is a disease characterized by low bone mass and deterioration of the

microarchitecture of bone tissue, leading to fragility fractures primarily in the hip, spine and

wrist [160]. Approximately one in three women and up to one in five men over the age of 50

will develop osteoporosis world-wide [3]. Although several factors have now been identified

as increasing the risk of osteoporosis, oxidative stress has now emerged as one of the most

important life style risk factor associated with loss of bone mass [161-163].

Antioxidants capable of counteracting this effect have demonstrated to be important in

decreasing the risk of osteoporosis [126, 164, 165]. There has been an increased interest in

the water-soluble antioxidant polyphenols as a result of in vitro and in vivo evidence

demonstrating that they may protect against oxidative damage, mainly due to their

antioxidative and free radical quenching properties, thus limiting the risk of various

degenerative diseases associated with oxidative stress, including osteoporosis [166-170].

Nutritional supplements such as greens+ bone builderTM contain not only polyphenol rich

plant constituents, but also other nutrients that were shown to be individually good for bone

health. The composition of bone builderTM includes the following: vitamins B6, B12, C, D3,

and folic acid; minerals such as calcium, magnesium, zinc, chromium, selenium, manganese,

boron, copper; and antioxidant lycopene. Previous in vitro results from our laboratory have

shown that greens+TM, a nutritional supplement rich in a variety of polyphenols is effective in

stimulating osteoblasts to form bone nodules in a dose-dependent manner [171]. Another

nutritional supplement, bone builderTM, which is rich in minerals, vitamins and nutrients, also

had a significant dose-dependent stimulatory effect on bone nodule formation [154]. When

greens+TM and bone builderTM were tested as combination, the effects were six times more

effective than either one alone [154].

This led us to believe that additive effects of greens+TM and bone builderTM may have a

beneficial effect in reducing oxidative stress and reducing the risk of osteoporosis. The aim

40

of this study, therefore, was to investigate the effects of the nutritional supplement

commercially known as greens+ bone builderTM, which combines the total polyphenols in

greens+TM with various vitamins, minerals and antioxidants present in bone builderTM, on

biomarkers of oxidative stress in postmenopausal women who are at risk for osteoporosis.

41

III. Methods

Statistics

All statistical analyses were performed using the latest version of SAS system (version

9.2; SAS Institute, Cary, NC, USA). Summary statistics of participant demographics such as

age and BMI were generated and presented as means ± standard errors of the mean (SEM).

Repeated-measures one-way analysis of variance (ANOVA), with Tukey’s multiple

comparison test was used to test for significant differences in oxidative stress parameters and

antioxidant status from the start of the treatment period to 4 and 8 weeks on the treatment. In

cases where data were not normally distributed, the Friedman test with Dunn’s multiple

comparison test was substituted. Percent change in oxidative stress parameters and

antioxidant status were calculated using an unpaired t-test, or the Mann-Whitney test for data

that were not normally distributed to analyze for significant differences between the Placebo

and Treatment groups. Significance was considered p<0.05.

42

IV. Results A total of 47 postmenopausal women, 24 in the Placebo group and 23 in the Treatment group

(one participant withdrew due to personal reasons), completed the study. Table 4.1 describes

the participant baseline characteristics. There were no significant differences among groups

with respect to age, BMI, years since menopause, or blood pressure (Table 4.1). Similarly,

there were no significant differences at baseline for average serum TAC (Treatment group,

1.45 ± 0.05 mM and Placebo group, 1.50 ± 0.05 mM) and protein oxidation (Treatment

group, 469.7 ± 18.1 µM and Placebo group, 490.1 ± 12.5 µM). However, there was a

significant difference between the Treatment group and Placebo group for TBARS at

baseline with the Treatment group showing higher levels of TBARS at 7.1 ± 0.3 nmol/mL

and 6.4 ± 0.2 nmol/mL, respectively (Table 4.1).

An interaction between time (4 and 8 weeks) and type of treatment (Treatment and Placebo

group) was detected (p<0.05) for TAC such that TAC was greater in the Treatment group

than in the Placebo group (Figure 4.1a) after treatment. Significant increases of 3.8 ± 1.4%

and 7.1 ± 1.9% from baseline to 4 and 8 weeks among this group was also significantly

different from the Placebo group (p<0.01, p<0.0001, Figure 4.1b).

There was an overall interaction between time (4 and 8 weeks) and type of treatment

(Treatment and Placebo group) (p<0.0001) on lipid peroxidation, such that consuming the

supplement rich in polyphenols and other micronutrients was lower than those who

consumed the placebo (Figure 4.2a) after treatment. The Treatment group also resulted in a

decrease of 6.6 ± 1.0% after 4 weeks, and to 10.0 ± 1.3% after 8 weeks in lipid peroxidation,

which was significantly opposite to the 2.6 ± 1.7% and 4.3 ± 2.9% increase at 4 and 8 weeks

of treatment seen among the Placebo group, respectively (p<0.0001, Figure 4.2b).

An interaction between time (4 and 8 weeks) and type of treatment (Treatment and Placebo

group) was detected (p<0.05) on protein thiols, such that the Treatment group was higher in

protein thiols, indicating a decrease in protein oxidation, compared to the Placebo group

43

(Figure 4.3a) after treatment. There was a 3.2 ± 1.1% and 5.3 ± 1.1% increase in protein

thiols from baseline to 4 weeks, and to 8 weeks among the Treatment group, which was

significantly different from the decrease in protein thiols of 3.5 ± 2.9% after 4 weeks and by

2.5 ± 1.8% after 8 weeks seen within the Placebo group (p<0.05, p<0.001, Figure 4.3b).

44

Table 4.1: Participant characteristics and baseline values for oxidative stress parameters and

antioxidant capacity for each group.

Parameters Measured Placebo

greens+ bone

builderTM

Age (yrs) 55.5 ± 0.3 56.2 ± 0.6

Weight (lbs) 144.4 ± 5.1 143.7 ± 5.4

Height (inches) 63.7 ± 0.5 64.6 ± 0.6

Pulse Rate (beats/min) 66.8 ± 1.8 65.9 ± 1.8

Blood Pressure - systolic (mmHg) 115.6 ± 3.2 118.9 ± 3.2

Blood Pressure - diastolic (mmHg) 75.1 ± 2.0 72.5 ± 2.3

BMI (kg/m3) 25.0 ± 1.0 23.8 ± 0.8

Years since Menopause 5.7 ± 0.6 4.6 ± 0.5

Total Antioxidant Capacity (TEAC)(mM) 1.50 ± 0.05 1.45 ± 0.05

TBARS (nmol/mL)1 6.4 ± 0.2 7.1 ± 0.3

Protein Thiols (µM) 490.1 ± 12.5 469.7 ± 18.1 1 Placebo values had significantly lower TBARS than the supplement group (unpaired t-

test, p<0.05)

45

0 4 81 .3

1 .4

1 .5

1 .6P la c e b o

T re a tm e n t

4 .1 a

T im e p e r io d fo r in te rv e n tio n (w k s .)

Tro

lox

(m

M)

-5

0

5

1 0P la c e b o

T re a tm e n t

*

* *

4 w e e k s 8 w e e k s

b

Ch

an

ge

in

Tro

lox

fro

m b

as

eli

ne

(%

)

Figure 4.1: a) Total antioxidant capacity (TAC) in the serum over 8 weeks, as determined using the TEAC assay. Time by treatment effect values are expressed as mean ± SEM and were compared within supplement group using a repeated-measures ANOVA with Tukey's Multiple Comparison test (p<0.05) b) The change in TAC was measured relative to baseline concentration and expressed as mean % change ± SEM for each supplement group. Significant differences between supplement groups at each time point were compared using an unpaired t-test (*p<0.01, **p<0.0001)

46

0 4 86 .0

6 .5

7 .0

7 .5P la c e b o

T re a tm e n t

4 .2 a

T im e p e r io d fo r in te rv e n tio n (w k s .)

TB

AR

S (

nm

ol/

ml)

-1 5

-1 0

-5

0

5

1 0P la c e b o

T re a tm e n t

**

4 w e e k s 8 w e e k s

b

Ch

an

ge

in

TB

AR

Sfr

om

ba

se

lin

e (

%)

Figure 4.2: a) Concentration of lipid peroxidation in the serum over 8 weeks, as determined by TBARS. Time by treatment effect values are expressed as mean ± SEM and were compared within supplement group using repeated-measures ANOVA with Tukey's Multiple Comparison test (p<0.0001). b) Change in lipid peroxidation was measured relative to baseline concentration and expressed as mean % change ± SEM for each supplement group. Significant differences between supplement groups at each time point were compared using an unpaired t-test (*p<0.0001)

47

0 4 84 0 0

4 5 0

5 0 0

5 5 0P la c e b o

T re a tm e n t

4 .3 a

T im e p e r io d fo r in te rv e n tio n (w k s .)

Th

iols

(m

M)

-1 0

-5

0

5

1 0P la c e b o

T re a tm e n t** *

4 w e e k s 8 w e e k s

b

Ch

an

ge

in

Th

iols

fro

m b

as

eli

ne

(%

)

Figure 4.3: a) Concentration of protein oxidation in the serum over 8 weeks as determined by thiols. Time by treatment effect values are expressed as mean ± SEM and were compared within supplement group using the Friedman’s test with Dunn’s Multiple Comparison test (p<0.01). b) Change in protein oxidation was measured relative to baseline concentration and expressed as mean % change ± SEM for each supplement group. Significant differences between supplement groups at each time point were compared using an unpaired t-test (*p<0.05, **p<0.001)

48

V. Discussion

Previous studies showing the antioxidant properties of polyphenols were based mainly on in

vitro and animal studies. Our laboratory is the first to evaluate the efficacy of the nutritional

supplement containing polyphenols and other micronutrients administered to postmenopausal

women who are at risk for osteoporosis. The Treatment group showed increased TAC after 8

weeks of treatment. Furthermore, there was a significant change at 4 and 8 weeks between

the Placebo and Treatment group. The TEAC method has been validated to measure the

antioxidant properties of both the lipid- and the water-soluble antioxidants in vivo [172]. This

assay is used primarily for its ability to assess many endogenous antioxidants in addition to

those found in the supplement and its capability to quench reactive oxygen species (ROS).

Although the polyphenols in greens+TM may have been the principle component of the

nutritional supplement contributing to the observed antioxidant effect, the combined effect

with other micronutrients and antioxidants in the bone builderTM may also have contributed

to this effect [173, 174]. Similar results obtained in our in vitro study showed that greens+

bone builderTM to be more effective than greens+TM or bone builderTM alone in stimulating

bone nodule formation. Our study was not designed to study the interactive mechanisms

between the various components of greens+TM and bone builderTM. It is therefore, not

possible to associate the antioxidant affects observed in the present study to any one

component of green+ bone builderTM.

In addition to measuring TAC, other more sensitive biomarkers such as lipid peroxidation

and protein oxidation to measure the effectiveness of the nutritional supplement in reducing

oxidative stress. Greens+ bone builderTM significantly reduced oxidative stress compared to

placebo groups after both 4 and 8 weeks. Previous in vitro, in vivo and clinical studies [166-

168, 170, 171] have also shown similar results where there was a decrease in oxidative stress

parameters after the consumption of polyphenols. In a recent study, the effectiveness of

lycopene, a fat-soluble antioxidant, on oxidative stress parameters in humans was

investigated. The results showed decrease in oxidized proteins and lipid perioxidation at 4

49

weeks of treatment [113]. This is similar to our finding where there was a significant

decrease in both protein oxidation and lipid peroxidation within the treatment group when

compared to placebo at 4 and 8 weeks of treatment.

A variety of dietary antioxidants have been shown to be capable of scavenging ROS directly.

Previous studies demonstrated that a diet high intake of foods rich in polyphenols and other

micronutrients, increasing the antioxidant capacity, has been linked to lowered risks of many

chronic diseases involving oxidative stress [175-177]. As well, an in-depth review suggesting

that the combination effect of carotenoids found in foods and supplements and may have a

greater reduction in the risk of chronic diseases such as osteoporosis when compared to a

single nutrient [178].

Since polyphenols are quickly metabolism in the human body, its concentration found in

blood is low (<1 µmol/L) [179-182]. This is such a low concentration to show any significant

and direct antioxidant activities, thus some researchers believe that it is unlikely that

polyphenols act as antioxidants in vivo [183, 184]. Thus, attention should be brought to the

additive effect of micronutrients and polyphenols. It has also been noted that polyphenols

may function as a co-antioxidant, and are involved in the regeneration of essential vitamins

[184].

The relationship between the antioxidant property of the nutritional supplement and its effect

on bone health was recently reported [171]. They demonstrated that supplementation with

greens+TM containing polyphenols such as quercetin, apigenin, kaempferol and luteolin was

shown to increase the proliferation of human osteoblast-like cells at early time points of

addition as well as to stimulate bone nodule formation in a dose- and time-dependent manner

[171]. These observations suggest a positive effect of ingesting polyphenol-rich foods and

supplements in reducing oxidative stress, stimulating the activity of osteoblast cells, which

may reduce the risk of osteoporosis.

In conclusion, our study has shown that daily supplementation of a combination of

polyphenols and other trace nutrients and minerals in the form of the nutritional supplement,

50

greens+ bone builderTM, can significantly increase the antioxidant capacity and decrease the

extent of oxidative damage in postmenopausal women at risk of osteoporosis. The beneficial

effects can be due to the additive effect of the polyphenols with other vitamins and minerals

found within the supplement. This observation along with previously observed bone-

protective effects of the individual polyphenols, nutrients and trace minerals, has generated

increased interest into further exploring them as a nutritional supplement that is beneficial to

bone health. Although there is evidence that implicate oxidative stress is an important

mediator of bone loss aiding in the development of osteoporosis, our laboratory is the first to

investigate and show the combined effect of a variety of polyphenols alongside other

micronutrients in healthy postmenopausal women, suggesting that this may be a good

alternative for the prevention or treatment of osteoporosis.

51

CHAPTER 5

Dietary polyphenols and nutritional supplements significantly

decreased oxidative stress parameters and the bone resorption

marker collagen type 1 cross-linked C-telopeptide in

postmenopausal women

This chapter has been submitted for publication to Osteoporosis International as a research

paper entitled: “Dietary polyphenols and nutritional supplements significantly decreased

oxidative stress parameters and the bone resorption marker collagen type 1 cross-linked C-

telopeptide in postmenopausal women” by N.N. Kang, A.V. Rao, R. Josse, H.

Vandenberghe, K. De Asis, L.A. Chan, L.G. Rao. It has been reformatted for this thesis.

52

I. Abstract

Purpose: Oxidative stress caused by reactive oxygen species has been associated with the

development of osteoporosis, which can be ameliorated via dietary antioxidants. Polyphenols

are water-soluble phytochemical antioxidants known to decrease the risk of many age-related

chronic diseases. However, the role of a variety of polyphenols in combination with

micronutrients in osteoporosis has not been studied.

Methods: Forty-seven postmenopausal women, 50-60 years old were recruited for a ten-

week clinical study. Week 1, the participants consumed a regular diet. Week 2, they refrained

from consuming polyphenol-rich foods, beverages and supplements. They were then

randomized to either Supplement group (N=23) or Placebo group (N=24) for 8 weeks.

Fasting blood and urine samples were collected and measured at baseline and 8 weeks for

serum total antioxidant capacity (TAC); the oxidative stress parameters, lipid peroxidation

and protein oxidation, and the bone turnover markers, C-terminal telopeptide of type I

collagen (CTX) and procollagen type I N-terminal propeptide (PINP). Urine samples were

measured for total polyphenol content.

Results: Statistical analysis showed that at 8 weeks, the serum total antioxidant capacity and

total urinary polyphenol content was increased while lipid peroxidation, protein oxidation

and CTX were decreased significantly in the Supplement group, none of which was seen in

the Placebo group. In fact, all measured parameters were altered in the Supplement group as

compared to Placebo.

53

Conclusions: Results suggest that daily supplementation with polyphenols and

micronutrients may be important in reducing oxidative damage by reducing bone resorption,

thus the risk for development of osteoporosis in postmenopausal women.

54

II. Introduction

Osteoporosis is a disease characterized by low bone mass and deterioration of the

microarchitecture of bone tissue and predisposition to fractures [160]. Postmenopausal

women, in particular, experience an accelerated phase of bone turnover and loss of bone

mass at the perimenopausal within the first few years after menopause, which can result in

increased fragility of bone, potentially leading to bone fractures [185, 186]. Nutritional

recommendations of a daily dietary intake of 1200 mg of calcium and 800-2000 IU of

vitamin D have been suggested to promote bone health and perhaps help to mitigate the

rapid bone loss at menopause [187]. Today, a vast amount of literature is focused on other

important micronutrients and their effects on bone metabolism [188-191]. However, further

assessments of other nutritional supplements, which may influence bone turnover, are

necessary to assess their benefit for the prevention of osteoporosis.

Although several factors are known to increase the risk of osteoporosis, oxidative stress has

been recognized as one of the most important risk factors associated with the loss of bone

mass, due to increased bone resorption [162, 163, 192]. Antioxidants have been shown to

counteract this increased bone resorption [126, 164, 165]. Specifically, there has been

increased interest in the water-soluble antioxidant polyphenols as a result of evidence

developed in studies done in vitro and in vivo, which shows that they may protect against

oxidative damage. This is due mainly to their antioxidative properties which quench free

radical formation. Polyphenols, therefore, have been suggested as agents to reduce the risk of

various diseases associated with oxidative stress, including osteoporosis [166-168, 193, 194].

55

Nutritional supplements such as greens+ bone builderTM contain not only polyphenol-rich

plant constituents, but also other nutrients that are considered beneficial for bone health.

Previous in vitro results from our laboratory have shown that greens+TM, a nutritional

supplement rich in a variety of polyphenols, is effective in stimulating osteoblasts to form

bone nodules in a dose-dependent manner in vitro [171]. Another nutritional supplement,

bone builderTM, which is rich in minerals, vitamins and other nutrients, also had a significant

dose-dependent stimulatory effect on bone nodule formation (Snyder et al., 2010). When

greens+TM and bone builderTM were tested as a combination, the effects were six times more

effective than either one alone [154]. This led us to believe that the combination effects of

polyphenols with minerals, vitamins and other nutrients may be beneficial in reducing

oxidative stress and may potentially decrease the risk of osteoporosis. The aim of this study,

therefore, was to investigate the effects of the nutritional supplement, which combines the

variety of polyphenols with various vitamins, minerals and antioxidants on biomarkers of

oxidative stress and bone turnover markers in postmenopausal women who are at risk for

osteoporosis.

56

III. Methods

Statistics

All statistical analyses were performed using GraphPad PRISM 5.00 for Windows

(GraphPad Software, California). Summary statistics of participant demographics such as age

and BMI were generated and presented as means ± standard errors of the mean (SEM). A

paired t-test was used to determine the overall effect of the supplement or placebo at 8 weeks

of treatment on urinary polyphenol content, biomarkers of bone turnover, parameters of

oxidative stress parameters, and antioxidant status. Percent change on the mentioned clinical

end-point markers and parameters were in the Supplement group was calculated and

compared to the change in the Placebo group using an unpaired t-test. Significance was set at

p<0.05.

57

IV. Results

A total of 47 postmenopausal women, 24 in the Placebo group and 23 in the Supplement

group (one participant withdrew for personal reasons), completed the study. Baseline

characteristics of the participants are described in Table 5.1. There were no significant

differences among groups with respect to age, BMI, years since menopause, or blood

pressure (Table 5.1). Similarly, there were no significant differences at baseline for average

serum TAC, total urinary polyphenol content, protein oxidation, and bone turnover markers.

However, there was a significant difference between the Supplement group and Placebo

group for TBARS at baseline with the Supplement group showing higher levels of TBARS

than the Placebo group at 7.1 ± 0.3 µM and 6.4 ± 0.2 µM, respectively (Table 5.1).

The Supplemented group had significantly decreased CTX over time. After 8 weeks of

supplementation, CTX decreased from the baseline concentration of 521.4 ± 51.7 µg/L to

462.1 ± 47.5 µg/L (p<0.05, Fig. 5.1a) or 11.6 ± 3.8% (Fig. 5.1b). There was no significant

change in participants consuming placebo from baseline (452.7 ± 42.9 µg/L) or 3.6 ± 4.4%

(Fig. 5.1a) to after the eight week supplement period (456.1 ± 41.5 µg/L) (Fig. 5.1b). There

were no changes in serum concentrations of PINP, a marker of bone formation, in either

group (Fig 5.1c,d).

The TPC significantly increased after 8 weeks of treatment in the Supplement group (p<0.05,

Fig. 5.2a), but was not shown in the Placebo group. The TPC measurements increased up to

58

almost 56% in the Supplement group, but only up to about 15% in the Placebo group

(p<0.05, Fig. 5.2b)

A significant increase in the TAC from the baseline concentration of 1.45 ± 0.05 mM to a

concentration of 1.54 ± 0.04 mM at 8 weeks (p<0.05, Fig. 5.3a) was observed for the

Supplement group. This increased approximately 7% after 8 weeks in the Supplement group,

which was significantly different from the Placebo group (p<0.0001, Fig. 5.3b). There were

no changes in TAC in the Placebo group (Fig. 5.3a).

Consuming the supplement significantly decreased lipid peroxidation from the baseline

concentration of 7.1 ± 0.3 µM to a concentration of 6.4 ± 0.2 µM at 8 weeks, respectively

(p<0.0001, Fig. 5.4a). This is equivalent to a decrease of approximately 10% after 8 weeks,

which was significantly opposite to the increase of approximately 4% seen in the Placebo

group (p<0.0001, Fig. 5.4b).

The Supplement group showed a significant increase in protein thiols, indicating a decrease

in protein oxidation, from the baseline concentration of 469.7 ± 18.1 µM to 495.0 ± 19.2 µM

after 8 weeks of treatment (p<0.0001, Fig. 5.5a). Protein thiols increased roughly 5% from

baseline among the Supplement group, which was significantly different from the decrease of

roughly 3% in the Placebo group (p<0.001, Fig. 5.5b).

59

Table 5.1 Participant characteristics and baseline values for oxidative stress parameters and

antioxidant capacity for each group

Parameters Measured Summary statistics (mean ± SEM)

Greens+ bone builderTM

(N=23)

Placebo (N=24)

Age (yrs) 56 ± 1 56 ± 0

Pulse rate (beats/min) 65.9 ± 1.8 66.8 ± 1.8

Blood pressure-systolic (mmHg) 118.9 ± 3.2 115.6 ± 3.2

Blood pressure-diastolic (mmHg) 72.5 ± 2.3 75.1 ± 2.0

BMI (kg/m2) 23.8 ± 0.8 25.0 ± 1.0

Years since menopause 4.6 ± 0.5 5.7 ± 0.6

Total urinary polyphenol content 39.0 ± 4.3 35.9 ± 4.8

Total antioxidant capacity

(TAC)(mM)

1.45 ± 0.05 1.50 ± 0.05

Bone Turnover

Markers

CTX (µg/L) 521.4 ± 51.7 452.7 ± 42.9

PINP (µg/L) 57.2 ± 4.9 55.7 ± 3.7

Oxidative Stress

Parameters

TBARS (µM)1 7.1 ± 0.3 6.4 ± 0.2

Protein thiols

(µM)

469.7 ± 18.1 490.1 ± 12.5

1 Significant difference between groups (p<0.05)

60

0 8 0 84 0 0

4 5 0

5 0 0

5 5 0

6 0 0P la c e b o

S u p p lem e n t

*

T im e p e r io d f o r in t e r v e n t io n ( w k s .)

Ser

um

b-C

ross

lap

s(p

g/m

L)

5 .1 a

-2 0

-1 5

-1 0

-5

0

5

1 0

*

P la c e b o

S u p p lem e n t

Ch

an

ge

fro

m b

as

eli

ne

at

8 w

ee

ks

(%

)

b

0 8 0 84 0

5 0

6 0

7 0P la c e b o

S u p p lem e n t

T im e p e r io d f o r in t e r v e n t io n ( w k s .)

PIN

P (m

g/L

)

c

-5

0

5

1 0P la c e b o

S u p p lem e n t

Ch

an

ge

fro

m b

ase

lin

ea

t 8

wee

ks

(%)

d

Figure 5.1: Concentration of bone resorption marker CTX in the serum for a period of 8 weeks. Values are mean ± SEM and were compared within supplement group using a paired t-test (*p<0.05) b Change in CTX for a period of 8 weeks and was measured relative to baseline concentration. Values are mean % change ± SEM for each supplement group and were compared using an unpaired t-test (*p<0.05) c Concentration of bone formation marker PINP in the serum for a period of 8 weeks. Values are mean ± SEM and were compared within supplement group using a paired t-test b Change in PINP for a period of 8 weeks and was measured relative to baseline concentration. Values are mean % change ± SEM for each supplement group and were compared using an unpaired t-test

61

0 8 0 82 0

3 0

4 0

5 0

6 0

7 0P la c e b o

S u p p lem e n t

5 .2 a

*

T im e p e r io d f o r in t e r v e n t io n ( w k s .)

To

tal

Uri

ne

Po

lyp

hen

ol

Co

nte

nt

( mg

GA

E/m

g C

rea

tin

ine)

- 5 0

0

5 0

1 0 0

* P la c e b o

S u p p lem e n t

b

Ch

an

ge

fro

m b

ase

lin

ea

t 8

wee

ks

(%)

Fig. 5.2: a Concentration of total polyphenol content in the urine over 8 weeks. 47 postmenopausal women between 50-60 years old were in the Supplement group (N=23) or Placebo group (N=24) for a period of 8 weeks. Values are mean ± SEM and were compared within supplement group using a paired t-test (*p<0.05). b Change in total polyphenol content over the 8 week supplement period and was measured relative to baseline concentration. Values are mean % change ± SEM for each supplement group and were compared using an unpaired t-test (*p<0.05)

62

0 8 0 81 .3

1 .4

1 .5

1 .6P la c e b o

S u p p lem e n t

*5 .3 a

T im e p e r io d f o r in t e r v e n t io n ( w k s .)

TA

C (

mM

)

- 5

0

5

1 0 * P la c e b o

S u p p lem e n t

b

Ch

an

ge

fro

m b

ase

lin

ea

t 8

wee

ks

(%)

Figure 5.3: a Total antioxidant capacity in the serum over 8 weeks, as determined using the TEAC assay. 47 postmenopausal women between 50-60 years old were in the Supplement group (N=23) or Placebo group (N=24) for a period of 8 weeks. Values are mean ± SEM and were compared within supplement group using a paired t-test (*p<0.05). b Change in total antioxidant capacity over the 8 week supplement period and was measured relative to baseline concentration. Values are mean % change ± SEM for each supplement group and were compared using an unpaired t-test (*p<0.0001)

63

0 8 0 85

6

7

8P la c e b o

S u p p lem e n t

*

5 .4 a

T im e p e r io d f o r in t e r v e n t io n ( w k s .)

TB

AR

S (

nm

ol/

ml)

- 1 5

-1 0

-5

0

5

1 0

*

P la c e b o

S u p p lem e n t

b

Ch

an

ge

fro

m b

ase

lin

ea

t 8

wee

ks

(%)

Figure 5.4: a Concentration of lipid peroxidation in the serum over 8 weeks, as determined by TBARS. 47 postmenopausal women between 50-60 years old were in the Supplement group (N=23) or Placebo group (N=24) for a period of 8 weeks. Values are mean ± SEM and were compared within supplement group using a paired t-test (*p<0.0001). b Change in lipid peroxidation over the 8 week supplement period and was measured relative to baseline concentration. Values are mean % change ± SEM for each supplement group and were compared using an unpaired t-test (*p<0.0001)

64

0 8 0 84 0 0

4 5 0

5 0 0

5 5 0P la c e b o

S u p p lem e n t*

5 .5 a

T im e p e r io d f o r in t e r v e n t io n ( w k s .)

Th

iols

(m

M)

- 5

0

5

1 0

*P la c e b o

S u p p lem e n t

Ch

an

ge

fro

m b

ase

lin

ea

t 8

wee

ks

(%)

b

Figure 4.5: a Concentration of protein oxidation in the serum over 8 weeks as determined by thiols. 47 postmenopausal women between 50-60 years old were in the Supplement group (N=23) or Placebo group (N=24) for a period of 8 weeks. Values are mean ± SEM and were compared within supplement group using a paired t-test (*p<0.0001). b Change in protein oxidation over the 8 week supplement period and was measured relative to baseline concentration. Values are mean % change ± SEM for each supplement group and were compared using an unpaired t-test (*p<0.001)

65

V. Discussion We have shown that intervention with a supplement containing polyphenols and other

nutritional components may decrease biomarkers for bone turnover meaning that the risk for

development of osteoporosis might be reduced as a consequence of antioxidant treatment.

This was tested by comparing bone turnover markers (CTX and PINP), oxidative stress

parameters (TBARS and protein thiols), TAC, and TPC in early postmenopausal women who

consumed a supplement rich in polyphenols and other micronutrients or a placebo. The

findings reported here demonstrate a decline in CTX, oxidative stress parameters as well as

an increase in TAC and total polyphenol content after 8 weeks of treatment among women

who were supplemented with the combination of polyphenols and micronutrients. The

changes were significantly different when comparing the two groups to one another. These

findings are important because no differences were observed in any of the outcome

measurements made in women who were on placebo for 8 weeks

Previous studies showing the antioxidant properties of polyphenols were based mainly on in

vitro and animal studies. Our laboratory is the first to evaluate the efficacy of the nutritional

supplement comprised of polyphenols, vitamins, minerals, and antioxidants administered to

postmenopausal women who are at risk for osteoporosis. Here, the Supplement group

demonstrated increased levels of TAC and total urinary polyphenols at 8 weeks of treatment,

which was significantly different when compared to the Placebo group. Although the

polyphenols in greens+TM may have been the principal component of the nutritional

supplement contributing to the observed antioxidant effect, the combined effect with other

66

micronutrients and antioxidants in the bone builderTM may also have contributed to this effect

[173, 174] but it was not possible to actually prove this one way or the other in this study.

The TEAC method has been validated to measure the antioxidant properties of both the lipid-

and the water-soluble antioxidants in vivo. This assay is used primarily for its ability to assess

many endogenous antioxidants in addition to those found in the supplement and its capability

to quench reactive oxygen species (ROS) [172]. Thus, the polyphenol-rich supplements

consumed by the participants may have been absorbed and metabolites excreted in the urine

as shown by the increase in the urinary concentrations of total polyphenols. This increased

absorption may have resulted in an increased ability to suppress oxidative stress. This

provides further credibility to the idea that it is the potent antioxidant capacity of the

supplement, which is responsible for the decreased oxidative stress and CTX shown in this

study.

Previous in vitro, in vivo and clinical studies [166-168, 171] on both lipid- and water-soluble

antioxidants done in our laboratory have also demonstrated similar results. For example, in a

recent study, the effectiveness of lycopene (a fat-soluble antioxidant) on oxidative stress

parameters in humans was investigated. Decreased oxidization of proteins and lipid

peroxidation following T2 months of treatment was shown [114]. This is similar to our

previous findings in our laboratory where a significant decrease in both protein oxidation and

lipid peroxidation was demonstrated within the Supplement group when compared to

Placebo following 8 weeks of treatment. The relationship between the antioxidant property of

the nutritional supplement and its effect on bone cells in vitro was recently reported.

67

Supplementation with greens+TM containing polyphenols such as quercetin, apigenin,

kaempferol and luteolin was shown to increase the proliferation of human osteoblast-like

cells at early time points of addition as well as to stimulate the formation of bone nodules in

vitro in both a dose- and time-dependent manner [171]. These observations might suggest a

positive effect of ingesting foods containing polyphenol and other supplements such as

greens+ bone builderTM in reducing oxidative stress, which may stimulate the activity of

osteoblast cells, thus may possibly be beneficial in bone formation. However, our study was

not able to show an effect on PINP, and longer treatment with greens+ bone builderTM may

be required.

The present intervention study demonstrated an association between increased urinary

polyphenols and decreased CTX in postmenopausal women. Studies have shown that high

bone turnover is correlated with a low BMD [195] and that increased bone turnover

correlates with increased bone fragility and deterioration of bone microarchitecture [196].

Our findings showed that with supplementation, there was a significant decrease in CTX

after 8 weeks of treatment. This is in keeping with another dietary intervention study, in

which supplementation with calcium and vitamin D resulted in a significant decrease in CTX

among postmenopausal women at risk of osteoporosis at 6 and 12 months of treatment [197].

More importantly, these changes in bone resorption markers are comparable to those seen in

postmenopausal women supplemented with calcium [198, 199], which together with

adequate vitamin D [55, 200], are currently recommended for maintenance of bone health

and the prevention of osteoporosis. A study by Meunier et al. showed that supplementation

68

with 596 mg/day of calcium significantly decreased serum CTX by 13% after 6 months in

postmenopausal women [198]. Furthermore, data from another study involving a combined

treatment of potassium citrate and calcium citrate are in line with this study [201]. They

showed that when compared to placebo, the combined treatment reduced all of the bone

resorption markers, thus suggesting bone protective properties through a synergistic effect.

There was no significant change found in PINP, a notable marker of bone formation, in these

participants. Although serum PINP gives an accurate description of changes in bone

turnover, particularly in postmenopausal osteoporosis [79, 202, 203], it is evident from other

studies on osteoporosis medications that changes in PINP tend to be smaller in magnitude

compared to those seen in CTX, and can take up to 6 months to be detected [72]. This

intervention study was carried out for only 2 months, and perhaps a longer period of

intervention would have resulted in significant changes in bone formation markers.

Nevertheless, in the present study the effect on CTX is still biologically important, regardless

of the lack of effect on PINP. Furthermore, a possible explanation for this result can be the

coupling effect of bone resorption and formation. When there is a decrease in the rate of bone

turnover, the bone first experiences decreases in bone resorption and subsequently bone

formation. Bone resorption at any given site takes approximately one month, followed by

bone formation in the same place the bone has been resorbed (coupling). Therefore, if the

rate of resorption is slowed down, bone formation may also be slowed down. Many current

therapies for osteoporosis target the increase in bone turnover markers by attempting to

decrease bone resorption [75, 80, 204]. Bone resorption markers tend to decrease rapidly in

response to potent anti-resorptive treatment, typically a 50-70% reduction in the telopeptides

69

within the first 12 weeks of treatment [71, 205]. Studies have shown that early antiresorptive-

induced reductions in biochemical markers have been correlated with subsequent increases in

BMD [206-209]. Notably, a smaller change of a 20% decrease in serum CTX within the first

6 months of therapy predicted significant increases in BMD at the total hip, greater

trochanter, intertrochanteric region, and spine after 2.5 years of therapy (p<0.05) [209]. In

this study, a similar anti-resorptive effect of supplementation for 8 weeks resulted in an

average decrease in CTX of 11.6 ± 3.8%. Despite the short study duration preventing us from

assessing the BMD of our participants, the present results on CTX suggest that long-term

supplementation with a variety of polyphenols and micronutrients may result in increased

BMD as long as the decreased CTX effect is sustained. Based on these findings, the results

support the hypothesis that supplements such as greens+ bone builderTM may decrease the

risk of osteoporosis as seen by the reduction of CTX. The effects of polyphenols on bone

resorption looks promising, and a longer-term study with BMD measurements may confirm

the bone-protective effects of total polyphenols in postmenopausal women at risk of

osteoporosis.

Limitations of this study are acknowledged. First, the present study was not specifically

designed to identify and quantify the individual types of polyphenols, minerals and other

micronutrients absorbed in the body. Secondly, we did not control for intake of all foods and

beverages that were rich in polyphenols, which could have also contributed to the changes in

antioxidant capacity. Third, based on previous studies, our a priori sample size was originally

intended to include 60 participants to show statistical power; however, with 47 participants,

we were able to see a significant difference in most outcome measures. Lastly, as mentioned

70

before, this study was short-term. A study showing the long-term effects of the supplement

on bone health should be considered along with measurements of BMD to document the

benefit to bone of the additive effect of the polyphenols and other nutrients.

In conclusion, our study has shown that daily supplementation of a combination of

polyphenols and other trace nutrients and minerals in the form of the nutritional supplement,

greens+ bone builderTM, can significantly increase the antioxidant capacity which results in

decreased bone resorption, thus decreasing the extent of oxidative damage in postmenopausal

women at risk of osteoporosis. The beneficial effects can be due to the combination of the

polyphenols in greens+ with the other nutrients and minerals contained in the bone builder.

This observation, along with previously observed bone-protective effects of the individual

polyphenols, nutrients and trace minerals, should generate an increased interest into

exploring further the potentially beneficial effects on bone of nutritional supplements such as

those studied here. Although there is evidence that implicates oxidative stress as an important

mediator of bone loss potentially enhancing the development of osteoporosis, our laboratory

was the first to investigate the combined effects of a variety of polyphenols alongside other

nutrients and minerals in healthy postmenopausal women, suggesting that greens+ bone

builderTM may prove to be a good alternative or complementary agent with other established

therapeutics, in this case, actual drugs approved for the prevention or treatment of

osteoporosis.

71

CHAPTER 6

GENERAL DISCUSSION AND CONCLUSIONS

72

I. Discussion of Results

Polyphenols are water-soluble phytochemical antioxidants that may protect bone against

oxidative stress-induced osteoporosis. In the past few years, polyphenols and their

antioxidant capabilities have been studied extensively. Studies have focused primarily on the

effects of individual polyphenols on various chronic diseases associated with oxidative stress

[166, 210-212]. Furthermore, there is convincing scientific evidence to support the important

role that oxidative stress plays in bone metabolism. There are several lines of evidence that

indicate oxidative stress increases differentiation and function of osteoclasts [85, 104, 119,

213] as well as inhibiting differentiation of osteoblasts [214, 215]. Therefore, based on these

findings, we hypothesized that consumption of polyphenols may be beneficial and could

prevent the development of osteoporosis due to antioxidant effects

Previous in vitro studies in our laboratory have demonstrated the beneficial effects of

greens+TM, bone builder TM and greens+ bone builderTM using systems of in vitro

osteogenesis. greens+ TM is a nutritional supplement rich in polyphenols whereas

greens+bone builderTM is rich in minerals, vitamins and micronutrients. greens+ bone

builderTM contains all the components of the first two supplements. greens+ TM and bone

builder TM were shown separately to have a favourable effect on osteoblasts by increasing the

number of bone nodules formed in vitro in a dose-dependent manner. Conversely, when

greens+TM and bone builderTM were tested together, the effects were greater than either one

alone suggesting either additive or synergistic effects. This finding suggested that there might

be a use for combining the contents of these agents into a single supplement for improvement

of bone-health. Therefore, a clinical study was conducted to determine whether greens+ bone

builderTM could reduce the risk indicators for osteoporosis in postmenopausal women as a

consequence of the supplement’s antioxidant properties.

73

Results obtained in this study are presented in two manuscripts (Chapters 4 and 5).

The key findings are as follows:

1. Postmenopausal women supplemented with greens+ bone builderTM for a period of 8

weeks had a significant interaction between time (4 and 8 weeks) and type of treatment

(Treatment and Placebo group) for total antioxidant capacity with a concomitant interaction

between time (4 and 8 weeks) and type of treatment (Treatment and Placebo group) effect in

the oxidative stress parameters for protein oxidation and lipid peroxidation.

2. Supplementation of greens+ bone builderTM for a period of 8 weeks resulted in an increase

in total urinary polyphenols and total antioxidant capacity as well as a decrease in oxidative

stress biomarkers in postmenopausal women. This decrease in oxidative stress corresponded

to a significant decrease in the bone resorption marker, CTX. It is possible that such

corresponding decreases may subsequently reduce the risk of osteoporosis. However, due to

the duration of the study period, intervention with greens+ bone builderTM did not lead to any

statistically significant differences for the bone formation marker between the two test

groups..

There is sufficient evidence to indicate that antioxidants provide defense against oxidative

stress in the body, and thereby protect the body against chronic diseases including

osteoporosis. A study has revealed that plasma total oxidative status (TOS) and oxidative

stress index (OSI) values were significantly higher, and plasma total antioxidant status (TAS)

levels were lower in postmenopausal females who were also osteoporotic, as compared to

healthy controls [126]. The health benefits of antioxidants depend on the amount consumed

and on their bioavailability [216]. In this thesis, the Supplemented group had an increase of

7.1 ± 1.9% from baseline to 8 weeks, which was significantly different from the Placebo

group, suggesting that the polyphenols and other micronutrients contributed to improving the

antioxidant status of individuals and consequently in mitigating the damaging effects of

oxidative stress. These results concur with the results of a study by Cao et al. wherein TEAC

values increased in elderly women after they consumed antioxidant-rich foods such as

strawberries, spinach or red wine [217].

74

Previous studies showing the antioxidant properties of polyphenols were based mainly on in

vitro and animal studies. Our laboratory is the first to evaluate the efficacy of the nutritional

supplement that comprised of polyphenols, vitamins, minerals, and antioxidants administered

to postmenopausal women who are at risk for osteoporosis. Here, the Supplement group

showed increased TAC and total urinary polyphenols by 8 weeks of treatment. This differed

significantly from the Placebo group. Although the polyphenols in greens+TM may have been

the principal component of the nutritional supplement contributing to the observed

antioxidant effect, the combined effect with other micronutrients and antioxidants in the bone

builderTM may also have contributed to this effect [173, 174].

Although polyphenols are the most abundant class of antioxidant phytochemicals in fruits

and vegetables, they also contain different vitamins and minerals. Many studies have shown

the advantageous role of these polyphenols, vitamins and minerals found in fruits and

vegetables against adverse effect of oxidative stress and related diseases [218, 219]. An

excellent review by Bouayed and Bohn suggested that the beneficial effects on human health

of antioxidants derived from fruits and vegetables can be explained by the additive or

synergistic effect of all of the ingredients. However, these findings have shown to be

controversial with the theory that individual compounds may be the major contributors

associated with proposed beneficial effects on health as previously postulated following

epidemiological studies and associated meta-analyses [220, 221].

Nevertheless, the most common polyphenols in the human diet are not inevitably the most

active in vivo, either because they have less intrinsic activity than others or because they are

inadequately absorbed from the intestine, highly metabolized, or rapidly eliminated [222].

The chemical nature of individual polyphenols influences their biological properties

including their bioavailability, antioxidant activity, specific interaction with cell receptors

and enzymes and other properties [148]. It is important to note that only a mix of

polyphenols was examined here and not individual polyphenols. Yet, an indirect evidence for

polyphenol absorption through the gut barrier is suggested by the rise seen in the antioxidant

capacity of the blood following consumption of polyphenol-rich foods [148]. The results

showed that the total antioxidant capacity of the supplemented group (i.e. with a variety of

polyphenols and other micronutrients) was greater than that shown in the Placebo group.

75

More direct evidence on the bioavailability of polyphenols has been acquired by determining

their concentrations in plasma and urine after the ingestion of polyphenol-rich products

[135]. The total concentration of polyphenol metabolites excreted in urine is roughly

proportional to maximum plasma concentrations, which are reached approximately 1.5 to 5.5

hours after ingestion of polyphenol-rich foods [148]. Typically, once the polyphenols are

absorbed, they are conjugated to glucuronide, sulphate and methyl groups in the gut mucosa

and inner tissues [223]. These reactions facilitate their excretion and prevent accumulation of

unsure levels [223]. Therefore, recovery of polyphenols and their metabolites in urine

indicate that polyphenols are being absorbed into the small intestine and then go to the liver

and other tissues to be metabolized, and are then excreted by the kidneys into the urine.

Thus, higher urinary total polyphenol levels indicate greater levels of serum polyphenols

which can be explained by absorption from the gut.

In this thesis, total polyphenol levels in the urine were used to estimate polyphenol intake and

absorption. Gallic acid was used as a standard because studies have shown that it is far better

absorbed than other polyphenols [135]. Total plasma polyphenols levels were not measured

because, in terms of the elimination half-lives, gallic acid appears to be one of the

polyphenols with a minimal chance of accumulating in plasma despite repeated ingestion.

The TEAC method has been validated to measure the antioxidant properties of both the lipid-

and the water-soluble antioxidants in vivo. This assay is used primarily for its ability to assess

many endogenous antioxidants in addition to those found in the supplement and its capability

to quench reactive oxygen species (ROS) [172]. Thus, the polyphenol-rich supplements

consumed by the participants may have been absorbed and metabolites excreted in the urine

as shown by the increase in the urinary concentrations of total polyphenols. This increased

absorption may have resulted in an increased ability to suppress oxidative stress. This

provides further credibility to the idea that it is the potent antioxidant capacity of the

supplement that is responsible for the decreased oxidative stress and CTX shown in this

study.

76

Previous in vitro, in vivo and clinical studies [166-168, 171] from our laboratory on both

lipid- and water-soluble antioxidant have also shown similar results. Thus, a recent study

investigated the effectiveness of lycopene, a fat-soluble antioxidant, on oxidative stress

parameters in humans. The results showed decreased oxidization of proteins and lipid

peroxidation at 2 months of treatment [114]. This is similar to our finding where there was a

significant decrease in both protein oxidation and lipid peroxidation within the Supplement

group when compared to Placebo at 8 weeks of treatment.

Furthermore, this thesis demonstrated an association between increased urinary polyphenols

and decreased CTX in postmenopausal women. Studies have shown that a high bone

turnover is correlated with a low BMD [195] and that an increased bone turnover correlates

with increased bone fragility and deterioration of bone microarchitecture [196]. Our findings

showed that with supplementation, there was a significant decrease in CTX after 8 weeks.

This is in keeping with another dietary intervention study, in which supplementation with

calcium and vitamin D resulted in a significant decrease in CTX at 6 and 12 months of

treatment among postmenopausal women at risk of osteoporosis [197].

More importantly, these changes in bone resorption markers are comparable to those seen in

postmenopausal women supplemented with calcium [198, 199], which together with

adequate vitamin D [55, 200], are currently recommended for maintenance of bone health

and the prevention of osteoporosis. A study by Meunier et al. showed that supplementation

with 596 mg/day of calcium significantly decreased serum CTX by 13% after 6 months in

postmenopausal women [198]. Furthermore, data from another study involving a combined

treatment of potassium citrate and calcium citrate are in line with this study [201]. They

showed that when compared to placebo, the combined treatment reduced all of the bone

resorption markers, thus suggesting bone protective properties through a synergistic effect.

The relationship between the antioxidant property of the nutritional supplement and its effect

on bone cells in vitro was recently reported. Supplementation with greens+TM containing

polyphenols such as quercetin, apigenin, kaempferol and luteolin was shown to increase the

proliferation of human osteoblast-like cells at early time points of addition as well as to

77

stimulate bone nodule formation in a dose- and time-dependent manner [171]. These

observations may suggest a positive effect of ingesting foods containing polyphenol and

other supplements such as greens+ bone builderTM in reducing oxidative stress, which may

stimulate the activity of osteoblast cells, thus may possibly be beneficial in bone formation.

However, our study was not able to show an effect on PINP, a notable marker of bone

formation, in these participants. Although serum PINP gives an accurate description of

changes in bone turnover, particularly in postmenopausal osteoporosis [79, 202, 203], it is

evident from other studies on osteoporosis medications that changes in PINP tend to be

smaller in magnitude compared to those seen in CTX, and can take up to 6 months to be

detected [72]. This intervention study was carried out for only 2 months, and perhaps a

longer period of intervention would have resulted in significant changes in bone formation

markers. Nevertheless, in the present study the effect on CTX is still biologically important,

regardless of the lack of effect on PINP.

Furthermore, a possible explanation for this result can be the coupling effect of bone

resorption and formation. When there is a decrease in the rate of bone turnover, the bone first

experiences decreases in bone resorption and subsequently bone formation. Bone resorption

at any given site takes approximately one month, followed by bone formation in the same

place the bone has been resorbed (coupling). Therefore, if the rate of resorption is slowed

down, bone formation may also be slowed down. Many current therapies for osteoporosis

target the increase in bone turnover markers by attempting to decrease bone resorption [75,

80, 204]. Bone resorption markers tend to decrease rapidly in response to potent anti-

resorptive treatment, typically a 50-70% reduction in the telopeptides within the first 12

weeks of treatment [71, 205]. Studies have shown that early antiresorptive-induced

reductions in biochemical markers have been correlated with subsequent increases in BMD

[206-209]. Notably, a smaller but significant change of a 20% decrease in serum CTX within

the first 6 months of therapy predicted significant increases in BMD at the total hip, greater

trochanter, intertrochanteric region, and spine after 2.5 years of therapy [209]. In this study, a

similar anti-resorptive effect of supplementation for 8 weeks resulted in an average decrease

in CTX of 11.6 ± 3.8%.

78

Despite the short study duration preventing us from assessing the BMD of our participants,

the present results on CTX suggest that long-term supplementation with a variety of

polyphenols and micronutrients may result in increased BMD as long as the decreased CTX

effect is sustained. Based on these findings, the results support the hypothesis that

supplements such as greens+ bone builderTM may decrease the risk of osteoporosis as seen by

the reduction of CTX. The effects of polyphenols on bone resorption looks promising, and a

longer-term study with BMD measurements may confirm the bone-protective effects of total

polyphenols in postmenopausal women at risk of osteoporosis. As well, since the nutritional

supplement contained minerals and vitamins known to be beneficial for bone health, in

addition to the phytochemical antioxidants, it is possible that mechanisms in addition to the

antioxidant may also be involved. Future studies should be directed towards studying these

interactions further.

An increase in serum antioxidant capacity following the consumption of greens+ bone

builderTM was expected since it contains several polyphenols and other micronutrients. The

combined effect of the supplement seen in this thesis could have contributed toward a

decrease in the status of oxidative stress as indicated by the reductions in lipid peroxidation

and protein oxidation. Previous studies have shown a causal relationship between oxidative

stress and the risk of osteoporosis. Results obtained in this study, therefore, support the

hypothesis that increased serum antioxidant capacity had a beneficial effect toward the

reduction of oxidative stress, which may have an association to the decrease seen in bone

resorption, thus reducing the risk of osteoporosis.

79

II. Implication of Findings

This thesis provides the rationale in that supplementation with a variety of polyphenols in

combination with other micronutrients may decrease the risk of osteoporosis via decreasing

oxidative stress parameters and bone resorption markers. The significant decrease seen in

CTX was similar to previous studies among postmenopausal women who were supplemented

with calcium and vitamin D [197-199]. Based on these findings, the consumption of diets and

nutritional supplements that are rich in polyphenols and other minerals, vitamins and

nutrients, by women at risk for osteoporosis, should be considered as a complementary

strategy, as it may improve overall bone health.

80

III. Limitations

The major limitation, as with many randomized controlled trials, was participant recruitment.

The sample of participants required had narrow inclusion criteria, thus limiting participant

eligibility. For instance, recruiting women between the ages of 50 and 60 who were not

taking any medications were able to refrain from consuming other vitamins and supplements,

as well as restricting high-polyphenol foods and beverages for 8 weeks happened to be very

challenging and difficult. Thus, many eligible participants did not enroll, which limited the

sample size to 48. Our original aim was to recruit 60 participants to show statistical power.

Nonetheless, we were able to show significant differences in most outcome measures with 47

participants..

Secondly, the present study was not specifically designed to identify and quantify the

individual types of polyphenols, minerals and other micronutrients absorbed in the body.

However, our previous in vitro study has shown the additive effect of the supplement on

bone health. Furthermore, we did not control for the intake of all foods and beverages that

were rich in polyphenols, which may have greatly contributed to the changes seen in the total

antioxidant capacity and total urinary polyphenol content.

Lastly, this study illustrated the short-term effects of the supplement on bone metabolism. A

study showing the long-term effects of the supplement should be considered along with

periodic measurements of BMD for several years to strengthen the efficacy of the additive

effects of polyphenols along with other micronutrients that are beneficial to the bone.

81

IV. Future Area of Research

A longitudinal prospective study where early postmenopausal women are given a supplement

containing polyphenols and other micronutrients and monitored for BMD and fracture risk

would further support the present findings. A strong correlation has been shown between

BMD, fracture risk and BTMs, and measuring all three parameters would provide strong

support for the findings shown in this thesis. Based on the paucity of various studies, further

research should consider potential confounders such as dietary (i.e. consumption of other

foods and nutrients) using the 7-day food records collected as well as lifestyle factors (i.e.

prior fracture incidences, exercise). Also, future studies should also consider the dosage,

type, duration, and frequency of consumption of polyphenols in addition to the

micronutrients. Complying with many of these guidelines may ultimately lead to the

development of evidence-based dietary recommendations for maintaining bone health.

As well, genomics is becoming an area of interest where future studies may consider the

beneficial effect of polyphenols as wells as micronutrients on bone health through genetics

and the gene-environment interaction.

Finally, as this thesis has shown a favourable outcome of the consumption of a nutritional

supplement rich in a variety of polyphenols and micronutrients in a randomized controlled

study, further research investigating these effects in conjunction with FDA-approved

medication to treat osteoporosis is warranted. If this additive product can provide a benefit to

currently approved methods for preventing and treating osteoporosis, this would result in a

more comprehensive understanding of bone health.

82

V. Conclusions

Osteoporosis is an enormous public health concern as fractures from this debilitating disease

occur more often than a heart attack, stroke and breast cancer combined [56]. Dietary

components are noteworthy aspects for the prevention of osteoporosis. Their incorporation

into the daily lifestyle is rather straightforward and inexpensive. This thesis demonstrated

that the additive effect of polyphenols with other micronutrients act as potent antioxidants to

decrease oxidative stress parameters and bone resorption, which suggests that it may also

decrease the risk of developing osteoporosis in postmenopausal women and may improve

their overall bone health.

83

References

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171. Rao, L.G., B. Balachandran, and A.V. Rao, Polyphenol Extract of Greens+ Nutritional Supplement Stimulates Bone Formation in Cultures of Human Osteoblast-like SaOS-2 Cells. Journal of Dietary Supplements, 2008. 5(3): p. 264-82.

172. Ghiselli, A., et al., Total antioxidant capacity as a tool to assess redox status: critical view and experimental data. Free Radical Biology and Medicine, 2000. 29(11): p. 1106-1114.

173. Jacobs, D.R., Jr., J. Mursu, and K.A. Meyer, The Importance of Food. Archives of Pediatrics & Adolescent Medicine, 2011. 166(2): p. 187-188.

174. Yeum, K.J., et al., Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma. Archives of Biochemistry and Biophysics, 2004. 430(1): p. 97-103.

175. Milner, J.A., Reducing the risk of cancer, in Functional foods: Designer foods, pharmafoods, nutraceuticals, I. Goldberg, Editor 1994, Chapman & Hall: New York. p. 39-70.

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176. Duthie, G.G. and K.M. Brown, Reducing the risk of cardiovascular disease in Functional foods: Designer foods, pharmafoods, nutraceuticals, I. Goldberg, Editor 1994, Chapman & Hall: New York. p. 19-38.

177. Basu, A. and V. Imrhan, Tomatoes versus lycopene in oxidative stress and carcinogenesis: conclusions from clinical trials. European Journal of Clinical Nutrition, 2007. 61(3): p. 295-303.

178. Halliwell, B., Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Archives of Biochemistry and Biophysics, 2008. 476(2): p. 107-12.

179. Manach, C. and J.L. Donovan, Pharmacokinetics and metabolism of dietary flavonoids in humans. Free Radical Research, 2004. 38(8): p. 771-85.

180. Williamson, G., et al., In vitro biological properties of flavonoid conjugates found in vivo. Free Radical Research, 2005. 39(5): p. 457-69.

181. Rechner, A.R., et al., The metabolic fate of dietary polyphenols in humans. Free Radical Biology and Medicine, 2002. 33(2): p. 220-35.

182. Prior, R.L., X. Wu, and K. Schaich, Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 2005. 53(10): p. 4290-302.

183. Williams, R.J., J.P. Spencer, and C. Rice-Evans, Flavonoids: antioxidants or signalling molecules? Free Radical Biology and Medicine, 2004. 36(7): p. 838-49.

184. Zhou, B., et al., Evidence for alpha-tocopherol regeneration reaction of green tea polyphenols in SDS micelles. Free Radical Biology and Medicine, 2005. 38(1): p. 78-84.

185. Riis, B.J., et al., Low bone mass and fast rate of bone loss at menopause: equal risk factors for future fracture: a 15-year follow-up study. Bone, 1996. 19(1): p. 9-12.

186. Recker, R., et al., Characterization of perimenopausal bone loss: a prospective study. J Bone Miner Res, 2000. 15(10): p. 1965-73.

187. Hanley, D.A., et al., Vitamin D in adult health and disease: a review and guideline statement from Osteoporosis Canada (summary). CMAJ, 2010. 182(12): p. 1315-9.

188. Dennehy, C. and C. Tsourounis, A review of select vitamins and minerals used by postmenopausal women. Maturitas, 2010. 66(4): p. 370-80.

189. Peters, B.S.E. and L.A. Martini, Nutritional aspects of the prevention and treatment of osteoporosis. Arq Bras Endocrinol Metab, 2010. 54(2): p. 179-185.

190. Hardcastle, A.C., et al., Dietary patterns, bone resorption and bone mineral density in early post-menopausal Scottish women. Eur J Clin Nutr, 2011. 65(3): p. 378-85.

191. Tucker, K.L., et al., Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women. American Journal of Clinical Nutrition, 1999. 69(4): p. 727-736.

192. Sanchez-Rodriguez, M.A., et al., Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized by antioxidant enzymes. BMC Musculoskeletal Disorders, 2007. 8: p. 124.

193. Salari Sharif, P., S. Nikfar, and M. Abdollahi, Prevention of bone resorption by intake of phytoestrogens in postmenopausal women: a meta-analysis. Age (Dordr), 2011. 33(3): p. 421-31.

194. Khennouf, S., et al., Effect of some phenolic compounds and quercus tannins on lipid peroxidation. World Applied Sciences Journal, 2010. 8(9): p. 1144-1149.

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195. Camacho, P.M. and N.A. Lopez, Use of biochemical markers of bone turnover in the management of postmenopausal osteoporosis. Clin Chem Lab Med, 2008. 46(10): p. 1345-57.

196. Seeman, E. and P.D. Delmas, Bone Quality — The Material and Structural Basis of Bone Strength and Fragility. New England Journal of Medicine, 2006. 354(21): p. 2250-2261.

197. Tenta, R., et al., Calcium and vitamin D supplementation through fortified dairy products counterbalances seasonal variations of bone metabolism indices: the Postmenopausal Health Study. Eur J Nutr, 2011. 50(5): p. 341-9.

198. Meunier, P.J., et al., Consumption of a high calcium mineral water lowers biochemical indices of bone remodeling in postmenopausal women with low calcium intake. Osteoporos Int, 2005. 16(10): p. 1203-9.

199. Bonjour, J.P., et al., Inhibition of bone turnover by milk intake in postmenopausal women. Br J Nutr, 2008. 100(4): p. 866-74.

200. Tang, B.M.P., et al., Use of calcium or calcium in combination with vitamin D supplementation to prevent fractures and bone loss in people aged 50 years and older: a meta-analysis. The Lancet. 370(9588): p. 657-666.

201. Sakhaee, K., et al., Effects of potassium alkali and calcium supplementation on bone turnover in postmenopausal women. J Clin Endocrinol Metab, 2005. 90(6): p. 3528-33.

202. Rogers, A., R.A. Hannon, and R. Eastell, Biochemical markers as predictors of rates of bone loss after menopause. J Bone Miner Res, 2000. 15(7): p. 1398-404.

203. Seibel, M.J., Molecular markers of bone turnover: biochemical, technical and analytical aspects. Osteoporos Int, 2000. 11 Suppl 6: p. S18-29.

204. Riggs, B.L. and A.M. Parfitt, Drugs used to treat osteoporosis: the critical need for a uniform nomenclature based on their action on bone remodeling. J Bone Miner Res, 2005. 20(2): p. 177-84.

205. Brown, J.P., et al., Comparison of the Effect of Denosumab and Alendronate on BMD and Biochemical Markers of Bone Turnover in Postmenopausal Women With Low Bone Mass: A Randomized, Blinded, Phase 3 Trial. Journal of Bone and Mineral Research, 2009. 24(1): p. 153-161.

206. Garnero, P., C. Darte, and P.D. Delmas, A model to monitor the efficacy of alendronate treatment in women with osteoporosis using a biochemical marker of bone turnover. Bone, 1999. 24(6): p. 603-9.

207. Ravn, P., B. Clemmesen, and C. Christiansen, Biochemical markers can predict the response in bone mass during alendronate treatment in early postmenopausal women. Alendronate Osteoporosis Prevention Study Group. Bone, 1999. 24(3): p. 237-44.

208. Ravn, P., et al., Biochemical markers for prediction of 4-year response in bone mass during bisphosphonate treatment for prevention of postmenopausal osteoporosis. Bone, 2003. 33(1): p. 150-8.

209. Greenspan, S.L., H.N. Rosen, and R.A. Parker, Early changes in serum N-telopeptide and C-telopeptide cross-linked collagen type 1 predict long-term response to alendronate therapy in elderly women. J Clin Endocrinol Metab, 2000. 85(10): p. 3537-40.

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210. Zern, T.L., et al., Grape polyphenols exert a cardioprotective effect in pre- and postmenopausal women by lowering plasma lipids and reducing oxidative stress. J Nutr, 2005. 135(8): p. 1911-7.

211. Osawa, T., Protective role of dietary polyphenols in oxidative stress. Mechanisms of Ageing and Development, 1999. 111: p. 133-139.

212. Ozgocmen, S., et al., Role of antioxidant systems, lipid peroxidation, and nitric oxide in postmenopausal osteoporosis. Molular Cellular Biochemistry, 2007. 295(1-2): p. 45-52.

213. Suda, N., et al., Participation of oxidative stress in the process of osteoclast differentiation. Biochim Biophys Acta, 1993. 1157(3): p. 318-23.

214. Bai, X.-c., et al., Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-κB. Biochemical and Biophysical Research Communications, 2004. 314(1): p. 197-207.

215. Mody, N., et al., Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Radical Biology and Medicine, 2001. 31(4): p. 509-519.

216. Manach, C., et al., Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. American Journal of Clinical Nutrition, 2005. 81(suppl): p. 230S-242S.

217. Cao, G., et al., Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine or vitamin C in elderly women. The Journal of nutrition, 1998. 128(12): p. 2383-2390.

218. Bouayed, J. and T. Bohn, Exogenous antioxidants - Double-edged swords in cellular redox state: Health beneficial effects at physiologic doses versus deleterious effects at high doses. Oxid Med Cell Longev, 2010. 3(4): p. 228-237.

219. Valko, M., et al., Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol, 2007. 39(1): p. 44-84.

220. Graf, B.A., P.E. Milbury, and J.B. Blumberg, Flavonols, flavones, flavanones, and human health: epidemiological evidence. J Med Food, 2005. 8(3): p. 281-90.

221. Arts, I.C. and P.C. Hollman, Polyphenols and disease risk in epidemiologic studies. Am J Clin Nutr, 2005. 81(1 Suppl): p. 317S-325S.

222. Medina-Remón, A., et al., Total polyphenol excretion and blood pressure in subjects at high cardiovascular risk. Nutrition, Metabolism and Cardiovascular Diseases, 2011. 21(5): p. 323-331.

223. Scalbert, A., et al., Absorption and metabolism of polyphenols in the gut and impact on health. Biomedicine and Pharmacotherapy, 2002. 56(6): p. 276-82.

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APPENDIX I GREENS+ BONE BUILDER+TM INGREDIENTS

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APPENDIX II INFORMED CONSENT PACKAGE

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Form A Selection, Inclusion and Exclusion Criteria

Oxidative stress and the risk of osteoporosis: the role of dietary polyphenols and

nutritional supplements (Genuine Health Inc, Toronto, Ontario) Selection and Inclusion Criteria:

• Healthy women whose menses have ceased at least one year prior to entry • Aged 50-60

The women who are willing to participate should agree to provide fasting blood and urine samples and maintain their dietary records when needed.

Exclusion Criteria:

• Those who smoke • Those who are on hormone replacement therapy • Those who take polyphenols, multivitamins or other supplements

containing antioxidants (i.e. vitamin C, or vitamin E) • Those who take medications for osteoporosis, coronary heart disease,

high blood pressure, diabetes and cancer • Those with other metabolic bone diseases • Those who are allergic to bees and/or pollen or ingredients listed on

Appendix to Consent Form 1 • Those who may still become/plan to be pregnant • Those who have a body mass index of ≥ 30 • Those who have a systolic blood pressure of ≥ 140 mm Hg or a diastolic

blood pressure of ≥90 mm Hg Ver Oct 27 08 Osteoporosis, Antioxidants and Nutritional Supplements, Form A, Page 1 of 1

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Forms B&C

INFORMED CONSENT DOCUMENTS – FORMS B & C

FORM B

Consent to Participate in a Research Study TITLE OF STUDY: Oxidative Stress And The Risk Of Osteoporosis: The Role Of Dietary Polyphenols And Nutritional Supplements Introduction: Before agreeing to take part in this research study, it is important that you read the information in this research consent form. It includes details we think you need to know in order to decide if you wish to take part in the study. If you have any questions, ask a study doctor or study staff. You should not sign this form until you are sure you understand the information. All research is voluntary. You may also wish to discuss the study with your family doctor, a family member or close friend. If you decide to take part in the study, it is important that you are completely truthful about your health history and any medications you are taking. This will help prevent unnecessary harm to you. Investigators:

The following investigators are involved in this study: 1. Principal Investigator: Dr. Leticia G. Rao, Associate Professor of Medicine and Co-Director,

Calcium Research Laboratory, Division of Endocrinology and Metabolism, St. Michael’s Hospital, 38 Shuter St. Annex, Toronto, Ontario M5B 1A6. Tel #: (416) 864- 5838, E-mail: leticia.rao@utoronto.ca Mon – Fri, 9:00 AM – 5:00 PM.

2. Co-investigator: Dr. A.V. Rao, Professor of Nutrition and Director, Program in Food Safety,

Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, Tel #: (416) 978-3621, Email: rao.v@utoronto.ca Tue – Thurs, 9:00 AM – 12:00 PM.

3. Co-investigator: Dr. Alan C. Logan, ND, FRSH, 50 Yonkers Terrace #8-J, Yonkers, NY

10704. 4. MSc student: Ms Nancy Kang, MSc student at the Department of Nutritional Sciences,

University of Toronto. Supervisor: Drs. A.V. Rao and L.G. Rao, Tel #: (416) 864-5838, Email: n.kang@utoronto.ca Mon – Fri, 9 AM - 5 PM.

5. Collaborator and Qualified Investigator: Dr. R.G. Josse, Professor and Director of the

Osteoporosis Centre and Associate Physician-In-Chief, St Michael’s Hospital and Department of Medicine, University of Toronto. 61 Queen St East. Toronto, Ontario.

Study Sponsor: Genuine Health, Inc., Canada Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 1 of 9 Ver 14 Sept 2010

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Forms B&C Conflicts of Interest: Dr. L.G. Rao is the principal investigator for the project. She gives educational talks to the public, and scientific conferences and is often invited to give talks at various Universities. The sponsor, Genuine Health, sometimes compensates her for related expenses. Drs. A.V. Rao and A. Logan provide advice on research-related matters to the company and are compensated for their time. However, they are not being financially compensated to conduct this research study. PURPOSE OF THE RESEARCH: St. Michael’s Hospital and the University of Toronto are carrying out research in order to understand the role of oxidative stress, the water-soluble antioxidant polyphenols, and nutritional supplements in osteoporosis (weakening of the bones). An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation reactions can produce free radicals (molecules), which start chain reactions that damage cells. Oxidative stress is the harmful condition that occurs when there is an excess of free radicals. Free radicals are molecules that produced when your body breaks down food, or by environmental exposures to tobacco smoke and radiation. Free radicals are also produced as a result of aging, poor nutrition or lifestyle, and a decrease in antioxidant levels such as polyphenols, or both. Known antioxidants include a number of enzymes and other substances such as vitamin C, vitamin E, lycopene and beta carotene (which is converted to vitamin A) that are capable of counteracting the damaging effects of oxidation. Studies suggest that oxidative stress may play a role in the development of osteoporosis. Although these studies suggest that the antioxidants vitamin C, E and beta-carotene may improve bone health, little is known of the role played by polyphenols. Polyphenols are water-soluble antioxidants found in green tea and a variety of fruits and vegetables, as well as in nutritional supplements. One such nutritional supplement is the greens+ bone builderTM. This is a new formulation which contains the original greens+ nutritional supplement combined with other supplements beneficial to bone, including folic acid, vitamins C, B and D; minerals calcium, zinc, selenium, silicon, boron, copper and maganese; amino acid L-lysine and the antioxidant lycopene. Recent studies have shown that high dietary intake of polyphenols may reduce the risk of several chronic diseases, and several studies suggest that green tea, a good source of polyphenols may also help in the reduction of the risk for developing osteoporosis. It is for this reason that our laboratory is studying whether polyphenols and nutritional supplements are beneficial to postmenopausal women who are at risk of osteoporosis. We are seeking postmenopausal, female participants aged 50-60 to take part in a 2-month study, in which we will supplement your diet with the nutritional supplement greens+ bone builder (Genuine Health, Inc.) or placebo (an inactive substance). Our major objectives are: (1) To determine whether the intake of greens+ bone-builder™ will increase with the antioxidant capacity or the total antioxidants present in the blood using vitamin E as standard, and decrease oxidative stress parameters and bone turnover markers (the blood parameters that will show whether the treatment is helping in the formation of bone and Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 2 of 9 Ver 14 Sept 2010

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Forms B&C decreasing the destruction of bone (2) To determine whether polymorphism, the genetic differences in enzymes that protect against oxidative stress, can explain any of the associations that are observed. This preliminary study should provide initial clinical data on which to base future studies of the effects of dietary antioxidants and nutritional supplements on the development of osteoporosis. Approximately 60 participants will be recruited from the greater Toronto area, each of whom will be enrolled in the study at St. Michael’s Hospital. DESCRIPTION OF THE RESEARCH: There will be 5 visits in total: On your first visit, the study will be explained to you and you will be given forms A to F to take with you to read and study. Once you agree to participate, the following schedules will be followed: Week 1 You will be asked to record your daily diet for one-week on Form E-2 of the package given to you. After 1 week, you will be asked to come to the hospital after 12 hours without food to give a fasting blood and urine sample. Week 2 (wash-out period) From week 2 until the end of the study (week 10) you will be asked to refrain from consuming green tea and nutritional supplements containing polyphenols and/or other antioxidants and vitamins. You will also be required to record your daily diet for one week, and at the end of Week 2, you will be asked to come to the hospital and give fasting blood and urine samples and hand in your food records. Week 3 to Week 6 For week 3, you will be randomized, that is selected by chance to receive either the greens+ nutritional supplement or placebo. The chance of receiving the placebo is 50:50. The section below will explain how the supplement is to be taken. You will be required to take your supplement every day. Record your daily food intake at start of Week 6, then come to the hospital to give a fasting blood and urine sample and hand in your food records. Weeks 7 to Week 10: Take your supplement every day. Record your daily food intake at the start of Week 10, then come to the hospital to give a fasting blood and urine sample and hand in your food records. At each of the clinic appointments we will take your height, weight and blood pressure. For the duration of the study we ask that you maintain your usual habits, including diet, exercise and lifestyle, as best as possible. Please remember to avoid consuming the foods listed on Form F from Weeks 2-10 of the study. Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 3 of 9 Ver 14 Sept 2010

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Forms B&C Total time commitment: Participation in this study requires 2 trips to our office (first visit and end of week 1) and 4 trips to the outpatient blood clinic of St. Michael’s Hospital; appointments will be scheduled to your convenience, between Monday-Friday from 7:30 AM to 9 AM. Each visit should take approximately half an hour. Additionally, participants will be required to record their diet for 7 days prior to each appointment. Participants will be contacted by telephone to confirm these appointments and to monitor progress during weeks 5 and 9 of the study. Additionally, if any new information relevant to the study is revealed during the study, participants will be contacted immediately by telephone. Supplement: Participants will be randomly assigned to take either greens+ bone builder supplement or placebo. The supplement or placebo is a powder which will be mixed with water and consumed daily with breakfast for a period of 8 weeks. If you forget or are not able to consume the supplement with your breakfast, please consume later in the day with a meal or a snack. If you do not remember until the following day that the supplement was missed please do not take extra supplement, however, please record the day that the supplement was missed, and advise the study coordinator at your next clinic appointment. Please return any unused study product to the study coordinator at the end of the study during the final clinic appointment (end of study week 10). Blood and urine samples: Blood samples will be taken at the outpatient lab of St. Michael’s Hospital by experienced nurses. A study coordinator will be present as well. Blood and urine samples are fasting samples; we ask that for 12 hours prior to the appointment you consume no food or beverages; however you may drink as much water as you like. Approximately 40mL (approximately 2.5 tablespoons) of blood will be taken. The blood and urine samples (urine is to be collected in the vial that will be provided to you) will be processed and stored for the analyses of antioxidant capacity, antioxidant polymorphisms, oxidative stress parameters and bone turnover markers. Samples will be frozen in a –80oC freezer at St. Michael’s Hospital for analyses as described above, all samples will be disposed off after 5 years. Availability of supplement: This supplement is currently available in health food stores and those wishing to continue taking it after the study period may purchase it quite easily. Placebo: As a participant in this study you may be randomly assigned to take a placebo; the chance of receiving a placebo is 50:50. A placebo is an inactive substance, and in this study it looks and tastes the same as the greens+ bone builder supplement, however it does not contain any polyphenols or other nutritional substances. We use a placebo in this study to ensure that any effects we see are due to the polyphenols and nutritional supplements contained within the greens+ bone builder supplement and not a result of the powder itself or any changes in your diet. Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 4 of 9 Ver 14 Sept 2010

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Forms B&C POTENTIAL HARMS (INJURY, DISCOMFORTS OR INCONVENIENCES): There are no known physical risks or injuries associated with consumption of greens+ bone builder supplement or its placebo as given in this study. However, first time users may experience temporary symptoms. These symptoms can include headaches, constipation, diarrhea, nausea and skin blemishes. There is no rescue medication required to relieve these symptoms, however you may treat these symptoms as you normally would. Please call the study coordinator if the symptoms persist, and for your safety you may be asked to withdraw from the study. There are no known drug interactions with greens+ bone builder supplement or its placebo; however those on prescription medication should consult their physician before taking this product. Those who are allergic to bees or bee pollen may experience a reaction to the supplement, which is why they are excluded from the study; there are no other known allergic reactions to this supplement. When blood is taken from the vein, a slight discomfort or redness may be experienced which will usually disappear in a few days. If a more severe reaction occurs, such as infection, please consult a physician and inform the study coordinator. We ask participants to record their diet - everything they eat and drink as well as quantities for 7 days prior to each appointment (weeks 1,2, 6 and 10 of the study) - and submit the completed records to the study coordinator at each appointment. We will provide you with blank diet records (Form E-2) on which to record this information. Some participants may find this process to be inconvenient as it may be time consuming. Withdrawal from the Study: Those wishing to withdraw from the study may do so at any time. For your safety, you may be asked to withdraw from the study if you experience persistent symptoms include headaches, constipation, diarrhea, nausea and skin blemishes. Any personal health information, dietary records, and blood and urine samples will be destroyed. POTENTIAL BENEFITS: You may receive no direct benefits from being in this study. However, results from this study may further medical and scientific knowledge on the role of nutritional supplements in bone health. Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 5 of 9 Ver 14 Sept 2010

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Forms B&C PROTECTING YOUR HEALTH INFORMATION: The study investigators, sponsor (Genuine Health, Inc,), coordinators, nurses and delegates (hereby referred to as “study personnel”) are committed to respecting your privacy. No other persons will have access to your personal health information or identifying information without your consent, unless required by law. Any medical records, documentation, study samples or information related to you will be coded by study numbers to ensure that persons outside of the study (i.e., sponsors) will not be able to identify you. No identifying information about you will be allowed off site. All information that identifies you will be kept confidential and stored and locked in a secure place that only the study personnel will have access to. In addition, electronic files will be stored on a secure hospital or institutional network and will be password protected. It is important to understand that despite these protections being in place, experience in similar studies indicates that there is the risk of unintentional release of information. The principal investigator will protect your records and keep all the information in your study file confidential to the greatest extent possible. The chance that this information will accidentally be given to someone else is small. By signing this form, you are authorizing access to your medical records by the study personnel, authorized representatives of the sponsoring company, Genuine Health, Inc., the St. Michael’s Hospital Research Ethics Board and by government regulatory authorities (i.e., Health Canada, the US Food and Drug Administration (FDA) and/or regulatory agencies from other countries). Such access will be used only for purposes of verifying the authenticity of the information collected for the study, without violating your confidentiality, to the extent permitted by applicable laws and regulations. National and Provincial Data Protection regulations, including the Personal Information Protection and Electronic Documents Act (of Canada) or PIPEDA and the Personal Health Information Protection Act (PHIPA) of Ontario, protect your personal information. They also give you the right to control the use of your personal information, including personal health information, and require your written permission for your personal information (including personal health information) to be collected, used or disclosed for the purposes of this study, as described in this consent form. You have the right to review and copy your personal information. However, if you decide to be in this study or chose to withdraw from it, your right to look at or copy your personal information related to this study will be delayed until after the research is completed. It is possible that a commercial product (device or pharmaceutical) may be developed as a result of this study. Neither the sponsor (Genuine Health, Inc,) nor the principal investigator (Dr. L. Rao) will compensate you if this happens, and you will have no rights nor receive royalties to any products that may be created as a result of this study or any future research studies. All study records will be kept confidential for 25 years. De-identified samples will be frozen in a –80oC freezer at St. Michael’s Hospital for analyses as described above, all samples will be disposed off after 5 years. Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 6 of 9 Ver 14 Sept 2010

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Forms B&C ALTERNATIVES TO PARTICIPATION: The alternative to participation in the study is non-treatment. There is no likely consequence to you if you decide not to participate in the study. Recruitment will be continued until the required number of participants is attained. STUDY RESULTS: Participants may contact the Study Coordinator or Principal Investigator by telephone for the results of the study, both individual and overall, once the study has been completed. Additionally, if requested, the participants will be given the information on any published manuscript related to the study. The results of this study will be published in peer-reviewed scientific journals and/or presented at conferences, seminars or other public forums without breaking the confidentiality and privacy as stated above. Your identity will not be disclosed in any presentations or publications of the results of the study. POTENTIAL COSTS OF PARTICIPATION AND REIMBURSEMENT TO PARTICIPANT: Those who participate will be provided $150.00 compensation for time and travel costs. The participant will be provided with $37.50 per visit to cover these costs (4 visits in total for the duration of the study), which will be given at one time, either at premature withdrawal or upon completion of the study. The participant will be asked to sign a receipt indicating the sum to be reimbursed and the hospital will issue a check within 2 months of study completion. COMPENSATION FOR INJURY: If you suffer a physical injury from taking the nutritional supplement and/or from participation in this study, medical care will be provided to you in the same manner as you would ordinarily obtain any other medical treatment. In no way does signing this form waive your legal rights nor release the study doctor(s), Genuine Health, Inc., the study sponsor or St. Michael’s Hospital from their legal and professional responsibilities. PARTICIPATION AND WITHDRAWAL: Participation in research is voluntary. If you choose not to participate, you and your family will continue to have access to customary care at St Michael’s Hospital. If you choose to participate in this study you can withdraw from the study at any time without any effect on the care you or your family will receive at St Michael’s Hospital. NEW FINDINGS OR INFORMATION: We may learn new things during the study that you may need to know. We can also learn about things that might make you want to stop participating in the study. If so, you will be notified about any new information in a timely manner. You may also be asked to sign a new consent form discussing these new findings if you decide to continue in the research study. Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 7 of 9 Ver 14 Sept 2010

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Forms B&C RESEARCH ETHICS BOARD CONTACT: The study protocol and consent form have been reviewed by a committee called the Research Ethics Board at St. Michael’s Hospital. The Research Ethics Board is a group of scientists, medical staff, and individuals from other backgrounds (including law and ethics) as well as members from the community. The committee is established by the hospital to review studies for their scientific and ethical merit. The Board pays special attention to the potential harms and benefits involved in participation to the research participant, as well as the potential benefit to society. This committee is also required to do periodic review of ongoing research studies. As part of this review, someone may contact you from the Research Ethics Board to discuss your experience in the research study. If you have any questions regarding your rights as a research participant, you may contact Dr. Julie Spence, Chair, and Research Ethics Board at 416-864-6060 ext. 2557 during business hours. STUDY CONTACTS: In case of questions or emergency please contact Dr. L.G. Rao at (416) 864-5838, Dr. A.V. Rao at (416) 978-3621 or Nancy Kang at (416) 864-5838. Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 8 of 9 Ver 14 Sept 2010

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Forms B&C

I.D. Code: FORM C

Signatures of Participants, Person Obtaining the Consent and Investigator

TITLE OF STUDY: Oxidative Stress and The Risk of Osteoporosis: The Role of Dietary Polyphenols and Nutritional Supplements Consent: The research study has been explained to me, and my questions have been answered to my satisfaction. I have been informed of the alternatives to participation in this study. I have the right not to participate and the right to withdraw without affecting the quality of medical care at St. Michael’s Hospital for me and for other members of my family. As well, the potential harms and benefits (if any) of participating in this research study have been explained to me. I have been told that I have not waived my legal rights nor released the investigators, sponsors, or involved institutions from their legal and professional responsibilities. I know that I may ask now, or in the future, any questions I have about the study. I have been told that records relating to me and my care will be kept confidential and that no information will be disclosed without my permission unless required by law. I have been given sufficient time to read the above information. I consent to participate. I have been told I will be given a signed copy of this consent form. I hereby consent to participate as shown below: I agree to participate

□ Yes □ No (initial) (initial) I agree to be contacted for other future studies related to bone health (initial): □ Yes □ No (initial) (initial)

Name of Participant (Print) Signature Date

Name of Person obtaining the consent Signature Date

Research Coordinator ________________

Osteoporosis, Antioxidants and Nutritional Supplements, Form B&C Page 9 of 9 Ver 14 Sept 2010

Form D

FORM D Participants Source Document

Oxidative stress and the risk of osteoporosis: the role of dietary polyphenols and

nutritional supplements (Genuine Health, Inc, Toronto, Ontario) Date

I.D. CODE

Health Status

Age Weight Height Pulse Rate Blood Pressure

Record of consumption or use of the following items:

Please check the appropriate line

Caffeine (coffee/tea/soda consumption in Supplements: vitamins, herbals,

cups/day): polyphenols:

0 No

1-2 Yes

3-4

5+ if yes, please include the amount, frequency

and brand in the 7-day food record

Green tea (cups/day):

Smoking: Alcohol consumption:

Non-smoker Beer glass/week

Ex-smoker. When did you stop Wine glass/week

smoking? Hard liquor oz/week

Smoker

½ pack/day

Hormone Replacement Therapy

1 pack/day

Never Stopped, when

2+ packs/day

Yes date started

Occasional. When did you

Other Estrogen users:

last smoke?

date started

6. Notes

109

APPENDIX III FOOD RECORD

110

Form E-2 FORM E-2 (day 1) Food Record

Oxidative stress and the risk of osteoporosis: the role of dietary polyphenols and nutritional supplements (Genuine Health Inc, Toronto, Ontario)

Is this a usual day? Yes No_ _ If “No”, please explain_

I.D. Code Date Day 1

Time eaten Quantity Food/Beverage and Description For Official Use

(cups, ml, g, Include vitamin supplements when applicable Only

tsp, etc)

111

Form F Foods to Avoid by Participants

Oxidative stress and the risk of osteoporosis: the role of dietary polyphenols and

nutritional supplements (Genuine Health, Inc, Toronto, Ontario) Please avoid consuming the following during the wash-out and treatment phases of the study: Green Tea Herbal Products containing polyphenols (i.e. Greens+ herbal supplements) Multivitamins or other supplements containing antioxidants (i.e. vitamin C, lycopene and vitamin E) Ver 14 Sept. 2007 Osteoporosis, Antioxidants and Nutritional Supplements, Form F Page 1 of 1