THE EFFECTS OF FORMULATION AND DOSING FREQUENCY OF PLANT

STEROLS ON PLASMA LIPID PROFILES AND CHOLESTEROL

KINETICS PARAMETERS IN HYPERCHOLESTEROLEMIC SUBJECTS

Suhad Sameer AbuMweis

School of Dietetics and Human Nutrition

McGill University, Montreal

August 2007

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy

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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada ABSTRACT

Plant sterol-containing food products are known to reduce low density lipoprotein (LDL) cholesterol, with the degree of placebo-adjusted reduction ranging from 5% to 15%.

Factors that affect plant sterol efficacy such as the type of formulation and dosing frequency are still to be determined. Thus, the objective of this thesis was to investigate the impact of novel plant sterol preparations, frequency of intake and time of intake of plant sterols on cholesterol kinetics and/or plasma lipid profile. Three randomized, placebo-controlled, single-blind, crossover, supervised feeding trials were conducted in

75 subjects with LDL levels of > 3.0 mmol/L. The results from the 3 studies showed that:

(i) traditional forms of plant sterols did not reduce LDL levels when given as a single

morning dose, thus the efficacy of the novel formulation of plant sterols could not be

confirmed; (ii) consumption of 1.8 g/d of plant sterols equally distributed over the day

lowered LDL, whereas the consumption of the same dose once a day with breakfast did

not lower LDL levels, in spite of a reduction in cholesterol absorption efficiency; (iii)

cholesterol fractional synthesis rate (FSR) increased when plant sterols were consumed 3

times/d, but not as a single morning dose, which could be attributed to feedback up-

regulation of cholesterol FSR inresponse to reduced cholesterol absorption; and (iv) no

overall reduction in LDL levels were observed when a 1.6 g/d of single dose of plant

sterols provided in yoghurt was consumed with breakfast or dinner; however, a reduction

in LDL was observed in subjects with low baseline cholesterol absorption efficiency

irrespective of time of intake. Furthermore, we performed a meta-analysis to further

identify any factors that could affect the efficacy of plant sterols and the results

demonstrated that: (i) consumption of plant sterols reduce LDL levels by 0.31 mmol/L; Ill

(ii) the reduction in LDL was greater in studies conducted on individuals with high baseline LDL levels; (iii) plant sterols incorporated into margarine, mayonnaise and salad dressing, milk and yogurt reduced LDL levels to greater extent than plant sterols incorporated into other food products; and (iv) plant sterols consumed as single dose in the morning did not reduce plasma LDL levels. In conclusion, the consumption of single morning dose of plant sterols may not be effective in reducing LDL levels, and that margarine, mayonnaise and salad dressing, milk and yoghurt are the best food carrier of plant sterols. In addition, subjects' baseline LDL levels and cholesterol absorption efficiency influence the response to plant sterols. Therefore, these findings could be used to improve plant sterol efficacy, as well as to establish future health claims. IV

RESUME

Les produits alimentaires contenant des sterols vegetaux sont des aliments fonctionnels connus pour leur effet reducteur sur le cholesterol LDL variant entre 5% et 15% par rapport au placebo. Cependant, les facteurs affectant l'efficacite des sterols des plantes, notamment le type de formulation et la frequence de dosage n'ont pas encore ete determines. L'objectif de la presente these est done d'etudier l'impact de nouvelles preparations de sterols vegetaux, ainsi que la frequence et le moment du dosage, sur la cinetique du cholesterol et/ou le profile lipidique du plasma. Trois etudes alimentaires, croisees en simple aveugle, controlees contre placebo ont ete effectuees incluant un echantillon de 75 hommes et femmes post-menopausees ayant des taux de cholesterol

LDL > 3 mmol/1. Les resultats des trois etudes demontrent que (i) les formes traditionnelles de sterols vegetaux ne reduisent pas les taux de cholesterol sanguin lorsqu'elles sont administrees en une dose matinale, l'efficacite des nouvelles preparations de sterols vegetaux ne peuvent done pas etre confirmees (ii) une dose de

1.8 g/jour de sterols vegetaux distribute en doses egales pendant lajournee reduit le taux de cholesterol LDL, contrairement a la meme dose consommee avec le petit-dejeuner malgre 1'effet reducteur de cette derniere sur l'absorption intestinale du cholesterol, (iii) la vitesse de synthese fractionnelle du cholesterol a tendance a augmenter lorsque les sterols vegetaux sont consommes avec chacun des trois repas, mais pas en une seule dose matinale, ce qui pourrait etre attribue a une regulation positive de la synthese du cholesterol par retroaction negative en reponse a une absorption de sterols limitee et (iv) aucune reduction des taux de cholesterol LDL n'a ete observee en reponse a une seule dose de 1.6 g/jour de sterols vegetaux, incorporee dans du yaourt et consommee avec le V petit-dejeuner ou le souper; cependant, une reduction du taux de cholesterol LDL a ete observee chez les sujets ayant une basse absorption intestinale de cholesterol de base, et cela independamment du moment d'administration du traitement. De plus, nous avons effectue une meta analyse afin de determiner les facteurs affectant Pefficacite des sterols vegetaux. Les resultats demontrent que: (i) la consommation des sterols/stanols vegetaux reduit les taux de cholesterol LDL de 0.31 mmol/1, (ii) de plus importants effets sont observes dans les etudes incluant des sujets ayant des taux de base plus el eves, (iii) les sterols vegetaux produisent des reductions du taux de cholesterol LDL plus importantes lorsqu'ils sont incorpores dans la margarine, la mayonnaise, la vinaigrette, le lait et le yaourt que dans le cas d'autres produits alimentaires et (iv) une seule dose matinale de sterols vegetaux ne reduit pas le taux de cholesterol LDL du plasma. En conclusion, nos resultats suggerent qu'une seule dose matinale de sterols vegetaux ne reduirait pas le taux de cholesterol LDL sanguin, et que les aliments gras comme la margarine et la mayonnaise, ainsi que le lait et le yaourt sont les vehicules les plus efficaces pour les sterols vegetaux. De plus, les taux de base de cholesterol LDL ainsi que Pefficacite de base de 1'absorption intestinale du cholesterol ont un effet significatif sur la reponse aux sterols vegetaux. Ces resultats pourraient done etre utilises pour ameliorer Pefficacite des sterols vegetaux ainsi que pour etablir des allegations de sante. VI

ADVANCE OF SCHOLARLY KNOWLEDGE

1. Original contribution to knowledge

The following are the contributions of this thesis to knowledge in the field of plant sterols and cholesterol metabolism:

- Examines for the first time the question of potential efficacy of novel plant

sterol preparations, i.e. plant sterols + fish oil fatty acids and plant sterols-fish

oil fatty acid ester, on plasma lipid profile and shows that a single morning dose

of traditional and novel forms does not dramatically affect LDL-cholesterol

levels.

Demonstrates the efficacy of plant sterols as cholesterol-lowering agents during

a very short-term period of consumption.

- Shows that dosing frequency is a critical factor in the cholesterol- lowering

potential of plant sterols. Plant sterols consumed with breakfast, lunch and

dinner was more efficacious than plant sterols as a single morning dose in

decreasing LDL-cholesterol levels, relative to control. This effect appeared to

be attributable to feedback up-regulation of cholesterol synthesis rate in

response to reduced cholesterol absorption.

- Examines for the first time the effect of time of intake of single dose of plant

sterols in yoghurt drinks consumed with a meal on plasma lipid profile and finds

that no overall reduction in LDL-cholesterol levels when a 1.6 g/d of single dose

of plant sterols provided in yoghurt was consumed with breakfast or dinner

- Illustrates for the first time that individuals with low baseline levels of

campesterol, an indicator of low cholesterol absorption efficiency, are more likely to benefit from consumption of single dose of plant sterol irrespective of

time of intake than individuals with higher baseline levels of campesterol.

- Conducts a systematic meta-analysis on effect of consumption of plant

sterol/stanol- containing food products on LDL-cholesterol levels.

- Provides original quantitative analysis of randomized clinical trials on plant

sterols and LDL-cholesterol levels and shows that consumption of plant

sterols/stanols reduced significantly LDL levels but that the reductions were

influenced by baseline LDL levels, food matrix to which plant sterols were

incorporated and frequency and time of intake of plant sterols.

Research publications in refereed scientific journals

Suhad S. AbuMweis, Catherine Nicolle, Peter J.H. Jones. Plant sterol-enriched

products for reducing blood cholesterol. Food Science and Technology Bulletin

2006; 2:101-110. (Manuscript 1/Review Article 1)

- Suhad S. AbuMweis, Catherine A. Vanstone, Naoyuki Ebine, Amira Kassis,

Lynne M. Ausman, Peter J.H. Jones, Alice H. Lichtenstein. Intake of a single

morning dose of standard and novel plant sterol preparations for 4 weeks does not

dramatically affect plasma lipid concentrations in humans. Journal of Nutrition.

2006 136:1012-1016. (Manuscript 2)

- Alvin Berger, Peter J.H. Jones, Suhad S. AbuMweis. Plant sterols: factors

affecting their efficacy and safety as functional food ingredients. Lipids in Health

and Disease. 2004; 3:5. (19 pages) (Review Article 2/ Appendix 1)

- Sylvia Santosa, Krista A Varady, Suhad S. AbuMweis, Peter J.H. Jones.

Relationship between human cholesterol absorption and biosynthesis: dietary and Vlll

physiological effects. Life Sciences 2007; 80:505-514. (Review Article 3/

Appendix 2)

3. Research manuscripts prepared for submitting to refereed scientific

journals

- Suhad S. AbuMweis, Catherine A. Vanstone, Alice H. Lichtenstein, Peter J.H.

Jones. Effect of plant sterol consumption frequency on plasma lipid levels and

cholesterol kinetics in humans. (In preparation for submitting to the European

Journal of Clinical Nutrition) (Manuscript 3)

- Suhad S. AbuMweis, Iwona Rudkowska, Peter J.H. Jones. Efficacy of plant sterol

- containing yoghurt consumed once a day with breakfast or dinner in

management of hypercholesterolemia in humans. (Manuscript 4)

- Suhad S. AbuMweis, Roula Barake, Peter J.H. Jones. Plant sterols/stanols as

cholesterol- lowering agents: A meta-analysis of randomized controlled trials. (In

preparation for submitting to the British Medical Journal) (Manuscript 5) IX

CONTRIBUTION OF CO-AUTHORS TO MANUSCRIPTS

The plan for Manuscript 1, Review Article 1, was developed by the candidate, who

collected articles and wrote the text. The candidate contributed substantially to the text

and tables, expanding the initial concept, and elaborating and updating specific themes of

the second review article. With regard to the third review article, the candidate wrote the

section on the circadian and genetic influences section and helped in editing and revising

the manuscript. The candidate co-designed and wrote the proposals for the second and the

third clinical trials. Additionally, the candidate submitted and followed up the documents

for the ethics committee for the second and the third clinical trials. The candidate

conducted and coordinated the 3 clinical trials with some help from other people. More

specifically, the candidate helped coordinate the second half of the first clinical trial,

completed over 8 months, and the third clinical trial that was completed over 10 months.

The second clinical trial was carried out extensively by the candidate who was

responsible for subject recruitment, subject screening, selection and randomization,

designing the experimental diets, subject supervision at the nutrition unit, preparing

isotopes, coding blood tubes, blood draw scheduling and processing of collected samples.

The candidate performed saponification and derivatization procedures, as well as gas

chromatography work for plasma plant sterols analysis for Manuscript 2. The candidate

analyzed all red blood cell samples and plasma samples for cholesterol absorption and

synthesis for Manuscript 3. Additionally, the candidate analyzed and interpreted the data

using statistical models for all the manuscripts. All manuscripts originated from the

human trials were written and revised in whole, by the candidate. Finally, the original

idea for the meta-analysis presented as Manuscript 5 came from the candidate who X designed and implemented the search strategy, assessed study quality, extracted data, performed all statistical analysis, interpreted the results and wrote and edited the manuscript.

Dr. Peter Jones, the candidate's supervisor, edited all manuscripts presented in this thesis.

Dr. Jones as principal investigator, contributed to the developing of the study protocols.

Dr. Jones conducted regular weekly meetings with the candidate to assess progress and provide guidance.

Dr. Alice H Lichtenstein was a co-investigator in the first clinical trial and also developed the study protocol. Dr. Lichtenstein's lab analyzed all lipid parameters for the first and the second clinical trials. In addition, Dr. Lichtenstein edited Manuscripts 2 (Authors: Suhad

S. AbuMweis, Catherine A. Vanstone, Naoyuki Ebine, Amira Kassis, Lynne M. Austrian,

Peter J.H. Jones, Alice H. Lichtenstein) and 3 (Authors: Suhad S. AbuMweis, Catherine

A. Vanstone, Alice H. Lichtenstein, Peter J.H. Jones) and provided insightful comments.

Catherine Vanstone, the Clinical Coordinator at the Mary-Emily Clinical Nutrition

Research Unit at McGill University, carried out all aspects involved in running the first half of the first clinical trial that was done before I started graduate studies. Ms. Vanstone contributed to the second study design by suggesting adding a stabilizing period. In addition Ms. Vanstone helped in subject screening and subject supervision of the second human trial. Finally, Ms. Catherine edited Manuscripts 2 Authors: Suhad S. AbuMweis,

Catherine A. Vanstone, Naoyuki Ebine, Amira Kassis, Lynne M. Ausman, Peter J.H. xi

Jones, Alice H. Lichtenstein) and 3 (Authors: Suhad S. AbuMweis, Catherine A.

Vanstone, Alice H. Lichtenstein, Peter J.H. Jones).

Dr. Lynne Ausman, a professor at Tufts University working with Dr. Lichtenstein, carried out the lipid parameters and C-reactive protein laboratory and statistical analyses for

Manuscript 2 (Authors: Suhad S. AbuMweis, Catherine A. Vanstone, Naoyuki Ebine,

Amira Kassis, Lynne M. Ausman, Peter J.H. Jones, Alice H. Lichtenstein).

Dr. Naoyuki Ebine, a postdoctoral fellow within Dr. Jones lab, provided technical

assistance during the analysis of plasma plant sterol levels. (Authors: Suhad S.

AbuMweis, Catherine A. Vanstone, Naoyuki Ebine, Amira Kassis, Lynne M. Ausman,

Peter J.H. Jones, Alice H. Lichtenstein)

Dr. Catherine Nicolle edited Manuscript 1 (Authors: Suhad S. AbuMweis, Catherine

Nicolle, Peter J.H. Jones) and added some comments.

Dr. Alvin Berger completed the first draft and tables of the second review article

(Authors: Alvin Berger, Peter J.H. Jones, Suhad S. AbuMweis) and updated the final text.

Amira Kassis, a graduate student in Dr. Jones lab, helped coordinate the last quarter of the

first clinical trial appeared as Manuscript 2. (Authors: Suhad S. AbuMweis, Catherine A.

Vanstone, Naoyuki Ebine, Amira Kassis, Lynne M. Ausman, Peter J.H. Jones, Alice H.

Lichtenstein) Xll

Roula Barake, a graduate student at the School and Dietetics and Human Nutrition of

McGill University, was involved in literature search, study selection and quality assessment, as well as data extraction for Manuscript 5 (Authors: Suhad S. AbuMweis,

Roula Barake, Peter J.H. Jones).

Sylvia Santosa, as a graduate student in Dr. Jones lab, wrote the main body of the third review article (Authors: Sylvia Santosa, Krista A Varady, Suhad S. AbuMweis, Peter J.H.

Jones) and integrated the sections written by the co-authors and coordinated revisions among the co-authors.

Krista Varady, as a graduate student in Dr. Jones lab, wrote the method section in the third review article (Authors: Sylvia Santosa, Krista A Varady, Suhad S. AbuMweis,

Peter J.H. Jones), and helped in editing and revising the manuscript.

Iwona Rudkowska, a graduate student in Dr. Jones lab, coordinated the third clinical trial appeared in Manuscript 4 (Authors: Suhad S. AbuMweis, Iwona Rudkowska, Peter J.H.

Jones) along with the candidate. xiii

ACKNOWLEDGEMENTS

First, I would like to thank my supervisor Dr. Peter Jones, for giving me the opportunity to conduct human clinical trials, and for his encouragement and guidance throughout my study period. I would also like to express my gratitude for the advice and support provided by my committee members, Dr. Hope Weiler, Dr. Stan Kubow and Dr. Louise

Thibault. I am also grateful to Catherine Vanstone for her amazing guidance during conducting my clinical trials. Additionally, I would like to thank all my labmates, especially Stephanie Jew, Amira Kassis, Sylvia Santosa, Yen Ming Chan, Chris

Marinangeli and Scott Harding for their support. Special thanks are due to my best friends, Roula Barake and Romaina Iqbal for their unlimited encouragement and support especially during the tough times. I would like to thank my parents for everything they did and are doing for me, without their support and prayers it would be impossible to be where I am today. Last, but not least, I would like to thank Sahel, my husband, for his enormous help and love during this extraordinary experience. DEDICATION

To Dad and Mom

And

To Sahel XV

TABLE OF CONTENTS

Abstract ii

Resume iv

Advance of scholarly knowledge vi

Contribution of co-authors to manuscripts ix

Acknowledgements xiii

Dedication xiv

Table of contents xv

List of tables xxi

List of figures xxiii

List of abbreviations xxv

Chapter 1. Introduction 1

1.1 Background and rationale 1

1.2 Thesis objectives 5

1.3 Null hypothesis 7

Chapter 2. Literature review 8

2.1 Introduction 8

2.2 Manuscript 1 9

Cholesterol-lowering action of plant sterol-enricried products

2.2.1 Abstract 10

2.2.2 Introduction 11

2.2.3 Plant sterols 11 2.2.4 Plant sterols and stands as cholesterol-lowering agents 12

2.2.4.1 Plant sterols versus plant stands 12

2.2.4.2 Effect of dietary cholesterol level on efficacy of plant sterols .... 14

2.2.4.3 Effect of dose frequency and time of day of plant sterol and

stanol intake on their efficacy 15

2.2.5 Mechanism of action of plant sterols 17

2.2.6 Safety of plant sterols 18

2.2.7 Use of vegetable oil-based spreads to reduce blood cholesterol

levels 20

2.2.7.1 Effects of plant sterol-enriched spreads on reduction of blood

cholesterol in different populations 20

2.2.7.2 Use of plant sterol/stanol-enriched spreads in combination with

other approaches for controlling blood cholesterol levels 22

2.2.8 Commercially available plant sterol-enriched spreads 24

2.2.9 Incorporation of plant sterols into low-fat foods 25

2.2.10 Summary and conclusion 30

Bridge 1 32

Chapter 3. Manuscript 2 33

Intake of a single morning dose of standard and novel plant sterol preparations for 4 weeks does not dramatically affect plasma lipid concentrations in humans xvii

3.1 Abstract 34

3.2 Introduction 35

3.3 Methods 37

3.3.1 Subjects 37

3.3.2 Protocol and diet 38

3.3.3 Analyses 39

3.3.4 Statistical analyses 40

3.4 Results 41

3.5 Discussion 42

3.6 Acknowledgement 47

Bridge 2 52

Chapter 4. Manuscript 3 53

Effect of plant sterol consumption frequency on plasma lipid levels and cholesterol kinetics in humans

4.1 Ab stract 54

4.2 Introduction 55

4.3 Subjects and methods 56

4.3.1 Subjects 56

4.3.2 Study design and protocol 56

4.3.3 Analyses 58

4.3.3.1 Plasma lipid profile 58

4.3.3.2 Cholesterol absorption determination 58

4.3.3.3 Endogenous cholesterol synthesis determination 60 xviii

4.3.3.4 Statistics 61

4.4 Results 62

4.5 Discussion 63

4.6 Acknowledgement 66

4.7 Figure legend 67

Bridge 3 75

Chapter 5. Manuscript 4 76

Efficacy of plant sterol - containing yoghurt consumed once a day with breakfast or dinner in management of hypercholesterolemia in humans

5.1 Abstract 77

5.2 Introduction 78

5.3 Subjects and methods 80

5.3.1 Subject selection 80

5.3.2 Study design and protocol 80

5.3.3 Analyses 82

5.4 Results 83

5.5 Discussion 85

5.6 Acknowledgement 90

5.7 Figure legend 91

Bridge 4 97

Chapter 6. Manuscript 5 98

Plant sterols/stanols as cholesterol- lowering agents: A meta-analysis of randomized controlled trials xix

6.1 Abstract 99

6.2 Introduction 101

6.3 Methods 105

6.3.1 Data collection 105

6.3.1.1 Search strategy 105

6.3.1.2 Selection of trials 105

6.3.1.3 Study quality assessment 106

6.3.2 Data extraction 106

6.3.3 Statistical analysis 107

6.4 Results 108

6.5 Discussion Ill

6.6 Acknowledgment 115

5.7 Figure legends 116

Chapter 7. Final conclusion and summary 149

References 161

Appendix 189

Appendix 1. Published version of Review Article 2: Plant sterols: factors

affecting their efficacy and safety as functional food ingredients 190

Appendix 2. Published version of Review Article 3: Physiological and

therapeutic factors affecting cholesterol metabolism: does a reciprocal

relationship between cholesterol absorption and synthesis really exist? 210

Appendix 3: Calculations used in the meta-analysis of plant sterols and

LDL-cholesterol levels 221 XX

Appendix 4. Human ethics certificates 226

Appendix 5. Consent forms 230

Appendix 6.1 Journal waiver for Manuscript 1 240

Appendix 6.2 Journal waiver for Manuscript 2 241

Appendix 6.3 Journal waiver for Review Article 2 242

Appendix 6.4 Journal waiver for Review Article 3 243

Appendix 6.5 Author wavier for Manuscript 2 246

Appendix 6.6 Author wavier for Manuscript 3 247

Appendix 6.7 Author wavier for Manuscript 4 249

Appendix 6.8 Author wavier for Manuscript 5 251 xxi

LIST OF TABLES

Table 3.1. Baseline characteristics of subjects 48

Table 3.2. Macronutrient composition of the study diet /12552 Kj (3000 Kcal) .... 49

Table 3.3. Major plant sterol concentration of the study treatments (% w/w) 50

Table 3.4. Plasma lipid, lipoprotein, apolipoprotein, CRP and sterol concentrations in subjects at the end of each dietary intervention 51

Table 4.1. Nutrient and plant sterol composition of control and sterol-enriched margarine 68

Table 4.2. Characteristics of the subjects at the time of screening 69

Table 4.3. Lipid and lipoprotein concentrations at the end of each experimental diet phase 70

Table 4.4. Cholesterol kinetics as measured from free cholesterol from RBCs in study subjects during each of dietary phase 71

Table 5.1. Nutritional composition of the placebo and plant sterol-containing yoghurt 92

Table 5.2. Baseline characteristics of the subjects 93

Table 5.3. Effect of study treatments on lipid profile 94

Table 5.4. LDL-cholesterol levels after 4 weeks of study treatments classified

according to basal cholesterol absorption efficiency measured with basal

campesterol levels 95

Table 6.1. Calculation of Jadad score to assess study quality 117

Table 6.2. Design and subject characteristics of randomized controlled studies of plant sterols/stanols 118 Table 6.3. Features of plant sterol intervention of randomized controlled studies of plant sterols/stanols 126

Table 6.4. Pooled estimates of treatment effect on LDL-cholesterol in subgroups

of trials defined by subject characteristics and study design features 135 xxiii

LIST OF FIGURES

Figure 4.1. Correlation between changes in the area under the curve (AUC (per

1 ^ mil x h)) of C-enrichment in RBCs- cholesterol and the changes in LDL- cholesterol concentrations (mmol/L) relative to control, at the end plant sterol single-BF (A) and 3 times/d phases (B) 73

Figure 4.2. Enrichment of' C in RBCs- cholesterol at the end of control phase, and plant sterol single-BF and 3 times/d phases 74

Figure 5.1. Individual differences in LDL-cholesterol levels between the end of the 4-week treatment with plant sterols enriched yoghurt consumed in the morning or in the evening and at the end of the 4-week control phase 96

Figure 6.1. Selection of randomized placebo-controlled studies for meta- analysis of plant sterols and circulating cholesterol levels 136

Figure 6.2. Effect size and 95% CI in LDL-cholesterol levels associated with consumption of plant sterol/stanol-containing food products 137

Figure 6.3. Effect size and 95% CI in LDL-cholesterol levels associated with

consumption of plant sterol/stanol-containing food products, analysis based on

subjects' age 138

Figure 6.4. Effect size and 95% CI in LDL-cholesterol levels associated with

consumption of plant sterol/stanol-containing food products, analysis based on

subjects' baseline LDL-cholesterol levels 140

Figure 6.5. Effect size and 95% CI in LDL-cholesterol levels associated with

consumption of plant sterol/stanol-containing food products, analysis based on

plant sterols dose 142 Figure 6.6. Effect size and 95% CI in LDL-cholesterol levels associated with consumption of plant sterol/stanol-containing food products, analysis based on carrier matrix 144

Figure 6.7. Effect size and 95% CI in LDL-cholesterol levels associated with consumption of plant sterol/stanol-containing food products, analysis based on

frequency of intake and time of intake of plant sterols 147 XXV

LIST OF ABBREVIATIONS

ABC: ATP binding cassette

Apo A-I: Apolipoprotein A-I

Apo B: Apolipoprotein B

AUC: Area under curve

BMI: Body mass index

CHD: Coronary heart disease

CRP : C-reactive protein

CVD: Cardiovascular disease d: day

DHA: Docosahexanoic acid

E : Energy

EPA: Eicosapentaenoic acid

FDA: Food and Drug Administration

FSR: Fractional synthesis rate g:gram

GC/ C/ IRMS: Gas chromatography/ combustion/ isotope ratio mass spectrometry

GC/ P/ IRMS: Gas chromatography/ pyrolysis/ isotope ratio mass spectrometry h: hour

HDL : High density lipoprotein

LDL: Low density lipoprotein

Lp(a): Lipoprotein a

MECNRU: Mary Emily Clinical Nutrition Research Unit xxvi

NCEP: National Cholesterol Education Program

NPC1L1: Niemann Pick CI Like 1

PDB: Pee Dee Belemnite

RBCs: Red blood cells

SD: Standard deviation

SMOW: Standard mean ocean water yr: years 1

CHAPTER 1.

INTRODUCTION

1.1 BACKGROUND AND RATIONALE

Cardiovascular disease (CVD) is a general diagnostic category that includes cerebrovascular event, coronary heart disease (CHD), hypertensive heart disease, peripheral arterial diseases, inflammatory heart disease, rheumatic heart disease, deep venous thrombosis and pulmonary embolism and other cardiovascular diseases (Mackay and Mensah, 2004). Cardiovascular diseases are a major health problem worldwide responsible for about 17 million death every year and cause about 10% and 18% of disability-adjusted life years in low and middle-income countries and in high income countries, respectively (Mackay and Mensah, 2004). High circulating level of cholesterol is a risk factor of CVD. In developed countries high cholesterol levels accounted for about 56% of CHD and 62% of ischemic stroke in 2002 (Mackay and

Mensah, 2004). It is estimated that a 10% reduction in serum cholesterol level produces a reduction in CHD of 50% at age 40, 40% at age 50, 30% at age 60, and 20% at age 70

(Lawetal, 1994).

The first step in the treatment of elevated blood lipids as recommended by the Third

Report of the National Cholesterol Education Program (NCEP) is through lifestyle changes that include exercise and dietary therapy (Cleeman et al., 2001; Fletcher et al.,

2005). However, if patients fail to correct their blood lipid levels through changing their lifestyle, if they have multiple risk factors or high cholesterol levels, then drug therapy should be initiated. Commonly prescribed drugs to manage hyperlipidemia include 2

statins, bile acid sequestrants, nicotinic acid, and fibric acids (Cleeman et al., 2001).

These drugs are associated with side effects such as myopathy, increased blood levels of

liver enzymes such as aminotransferase, hepatotoxicity, gastrointestinal distress,

constipation, flushing, and gallstone (Cleeman et al., 2001) that might result in

intolerance and low compliance to drug therapy. As such, it is of great importance to have

dietary compounds capable of improving the dietary therapy component of lifestyle

approach for the prevention of CVD.

Recent dietary suggestions for decreasing risk of CVD include increasing the intake of plant sterols/stanols (National Cholesterol Education Program Expert Panel, 2002) and

fish oil (Kris-Etherton et al., 2002) in the diet of hyperlipidemic individuals. Plant sterols

and plant stanols, hydrogenated form of plant sterols, are compounds having a similar

chemical structure to and biological function as cholesterol (Piironen et al., 2000) in that they reduce cholesterol absorption and thus lower levels of low density lipoprotein (LDL) cholesterol (Vanstone et al., 2002). On the other hand, intakes of long chain unsaturated

fatty acids from fish oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduce CVD mortality possibly by possessing many beneficial actions including triacylglycerol lowering action, anti-inflammatory and anti-thrombogenic actions, as well

as prevent against arrhythmia and induce nitric oxide-induced endothelial relaxation

(Connor, 2000).

Plant sterol esterification with fatty acids enhances their solubility in food matrices as well as their dispersion within the intestine, consequently improving their efficacy (Katan et al., 2003). To date, human studies on sterol esters have been carried out using sterols 3 esterified to fatty acids from plant oils, mainly rape seed (Hallikainen et al., 2000a; Jones et al., 2000; Plat and Mensink, 2000), sunflower (Neil et al, 2001; Weststrate and Meijer,

1998) or soybean oil (Maki et al., 2001; Nestel et al., 2001). Alternatively, a novel approach to enhance plant sterol solubility in food matrices is to esterify plant sterols to

EPA and DHA. Esterifi cation of plant sterols to EPA and DHA could also help increase the intake of these healthy fatty acids through the consumption of plant sterol-containing food products. The efficacy of plant sterols as cholesterol-lowering agents when given in combination with fish oil fatty acids remains to be investigated in humans.

In addition to proper solublization of plant sterols in the food matrix, other factors related to frequency and time of intake of these products may also be important in determining efficacy of plant sterols as cholesterol-lowering agents as the exact mechanism by which plant sterol reduce cholesterol absorption has not been fully elucidated. The conventional proposed mechanism of action involves plant sterols competing with cholesterol for incorporation into the micelles (Heinemann et al., 1991), which suggests that plant sterols should be consumed at each meal to achieve full potential cholesterol-lowering effect

(Plat et al., 2000).The efficacy of plant sterols as a cholesterol-lowering agent may demonstrate a time-of-day variation, possibly coinciding with the diurnal rhythm of cholesterol metabolism. Diurnal rhythm in cholesterol synthesis has been shown in humans (Cella et al., 1995; Jones et al., 1992; Jones and Schoeller, 1990). The diurnal rhythm of cholesterol biosynthesis in humans is affected by food intake. Delaying the meal time by a 6.5 h resulted in a 8.6 h and 6.5 h delay in maximum and minimum

cholesterol synthesis rate, respectively (Cella et al., 1995). Moreover, bile acid synthesis in humans has also a diurnal rhythm that is opposite from the diurnal rhythm of 4 cholesterol synthesis (Galman et al., 2005). The diurnal variation in cholesterol absorption has not been studied. However, since cholesterol synthesis and absorption are inversely related, as when one increases the other decreases and vise versa (Appendix 2), one can speculate that cholesterol absorption is low early in the morning and increases during the daytime period.

The majority of studies have shown the efficacy of plant sterols incorporated into margarine distributed in two (Hallikainen et al., 2000a; Maki et al., 2001; Ntanios, 2001;

Weststrate and Meijer, 1998) or three (Jones et al, 2000; Plat and Mensink, 2000;

Vanstone et al., 2002) daily meals. However, a single dose of plant sterol has been shown to lower cholesterol levels when consumed with lunch and it was hypothesized that plant stanols remain in the intestinal lumen for extended periods where they decrease cholesterol absorption, thus it is not necessary to consume plant stanol ester products with every meal (Plat et al., 2000). Therefore, whether consumption of single dose plant sterols early in the day; versus consumption of the same dose throughout the day will reduce

LDL-cholesterol levels remains to be investigated.

Plant sterols are being incorporated into various products including breakfast foods. New plant sterol enriched products such as yoghurt are being marketed to be consumed once a day in an effort to increase individual's compliance. However, a factor that remains to be assessed is identification of the best time of day to consume these products. For example,

Doornbos et al {Doornbos et al., 2006) showed that intake of a single dose of plant sterols provided in yoghurt drinks with lunch resulted in a larger decrease in LDL-cholesterol levels than intake of same dose of plant sterols XA h before breakfast. This lower efficacy 5 was attributed to the fact that morning yoghurt was ingested in a fasted state versus with a meal. On the other hand, the efficacy of plant sterols consumed in the morning may be

influenced by diurnal variation in cholesterol metabolism seen in cholesterol synthesis

(Jones and Schoeller, 1990) and bile acid synthesis (Galman et al., 2005). The efficacy of

new plant sterol enriched products consumed with a meal and at different times of the day

remains unclear.

Seeing that studies with different characteristics have reported a wide range of changes in

LDL-cholesterol levels due to consumption of plant sterol enriched products (Devaraj et

al., 2006; Jones et al., 1999; Maki et al., 2001; Mussner et al., 2002; Volpe et al., 2001),

factors other than frequency of consumption and time of intake of plant sterols may also

affect the efficacy of plant sterols as cholesterol- lowering agents. The degree of placebo-

adjusted reduction in LDL-cholesterol levels caused by plant sterols has ranged from 5%

to 15% in different studies (Devaraj et al, 2006; Jones et al., 1999; Maki et al., 2001;

Mussner et al., 2002; Volpe et al., 2001). To more comprehensively determine the effect

of plant sterols on LDL-cholesterol levels, a meta-analysis approach can be utilized. The

two previous meta-analyses (Katan et al., 2003; Law, 2000) on efficacy of plant sterols as

a cholesterol- lowering agents were not performed systematically and did not look into

factors that may affect plant sterols efficacy. Thus, a systematic meta-analysis that

quantifies the effect of plant sterols on LDL-cholesterols levels has yet to be performed.

1.2 THESIS OBJECTIVES

The overall objectives of this thesis were to investigate the impact of novel plant sterol preparations, frequency of intake and time of intake of plant sterols on cholesterol 6 kinetics and/or plasma lipid profile. In addition, a further objective was to conduct a

systematic meta-analysis to assess the effect of plant sterol enriched products on LDL-

cholesterol levels.

Accordingly, the specific objectives of the present thesis were:

1. To compare the effects of two novel forms of plant sterols (plant sterols esterified to

fish oil, and a combination of plant sterols and fish oil) to traditional forms of plant

sterols (free plant sterols and plant sterols esterified to sunflower oil) on:

a. Lipid profiles

b. Apolipoproteins A and B, and lipoprotein (a) levels

c. C-reactive protein as an inflammatory marker associated with CVD

2. To investigate under controlled conditions the effect of consumption frequency of

plant sterol enriched spread on:

a. Lipid profiles

b. Cholesterol absorption

c. Cholesterol synthesis

3. To examine the efficacy of a new plant sterol-containing yoghurt consumed once a

day with breakfast or dinner as a cholesterol-lowering functional food

4. To conduct a meta-analysis that will systematically evaluate the effect of plant sterol

enriched products on LDL-cholesterol levels by pooling the results from randomized 7

controlled trials and that will also explore the potential factors affecting the efficacy

of plant sterols as cholesterol-lowering agents.

1.3 NULL HYPOTHESES

1. Traditional and novel forms of plant sterols have the same effect on blood lipid

profile, apo A-I, apo B and Lp(a) levels.

2. The frequency of consumption of plant sterols does not influence their effect on

lipid profile and cholesterol absorption and synthesis.

3. The timing of intake of single dose of plant sterols consumed with meal does not

affect the magnitude of reduction in circulating LDL-cholesterol levels. 8

CHAPTER 2.

LITERATURE REVIEW

2.1 INTRODUCTION

This chapter of the thesis is presented in a modified form of a published manuscript that provides background information on plant sterol chemical structure, daily dietary intakes, absorption rates and circulating levels, safety issues, and mechanism of action. The manuscript discusses in detail the efficacy of traditional and novel plant sterol-enriched products as well as factors that affect plant sterols efficacy. A more comprehensive review of plant sterols, their various physiological effects and factors affecting their efficacy and safety is presented as a second review article recently published by the principal author in Appendix 1. 9

2.2 Manuscript 1. Published in Food Science and Technology Bulletin: Functional

Foods 2006,2:101-110

CHOLESTEROL-LOWERING ACTION OF PLANT STEROL-ENRICHED

PRODUCTS

Suhad S. AbuMweis1, Catherine Nicolle2, Peter J.H. Jones1

Author affiliation:

1 School of Dietetics and Human Nutrition

McGill University, Ste-Anne-de-Bellevue, Quebec, H9X 3V9, Canada

2 Danone Vitapole, Route Departementale 128, 91767 PALAISEAU Cedex - France

Corresponding Author:

Peter Jones, PhD

School of Dietetics and Human Nutrition

McGill University

21,111 Lakeshore Road

Ste-Anne-de-Bellevue, Quebec

H9X 3V9, Canada

Tel: 514-398-7547

Fax; 514-398-7739 peter.iones(a>mcgill.ca 10

2.2.1 Abstract

Plant sterols are naturally occurring compounds that interfere with cholesterol absorption and thus reduce blood cholesterol levels. The objective of this review is to present recent

advances in knowledge of the cholesterol-lowering action of plant sterols, focusing on the

efficacy of plant sterol-enriched products, including full-fat, low-fat and non-fat food

products. Although the cholesterol-lowering efficacy of plant sterols is well acknow­

ledged, additional studies are needed to determine whether dietary cholesterol levels

affect the cholesterol-lowering action of plant sterols, to establish the best time of day to

consume plant sterol-enriched products, and to assess the optimal dose frequency of plant

sterol intake. Fat spreads enriched with plant sterols/stanols have been shown to be

effective in reducing circulating cholesterol levels in healthy adults with both normal and

high cholesterol levels, as well as in children with hypercholesterolemia. Such spreads

can be used in combination with other approaches including a healthy diet, statin therapy

and exercise for controlling blood cholesterol levels. Originally, plant sterols/stanols have

been incorporated into fat spreads and, more recently, into low-or non-fat food products.

However, the efficacy of plant sterols as cholesterol-lowering agents depends on their

proper solubilization, while the majority of studies to date have incorporated plant sterols

into vegetable oil-based spreads. Therefore, until more information is available, data from

older studies using full-fat spreads cannot be used as a platform to promote novel and, as

of yet, inadequately tested low-fat and non-fat plant sterol-enriched products.

Keywords: plant sterols, hypercholesterolemia, functional spreads 11

2.2.2 Introduction

Coronary heart disease (CHD) is a major health problem worldwide (Sleight 2003). A high level oflow-density lipoprotein (LDL) cholesterol in the blood is associated with increased risk of CHD (Sniderman et al., 2003) . Recently, the National Cholesterol

Education Panel (NCEP) has recommended the use of plant sterols as a dietary approach for controlling blood cholesterol levels in addition to changes in diet and lifestyle (Krauss et al., 2001). The consumption of about 2 g/d of plant sterols reduces LDL-cholesterol levels by approximately 10% (Katan et al., 2003). The objective of this review is to present recent advances in knowledge of the cholesterol-lowering action of plant sterols and to discuss the efficacy of plant sterol-enriched products, including a comparison of the original matrix of plant sterol delivery, i.e. full-fat spreads, with more novel plant sterol-enriched food products.

2.2.3 Plant sterols

Plant sterols, or phytosterols, are steroid alcohols which have similar chemical structures to cholesterol (Piironen et al., 2000). Compared to cholesterol, plant sterols contain an extra methyl or ethyl group and one or two carbon-carbon double bonds (Moreau et al.,

2002); saturation of plant sterols at the 5 a-ring position leads to the formation of plant

stanols (Jones et al., 1997). The most abundant plant sterols are ^-sitosterol, campesterol

and stigmasterol (Moreau et al., 2002), which, when hydrogenated, form P-sitostanol, campestanol and stigmastanol, respectively. Since crystalline forms of plant sterols have limited applications in food, plant sterols are esterified with fatty acids to create a

substance with fat-like properties; these can then be incorporated into a range of food products (Wester, 2000) according to hydrophobicity level. 12

The daily dietary intake of plant sterols ranges from 167 to 437 mg among different populations (Ahrens and Boucher, 1978; de Vries et al., 1997; Hirai et al., 1986; Jones et al., 1997; Miettinen et al., 1989; Morton et al., 1995) and originates from the consumption of vegetable oils, nuts, seeds and grains (Piironen et al., 2000). Absorption of plant sterols in humans is considerably less than that of cholesterol, with percentage absorption of different plant sterols and stanols occurring between <1 and 16% (Ostlund, 2002), compared to 40-60% for cholesterol (Bosner et al., 1999). The extra carbon groups present in the plant sterol structure are responsible for the less efficient absorption of these compounds (Ros, 2000). Consequently, blood levels of plant sterols in humans are

only 0.1-0.14% of those of cholesterol (Miettinen et al., 1990). Plant sterols are

eliminated from the body through bile (Robins et al., 1996), in a similar way to

cholesterol.

2.2.4 Plant sterols and stanols as cholesterol-lowering agents

2.2.4.1 Plant sterols versus plant stanols

It is now well known that intake of plant sterols or stanols reduces total cholesterol and

LDL-cholesterol levels (Katan et al., 2003; Law, 2000; Lichtenstein, 2002; Neil and

Huxley, 2002). The efficacy of sterol esters versus stanol esters as cholesterol-lowering

agents has been previously studied using clinical trials. Weststrate and Meijer (Weststrate

and Meijer, 1998) studied normocholesterolemic and mildly hypercholesterolemic

subjects consuming approximately 1.5-3.3 g/d of either sitostanol or soybean sitosterol

esterified to fatty acids from plant oil. LDL-cholesterol levels decreased by 13% in both

plant sterol and stanol groups, compared to controls. With respect to their total cholesterol

and LDL-cholesterol- lowering efficiency, sitostanol and soybean sitosterol esters showed 13 negligible differences. A similar efficiency of sterol and stanol esters extracted from rapeseed oils was also reported in hypercholesterolemic subjects (Hallikainen et al.,

2000b). A 2 g daily dose of sterol and stanol esters was offered to subjects in enriched margarine for 4 weeks and respective reductions in LDL-cholesterol were 10.4 and 12.7% compared to the control group. Noakes et al. (Noakes et al., 2002) investigated the efficiency of sterol (2.3 g/d) and stanol esters (2.5 g/d) in hypercholesterolemic individuals in a crossover comparison. After 3 weeks of intervention, reductions in LDL- cholesterol were 7.7 and 9.5% for sterol and stanol ester groups, respectively, relative to controls. Jones et al. (Jones et al., 2000) also examined the efficiency of sterol and stanol

esters as cholesterol-lowering agents in a crossover trial. Hypercholesterolemic males consumed a margarine containing 1.84 g of either sterol or stanol esters extracted from rapeseed oil as part of a fixed diet for 3 weeks. The plant sterol and stanol dose was

divided equally in the three meals provided. Decreases in LDL-cholesterol levels were

13.2 and 6.4% for the sterol and stanol ester groups, respectively, relative to controls.

Although sterol esters were seen to reduce LDL-cholesterol to a greater extent than did

controls, there was no difference in LDL-cholesterol reduction between the stanol ester treatment and the control; however, there was also no difference between sterol and stanol

ester groups in terms of their efficacy in lowering blood cholesterol. In another study, the

efficiency of free sterols and stanols in lowering blood cholesterol levels was compared in

a crossover double blind feeding clinical trial. Vanstone et al. (Vanstone et al., 2002)

compared the effect of ingesting 1.8 g/d of free sterols, stanols and a 50:50 mixture of both on the lipid profiles of hyperlipidemic subjects. Relative to the control group, reductions in LDL-cholesterol levels were 11.3,13.4 and 16.0% in the sterol, stanol and

50:50 mixture groups, respectively. There were no statistically significant differences 14 observed among the three treatment groups. Similarly, a meta-analysis of studies on plant sterol efficacy as cholesterol-lowering agents concluded that the reductions in LDL- cholesterol levels were in the ranges 8.9-11.3% and 8.5-10.8% in clinical trials testing stanols and sterols, respectively (Katan et al., 2003). Since the comparison lacked the statistical power to detect a difference in efficacy of stanols and sterols as cholesterol- lowering agents, there is currently no support to claim that plant sterols are better than plant stanols or that plant stanols are better than plant sterols (Katan et al. 2003) in reducing cholesterol levels.

2.2.4.2 Effect of dietary cholesterol level on efficacy of plant sterols

Effects of plant sterol administration on blood lipids and cholesterol kinetics have been

examined at different dietary cholesterol intake levels. Plant sterol intake as a part of a

low-cholesterol diet which provides 200-300 mg of cholesterol/d has been shown to

lower LDL-cholesterol levels by 5-14% over 4-8 weeks compared to controls (de Graaf

et al., 2002; Hallikainen et al., 2000b; Hallikainen and Uusitupa, 1999; Homma et al.,

2003; Maki et al., 2001; Nestel et al., 2001). Only one study failed to show any effect of plant sterols when taken as an adjunct to a low-cholesterol diet (Denke, 1995). However,

in this study, plant sterols were administered in capsules and not blended with a fatty

matrix, which limited their cholesterol-lowering action. In addition, subjects' compliance

to plant sterol intake was monitored by capsule counting and not by direct supervision.

Other studies that have shown that plant sterols are effective in lowering blood

cholesterol levels have reported a habitual cholesterol intake between 220 and 410 mg/d

(Cleghorn et al., 2003; Hendriks et al., 2003; Judd et al., 2002; Matvienko et al., 2002; 15

Mensink et al., 2002; Noakes et al., 2002; Ntanios et al., 2002; Pelletier et al., 1995; Plat and Mensink, 2000; Sierksma et al., 1999; Weststrate and Meijer, 1998). Mussner et al.

(Mussner et al., 2002) reported that plant sterol consumption resulted in a marked decrease in cholesterol levels in hypercholesterolemic subjects with a high dietary intake of cholesterol. However, the range of high dietary cholesterol intake in the Mussner et al.

(Mussner et al., 2002) study was between 273 and 843 mg/d; this represents a wide variation and does not distinguish precisely the efficacy of plant sterols in lowering blood cholesterol as a function of low versus high intakes of dietary cholesterol. Effects of plant

sterols on blood lipids and cholesterol absorption/synthesis at high-and low-cholesterol

intakes have not been assessed concurrently in hypercholesterolemic individuals. Studies

of the cholesterol-lowering effect of plant sterols at different dietary cholesterol intakes

are needed to support recent recommendations of adding plant sterols to a healthy diet that is low in dietary fat and cholesterol as a therapeutic approach to lowering blood

cholesterol levels.

2.2.4.3 Effect of dose frequency and time of day of plant sterol and stanol

intake on their efficacy

The majority of studies have shown the efficacy of plant sterols distributed in two

(Hallikainen et al., 2000b; Maki et al., 2001; Ntanios et al., 2002; Weststrate and Meijer,

1998) or three (Jones et al., 1999; Mensink et al., 2002; Plat and Mensink, 2000;

Vanstone et al., 2002) meals given over the day. However, a single dose of plant sterols

has also been shown to lower blood cholesterol (Matvienko et al., 2002; Plat et al., 2000).

In a study by Plat et al. (Plat et al., 2000), 2.5 g of plant stanol ester incorporated into 16 margarine or shortening and consumed once only at lunch or three times per day for 4 weeks decreased LDL-cholesterol levels by 9.4 and 10.4%, respectively, in comparison to the control. During the three doses per day period, 0.42, 0.84 and 1.25 g of plant stanols were consumed at breakfast, lunch, and dinner, respectively. In another study, a 2.7 g dose of plant sterols added to ground beef and consumed only at lunch for 4 weeks reduced LDL-cholesterol by 10% in comparison to the control group (Matvienko et al.,

2002). Plat et al. (Plat et al., 2000) hypothesized that plant stanols remain in the intestinal lumen for extended periods where they decrease cholesterol absorption; thus it is not necessary to consume plant stanol ester products with every meal. Moreover, Plat et al.

(Plat et al., 2000) suggested that consumption of plant stanols will increase consumers' compliance to plant sterol-enriched products and will provide a greater variety of products. For instance, if a single dose of plant sterols reduces blood cholesterol as effectively as more frequent doses; this would mean that plant sterols could be incorporated into a wide variety of products including those that are often consumed once per day. In the two studies by Plat et al. (Plat et al., 2000) and Matvienko et al.

(Matvienko et al., 2002), which showed that a single dose of plant sterol or stanol reduced blood cholesterol, plant sterols were consumed at lunch. However, the efficacy of a single dose of plant sterols taken with breakfast or dinner should also be considered, as some plant sterol-containing food products, such as orange juice and yoghurt, are more often consumed with breakfast meals. 17

2.2.5 Mechanism of action of plant sterols

The primary mechanism by which plant sterols decrease total cholesterol and LDL- cholesterol levels is by reducing cholesterol absorption in the gut. Different forms of plant sterols (including free or esterified sterols) and stands have been found to reduce cholesterol absorption in humans by 26-56% (Jones et al., 2000; Miettinen et al., 2000;

Normen et al., 2000; Vanstone et al., 2002). Although a compensatory response to the de­ crease in cholesterol absorption is an associated increase in body cholesterol biosynthesis

(Jones et al., 2000; Miettinen et al., 2000; Vanstone et al., 2002), the net effect is a decrease in total cholesterol and LDL-cholesterol levels (Jones et al., 2000; Vanstone et al, 2002).

Plant sterols may reduce cholesterol absorption either by preventing incorporation of free cholesterol into micelles and thus preventing micellar absorption, or preventing esterification of the free cholesterol into cholesterol esters (St-Onge and Jones, 2003).

Another proposed mechanism by which plant sterols reduce cholesterol levels is by affecting the intestinal gene expression of transporters involved in sterol homeostasis, including Niemann Pick CI Like 1 (NPC1L1) and ATP binding cassette (ABC) transporters. NPC1L1 exists as an intestinal sterol transporter protein (Davis et al., 2004), while ABC transporters enhance the excretion of sterols back into the intestinal lumen

(Chen, 2001). Data from animal studies have shown that plant sterol/stanol-enriched diets do not change intestinal ABC or NPC1L1 gene expression levels (Calpe-Berdiel et al.,

2005; Field et al, 2004; Repa et al., 2002). However, in a cell culture study, Plat et al.

(Plat and Mensink, 2002a) showed that stanols were potent inducers of ABC Al gene 18 expression, which transports cholesterol from the enterocyte into the lumen. Thus, the exact mechanism by which plant sterols reduce cholesterol absorption remains to be precisely established.

2.2.6 Safety of plant sterols

Use of plant sterols and stands has been recognized as safe for humans by the US Food and Drug Administration (FDA) and the Scientific Committee on Foods of the European

Union (Katan et al., 2003). However, some potentially adverse biological effects have been noted. Since plant sterols decrease the absorption of cholesterol, they might also affect absorption of fat-soluble vitamins. Studies have shown that plant sterols do not affect levels of vitamins D or A (Hallikainen and Uusitupa, 1999; Hendriks et al., 2003;

Maki et al., 2001; Raeini-Sarjaz et al., 2002). In some studies, plant sterol consumption

for 3-52 weeks was shown to significantly reduce circulating levels of carotenoids

(Hallikainen et al., 2000b; Hendriks et al., 2003; Maki et al., 2001; Mensink et al., 2002;

Ntanios et al., 2002; Weststrate and Meijer, 1998), tocopherols (Hallikainen et al., 2000b) and lycopene (Maki et al., 2001; Weststrate and Meijer, 1998). However, other studies have reported that consumption of plant sterols does not affect circulating levels of carotenoids (Christiansen et al., 2001; Hallikainen et al., 1999; Nestel et al., 2001), tocopherols (Christiansen et al., 2001; Hendriks et al., 1999) and lycopene (Hendriks et

al., 1999). In most of the studies demonstrating a reduction in carotenoid plasma levels, the decrease was often within an inter-individual and inter-seasonal range. Nevertheless,

increasing the intake of fruits and vegetables to 5 servings during plant sterol consumption and including one or more carotenoid-rich source (Noakes et al., 2002), or consuming products that are enriched with both plant sterols and carotenoids (Quilez et 19 al., 2003) has been demonstrated to reverse any suppression in carotenoid levels resulting from sterol intake. In this case, fat-soluble vitamin plasma levels can be maintained within a normal range by following dietary recommendations including increasing the consumption of carotenoid-rich fruit and vegetables.

Another concern regarding the safety of plant sterols relates to the fact that sterol intake leads to an increase of it in plasma; whether this increase in plasma sterol levels has any adverse consequences has not yet been determined. In a recent paper, Jones et al. (Jones et al., 2005) demonstrated a lack of effect of an intensive diet known as the portfolio diet, which involves low intakes of saturated fat, trans fatty acids and cholesterol, and emphasizes consumption of soy protein, soluble fiber, plant sterols and almonds, on red blood cell fragility in hypercholesterolemic subjects; it was thought that an increase in plant sterol plasma levels could directly affect red blood cell fragility as well as the cell membrane composition of fatty acids. The Jones et al. (Jones et al., 2005) study demonstrated that the portfolio diet induced a moderate increase in plant sterol plasma levels, although changes in plasma lipids were not related to osmotic fragility of red blood cells and did not promote haemorrhagic events. Furthermore, it is believed that the effects of increased plasma sterol levels are compensated for by a decrease in LDL- cholesterol levels (Katan et al., 2003). Some experts believe that the use of plant stanols to reduce blood cholesterol may be safer than the use of plant sterols because plant stanols are absorbed to a lesser extent than plant sterols (Miettinen and Gylling, 1999).

On the other hand, feeding plant stanols instead of plant sterols to avoid the increase in plasma plant sterol levels may hinder other potentially beneficial roles of plant sterols, including anticancer (Awad and Fink, 2000) and anti-inflammatory (Bouic, 2002) actions. 20

2.2.7 Use of vegetable oil-based spreads to reduce blood cholesterol levels

2.2.7.1 Effects of plant sterol-enriched spreads on reduction of blood cholesterol in different populations

To date, the most accepted method used to optimize the effect of plant sterols on cholesterol absorption has been to dissolve or suspend them in food fats (Ostlund, 2004).

The majority of studies have incorporated plant sterols/ stands into either regular or low- fat spreads. A meta-analysis showed that intake of fatty spreads enriched with plant sterols/stanols decreased blood LDL-cholesterol by an average of 0.54 mmol/L for subjects aged 50-59 years, and 0.43 mmol/L in subjects aged 40—49 years, relative to a placebo (Law, 2000). The meta-analysis of (Law, 2000) suggested that plant sterols/stanols would reduce LDL-cholesterol more effectively at a given dose in older rather than younger people. However, it should be taken into consideration that older people possess higher starting levels of cholesterol; consequently, the percentage change in LDL-cholesterol levels remains constant across age ranges. A reduction of 0.5 mmol/L in LDL-cholesterol, which can be achieved by consumption of plant sterols, is expected to reduce the risk of heart diseases by 25%. However, to date, no long-term randomized controlled studies have directly tested the effect of plant sterols/stanols on heart disease risk.

Studies on fatty spreads enriched with plant sterols/stanols have been carried out across different parts of the world, including North America (Jones et al., 2000; Jones et al.,

1999; Maki et al., 2001), Europe (Hendriks et al., 2003; Mussner et al., 2002; Naumann et al., 2003; Plat and Mensink, 2000), Japan (Homma et al., 2003; Ntanios et al., 2002),

Brazil (Lottenberg et al., 2003) and New Zealand (Cleghorn et al., 2003). It therefore 21 seems that plant sterol-enriched spreads reduce blood cholesterol levels regardless of a population's genetic make-up or dietary pattern.

The cholesterol-lowering action of plant sterols incorporated into spreads has been reported in healthy adults with both normal (Plat and Mensink, 2000; Plat et al., 2000;

Weststrate and Meijer, 1998) and high (Cleghorn et al., 2003; Hendriks et al., 2003;

Homma et al., 2003; Jones et al., 2000; Lottenberg et al., 2003; Maki et al., 2001;

Mussner et al., 2002; Naumann et al., 2003; Ntanios et al., 2002) cholesterol levels, as well as in adults with familial hypercholesterolemia (Neil et al., 2001; Vuorio et al., 2000) or type II diabetes (Gylling and Miettinen, 1994; Lau et al., 2005; Lee et al., 2003). In addition, plant sterols incorporated into spreads have been shown to decrease blood cholesterol levels in children with hypercholesterolemia ((Amundsen et al., 2002;

Ketomaki et al., 2003; Tammi et al., 2002). Plant sterol-enriched spreads can therefore be recommended for cholesterol reduction in healthy, hypercholesterolaemic and diabetic adults. However, the recommendation of using plant sterol-enriched spreads to control blood cholesterol levels in hypercholesterolaemic children should be carried out with caution since more studies are needed to verify the safety of plant sterol consumption in children. 22

2.2.7.2 Use of plant sterol/stanol-enriched spreads in combination with other approaches for controlling blood cholesterol levels

Intake of spreads enriched with plant sterols/stanols reduces blood LDL-cholesterol even in combination with a low-fat and cholesterol-reduced diet. Spreads containing stanol esters extracted from both wood and vegetable oil were introduced into the diets of subjects who followed a diet similar to the NCEP Step I diet for 8 weeks (Hallikainen and

Uusitupa, 1999). Reductions in LDL-cholesterol levels were 13.7 and 8.6% greater in the groups consuming wood stanol ester-and vegetable oil stanol ester-containing spreads, respectively, than in those who followed the Step I diet alone (Hallikainen and Uusitupa,

1999). In another study, plant sterol-enriched low-fat spreads were a component of a Step

I NCEP diet for 5 weeks (Maki et al., 2001). Compared to controls, LDL-cholesterol levels were reduced by 7.6 and 10.2% when target daily intakes of plant sterols were 1.1 and 2.2 g, respectively; reductions in LDL-cholesterol were not different between the low and high sterol groups (Maki et al., 2001). Thus, even daily intakes of approximately 1 g of plant sterols as a component of the NCEP Step I diet reduced LDL-cholesterol levels.

Approximately 1.7 g/d of a mixture of plant sterols and stanols blended in margarine and consumed for 4 weeks lowered LDL-cholesterol levels by 15%, compared to a prudent diet that was high in mono-and polyunsaturated fats (Jones et al., 1999). As part of a

Japanese diet that contained <200 mg/d of cholesterol and 23-27% of dietary energy from fat, intake of 2 g/d of plant stanols in a spread for 4 weeks reduced LDL-cholesterol levels by approximately 10% (Homma et al., 2003). Meanwhile, in subjects who followed a self-selected low-fat diet, with fat comprising approximately 30% of dietary energy and dietary cholesterol intake being <300 mg/d, intake of 2 g/d of plant sterols for 4 weeks reduced LDL-cholesterol by 7% compared to a standard spread (Cleghorn et al., 2003). 23

Overall, plant sterols/stanols combined with a low-fat cholesterol-reduced diet decreased

LDL-cholesterol levels at least twice as much as dietary means alone. Dietary intake of plant sterol-enriched margarines in combination with statin therapy has been shown to have a cumulative effect on reduction of LDL-cholesterol levels. In a study by Simons

(Simons, 2002), 400 mg of cerivastatin reduced LDL-cholesterol levels by 32% compared to a placebo, whereas 2 g/d of plant sterol margarine consumed for 4 weeks reduced

LDL-cholesterol levels by 8% compared to a placebo. However, the intake of both plant sterol margarine and cerivastatin reduced LDL-cholesterol levels by 39% compared to a placebo (Simons, 2002). Similarly, subjects who consumed plant stanols incorporated into a canola oil-based margarine in combination with statin therapy, showed a 10% greater reduction in LDL-cholesterol than did those who consumed a placebo spread combined with statins for 8 weeks (Blair et al., 2000). This reduction in LDL-cholesterol levels was greater than could be achieved by doubling the dose of statin, which would normally produce a further reduction of about 6% in LDL-cholesterol levels (Blair et al.,

2000). Such use of plant sterol/stanol spreads in combination with statin therapy serves as an alternative approach to doubling the statin dose, without producing the associated increase in side effects. Moreover, the addition of plant sterols/stanols to statin therapy is considered a cost-saving strategy in the reduction of LDL-cholesterol levels (Vorlat et al.,

2003).

Recently, plant sterol-enriched spreads have been used with other strategies known to improve lipid profiles. A portfolio diet reduced LDL-cholesterol levels in hypercholesterolemic individuals by 28% (Jenkins et al., 2003). However, this diet could be difficult to follow for an extended time period, reducing its potential long-term 24 benefits. It should be noted that the portfolio diet, which contains 1 g of plant sterols/1000 kcal/d, is considered to be close to what humans consumed several thousand years ago

(Jenkins et al., 2001). In another recent study, the combination of consuming a plant sterol-enriched spread with exercise for 8 weeks brought about the most favorable modification in lipid profiles compared to intake of plant sterols or performing exercise alone (Varady et al., 2004). Combining a plant sterol-enriched spread and exercise reduced TC levels by 8.3% and triacylglycerol levels by 13.3%, and increased high- density lipoprotein cholesterol levels by 7.5% from baseline (Varady et al., 2004). Plant sterols can thus be used in combination with a healthy diet, statin therapy or physical activity to improve lipid profiles. The key message that must be communicated to the public by health professionals is that plant sterol/stanol-enriched spreads should be part of a healthy diet and not a substitute for it. There is no one easy step for reducing blood cholesterol; it requires an overall improvement in dietary habits and regular physical activity (Krauss et al., 2001).

2.2.8 Commercially available plant sterol-enriched spreads

Plant sterol/stanol-enriched spreads currently available on the market include Logicoll

(Meadow Lea), Smart Balance spread (Peerless), Take Control and Proactive (Unilever), and Benecol (Raisio Group). In addition, an olive oil-based spread containing plant sterols is being developed (Berger et al., 2004). In general, daily consumption of 20-25 g of plant sterol-enriched spread is required to achieve a daily intake of about 1.6-2.0 g of free plant sterols/stanols; this daily intake of spread will supply approximately 180-225 kcal of energy. Individuals who are willing to use plant sterol-enriched spreads should be 25 educated to replace their regular spread or butter with the plant sterol/stanol-enriched spread instead of simply adding the enriched spread to their diet. If plant sterol-enriched spreads are not used as a substitute for consumers' habitual spreads, they will probably cause increased body weight. For example, consumption of 20-25 g/d of a regular spread for 30 days can increase body weight by 700-877g. Alternatively; low-fat spreads enriched with plant sterols/stanols are also available on the market and can help minimize increases in caloric intake. However, since the stability of plant sterols/stanols during frying is unknown (Katan et al., 2003), it is recommended that plant sterol/stanol- enriched spreads not be used in cooking.

2.2.9 Incorporation of plant sterols into low-fat foods

Since it appears counterintuitive to use a high-fat food product to deliver a cholesterol- lowering agent, clinical trials have been conducted to test the efficacy of plant sterols that have been incorporated into low-fat products (St-Onge and Jones, 2003); such foods include low-fat milk (Noakes et al., 2005; Thomsen et al., 2004), low-fat yoghurt

(Mensink et al., 2002; Noakes et al., 2005; Volpe et al., 2001), bakery products (Quilez et al., 2003), orange juice (Devaraj et al., 2004) and low-and non-fat beverages (Jones et al.,

2003). Results from these trials have shown reductions in LDL-cholesterol levels ranging between 5 and 14% after 3-8 weeks of consumption, relative to controls, which are close to the reductions in LDL-cholesterol levels reported in studies that used full-fat spreads as a vehicle for delivering plant sterols. However, plant sterols incorporated into non-fat or reduced-fat foods do not always reduce blood cholesterol. A controlled feeding clinical trial has shown that the intake of 1.8 g/d of free plant sterols provided in low-fat and non­ fat beverages and consumed for 3 weeks did not affect the lipid profile of moderately 26 hypercholesterolaemic individuals (Jones et al., 2003). In another clinical trial, however, intake of orange juice fortified with 2 g/d of plant sterols for 8 weeks reduced LDL- cholesterol levels by 7%, relative to controls, in healthy hypercholesterolaemic individuals (Devaraj et al., 2004). In the study by Jones et al. (Jones et al., 2003), free plant sterols were added directly into beverages, while in the study by Devaraj et al.

(Devaraj et al., 2004), plant sterols were suspended in orange juice, although the exact process of suspension was not described. Furthermore, plant sterols did not reduce blood cholesterol levels when administered in capsules rather than blended with a fatty matrix

(Denke, 1995), which indicates that the efficacy of plant sterols incorporated into non-fat products depends on their proper solubilization. In another study where plant sterol esters were provided in a reduced-fat spread and/or salad dressing for 8 weeks, total cholesterol

and LDL-cholesterol did not differ significantly from the control (Davidson et al., 2001).

The authors attributed this lack of efficacy to low baseline cholesterol levels of the study

subjects (Davidson et al., 2001). However, it is known that plant sterols reduce blood

cholesterol even in subjects without hypercholesterolemia (Plat and Mensink, 2000; Plat

et al., 2000; Weststrate and Meijer, 1998); thus, it is most likely that the lack of efficacy

of plant sterols in the study by Davidson et al. (Davidson et al., 2001) is due to improper

solubilization of the plant sterols in the reduced-fat products. Plant sterol-enriched low-fat

yoghurts have resulted in different magnitudes of LDL-cholesterol reduction in various

clinical trials. Intake of 1 g/d of plant sterols in a low-fat yoghurt drink for 4 weeks

reduced LDL-cholesterol levels by 11% relative to baseline (Volpe et al., 2001). Relative

to the control, the plant sterol-containing yoghurt drink reduced LDL-cholesterol levels

by 5% (Volpe et al., 2001). However, the authors did not report whether there was

difference in LDL-cholesterol reduction between the plant sterol-containing yoghurt 27 product and the placebo. In addition, Volpe et al. (Volpe et al., 2001) did not report the time of day when the subjects took the yoghurt drink, how frequently it was consumed, and whether the yoghurt was drunk with or without a meal. In another study, 3 g/d of plant stanols provided in a low-fat yoghurt drink for 4 weeks reduced LDL- cholesterol by 14% relative to controls (Mensink et al., 2002). The subjects in this study were asked to consume 3 cups of a placebo yoghurt for 3 weeks in the run in period; yoghurt cups were either consumed with each meal or one with breakfast and two with dinner. It is

assumed that the experimental yoghurt was consumed in the same pattern. More recently,

the effect of 1.8 g/d of plant sterols administered as sterol/stanol ester-enriched low-fat

yoghurts for 3 weeks has been examined (Noakes et al., 2005). Subjects consumed one

serving of yoghurt twice a day without particular instructions on either the time of intake

or whether to consume the yoghurt with or without a meal. Compared to controls,

reductions in LDL-cholesterol levels were 6 and 5% with plant sterol ester-and plant

stanol ester-enriched low-fat yoghurts, respectively (Noakes et al., 2005). The greater

reduction in LDL-cholesterol levels in the study by Mensink et al. (Mensink et al., 2002),

as compared to studies by Volpe et al. (Volpe et al., 2001) and Noakes et al. (Noakes et

al., 2005) may be due a number of factors. First, the amount of plant sterols consumed

throughout the day varied across the different studies. The total daily intakes of plant

sterols were 3,1 and 1.8 g in the studies of Mensink et al. (Mensink et al., 2002), Volpe et

al. (Volpe et al., 2001) and Noakes et al. (Noakes et al., 2005), respectively. It is possible

that the total daily intake of plant sterols in enriched yoghurt must be >2 g/d to achieve

the maximum possible reduction in LDL-cholesterol levels. Second, the time of day and

the frequency of administration of plant sterol-enriched yoghurt may influence its

efficacy. In the work of Mensink et al. (Mensink et al., 2002), yoghurt cups were either 28 consumed with each meal, or one at breakfast and two with dinner; thus, the plant sterols were distributed throughout the day, or the larger portion of plant sterols was taken with dinner, a meal which usually contains more dietary fat and cholesterol. In the work of

Noakes et al. (Noakes et al., 2005), subjects consumed one serving of yoghurt twice a day. The exact time of consumption was not mentioned; hence it is possible that the two servings were consumed in the morning and not with lunch or dinner. Volpe et al. (Volpe et al., 2001) did not report the time of day when the plant sterol-enriched yoghurt was consumed or how many times per day it was taken. Since the 1 g dose of plant sterols was provided in 100 ml of yoghurt, which is a typical serving size, it is possible that the 1 g of plant sterols was consumed at one sitting, and not throughout the day. Third, the efficacy of plant sterol-enriched yoghurt in lowering blood cholesterol may depend on whether the yoghurt is taken with or without a meal. The gastric emptying rate of the liquid component of a meal is faster than the solid component, with this rate being slowed by simultaneous ingestion of solids (Fisher et al., 1982). Therefore, it is possible that plant sterols provided in liquid foods have less time to mix with other gastrointestinal contents including micelles in the small intestine (Jones et al., 2003). If plant sterol-enriched liquid products are consumed without a meal or as a snack, they may leave the gastrointestinal tract rapidly without having the chance to be mixed with gastrointestinal contents from the next meal, and thus will not coexist to compete with cholesterol for absorption.

Moreover, cholecystokinin concentration in humans is higher at 12 noon compared with

8.00 a.m (Lundberg et al., 2007) and therefore gallbladder emptying may be slower in the morning than in the afternoon which in turn could affect cholesterol absorption. 29

Most studies incorporating plant sterols into low-fat foods have been carried out to test a single plant sterol-enriched product against a control without direct comparison to plant sterol-enriched full-fat spreads. In one study, a plant sterol dose of 2.4 g/d was provided from breakfast cereal, bread and margarine in a 1:1:1 ratio for 4 weeks (Nestel et al.,

2001). However, information concerning which of these different products contributed more to the LDL-cholesterol reduction was not provided. Another study compared effects on plasma lipids of plant stanols consumed for 3 weeks as an ester in a number of food matrices including bread, breakfast cereal, milk and yoghurt (Clifton et al., 2004). Results showed that the efficacy of plant stanol esters may differ according to the food matrix in which they are incorporated. Plant stanols provided in low-fat milk were almost three times more effective than in bread and cereal for lowering plasma cholesterol levels

(Clifton et al., 2004). However, Clifton et al. (Clifton et al., 2004) did not compare the

efficacy of sterol-enriched low-fat milk, bread and cereal directly to a sterol-containing

fatty spread. Noakes et al. (Noakes et al., 2005), however, simultaneously assessed the

efficacy of plant sterol ester-enriched low-fat milk and plant sterol ester-enriched spread.

A 2 g/d dose of plant sterol ester incorporated into low-fat milk or spread was equally

efficacious in lowering LDL-cholesterol levels (Noakes et al., 2005). Relative to the

control, the reductions in LDL-cholesterol were 8 and 10% for the plant sterol ester-

enriched low-fat milk and spread, respectively (Noakes et al., 2005).

Studies have shown that plant sterols incorporated into low-fat foods do not always

reduce blood cholesterol and that the same food matrix tested in different trials does not

always result in the same magnitude of LDL-cholesterol reduction. 30

In addition, there is still uncertainty as to whether all low-fat plant sterol/stanol-enriched food matrices are as efficacious as plant sterol/stanol-enriched fat spreads in lowering blood cholesterol. Simultaneous comparison of efficacy of plant sterols incorporated into other low-fat foods versus a fatty spread is of utmost priority since the efficacy of plant sterols as cholesterol-lowering agents depends on their proper solubilization, allowing them to become incorporated into micelles so that they may interfere with cholesterol absorption. Although some studies have shown that non-fat plant sterol-enriched products reduce cholesterol levels, the efficacy of plant sterols added to these products has not been satisfactorily tested. Thus, caution should be exercised when extrapolating data from studies on plant sterol-enriched spreads to promote new plant sterol-enriched food matrices.

2.2.10 Summary and conclusion

Although the cholesterol-lowering effect of plant sterols/ stands is well known, the exact mechanisms by which plant sterols reduce cholesterol absorption remains to fully be defined. Moreover, further studies are needed to determine whether dietary cholesterol level affects the cholesterol-lowering action of plant sterols. In addition, there is a need to establish the best time of day and the optimal frequency of intake of plant sterol-enriched products for maximal effectiveness, and to determine whether plant sterol-enriched products can reduce blood cholesterol levels even if not taken with meals. Plant

sterol/stanol-enriched spreads are available on the market and are recommended as part of a healthy diet to reduce blood cholesterol levels; additionally, plant sterols and stanols are increasingly being incorporated into low-fat foods and may also form part of a healthy 31 diet. Given that the efficacy of plant sterols as cholesterol-lowering agents depends on their proper solubilization, additional studies are needed to compare the effects of plant sterols/stanols when incorporated into low-fat and non-fat foods versus full-fat spreads.

Hence, until more information surrounding these aspects of efficacy of plant sterols as cholesterol-lowering agents is available, data supplied by older studies using full-fat spreads should not be used as a platform to promote novel, and as of yet inadequately tested, low-fat and non-fat plant sterol-enriched products. 32

BRIDGE 1.

In reviewing the literature on plant sterols as cholesterol-lowering agents, it is apparent that human studies on sterol esters have been carried out using sterols esterified to fatty acids from plant oils, mainly rapeseed, sunflower or soybean oils. Instead, a novel approach to enhance plant sterol solubility in food matrices is to esterify plant sterols to fatty acids from fish oil, EPA and DHA. Esterification of plant sterols to EPA and DHA will assist increase the intake of these healthy fatty acids. Since to date no human trial has examined the efficacy of plant sterols esterified to fatty acids derived from fish oil, the objective of the first clinical trial was to examine the effects on plasma lipids of plant

sterols combined with, or esterified to, fish-oil fatty acids. 33

CHAPTER 3. Manuscript 2 (Study # 1). Published in Journal of Nutrition 2006.

136:1012-1016.

INTAKE OF A SINGLE MORNING DOSE OF STANDARD AND NOVEL

PLANT STEROL PREPARATIONS FOR 4 WEEKS DOES NOT

DRAMATICALLY AFFECT PLASMA LIPID CONCENTRATIONS IN HUMANS

Suhad S. AbuMweis1, Catherine A. Vanstone1, Naoyuki Ebine1, Amira Kassis1, Lynne M.

Ausman2, Peter J.H. Jones*'1, Alice H. Liechtenstein2

Author affiliation:

'School of Dietetics and Human Nutrition McGill University, Ste-Anne-de-Bellevue,

Montreal, Quebec, Canada

Cardiovascular Nutrition Laboratory, Jean Mayer USDA Human Nutrition Research

Center on Aging, Tufts University

Correspondence and reprint requests:

Peter J.H. Jones, PhD,

School of Dietetics and Human Nutrition, McGill University,

21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9. Phone (514)

398-7547.

Fax:(514)398-7739.

e-mail: peter.jones(5),mcgill.ca 34

3.1 ABSTRACT

Recommendations for decreasing the risk of developing cardiovascular disease include increasing the intake of plant sterols and fish oil. The cholesterol-lowering action of plant sterols, when provided in a fish-oil fatty acids vehicle, remains to be investigated in humans. A randomized, crossover-feeding, single-blind trial was conducted in 30 subjects with mild-to-moderate hypercholesterolemia to study the effects on plasma lipids of 2 novel forms of plant sterols: those combined with, or esterified to, fish-oil fatty acids. The treatments were margarine (control), free plant sterols, plant sterols esterified to fatty

acids from sunflower oil, plant sterols esterified to very long-chained fatty acids from fish oil, and plant sterols combined with the same amount of very long-chain fatty acids from

fish oil. Each sterol- containing food (1.0-1.8 g plant sterols/d) was consumed for 29 d as

a single dose with breakfast under staff supervision. Compared with the control treatment, none of the plant sterol preparations reduced plasma total cholesterol or LDL-cholesterol, triacylglycerol, apolipoprotein A-I, apolipoprotein B, lipoprotein (a), or C-reactive protein concentration. Relative to the control phase, all plant sterols treatment increased the plasma HDL-cholesterol concentration (P< 0.05) by 8%. In conclusion, because

standard forms of plant sterols did not reduce plasma cholesterol concentrations, the

efficacy of the new formulation of plant sterols cannot be confirmed from the present

study design, where plant sterols were given as a single morning dose.

Key words: plant sterols, fish oil, plant sterol-fish-oil ester, single dose 35

3.2 INTRODUCTION:

Cardiovascular disease (CVD) remains the major cause of mortality and morbidity in developed countries (Mackay and Mensah, 2004; Sleight, 2003). Hyperlipidemia is a significant risk factor for CVD (Bruckner, 2000). Recent recommendations to decrease

CVD risk include using plant sterols (phytosterols) in hypercholesterolemic individuals

(Cleeman et al., 2001) and increasing intake of long chain (n-3) fatty acids offish oil

(Kris-Etherton et al., 2002).

Plant sterols are compounds that have a chemical structure similar to cholesterol with the exception of an extra methyl or ethyl group or a double bond on carbon 24 of the side chain (Piironen et al., 2000). Plant sterol consumption has been reported to reduce cholesterol absorption and thus to decrease circulating cholesterol levels. Intake of plant sterols in the range of 1.5 - 2.5 g/d reduces LDL- cholesterol levels by 8.5-10% (Katan et al., 2003). However, the efficacy of new formulations prepared to enhance plant sterol solubility and/or blood lipid - lowering effects, as well as the frequency at which plant sterols are given throughout the day remain to be investigated.

Increasing the intake offish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), to 0.5-1.8 g/d is recommended by many experts to reduce subsequent cardiac and all-cause mortality (Kris-Etherton et al., 2002). A novel approach to increase the intake of EPA and DHA is to combine them with plant sterols. Animal studies have examined the effect of plant sterols esterified to these fatty acids. Adult guinea pigs fed a diet supplemented with plant sterols - fish oil ester and corn oil have lower circulating concentrations of triacylglycerol, total cholesterol and non-HDL 36 cholesterol compared to controls (Ewart et al., 2002). In insulin-resistant rats, supplementation with the plant sterol - fish oil ester significantly reduced plasma triacylglycerols and improved endothelial and vascular smooth muscle cell function

(Russell et al., 2002). To date, human studies that examine the effects of plant sterols on circulating lipid concentrations have been carried out using sterols esterified to fatty acids from plant oils, mainly rapeseed (Hallikainen et al., 2000b; Jones et al., 2000; Mensink et al., 2002; Plat and Mensink, 2000), sunflower (Neil et al., 2001; Weststrate and Meijer,

1998) or soybean oil (Maki et al., 2001; Nestel et al., 2001). The efficacy of plant sterols

as a cholesterol- lowering agent, when given in combination with fish oil fatty acids, remains to be investigated in humans.

The objective of this study was to examine the effects of plant sterols combined with, or

esterified to, fish oil fatty acids, on blood lipid concentrations and recent factors

associated with CVD, i.e. apolipoproteins A-I (apo A-I) and B (apo B), lipoprotein (a)

(Lp(a)) (Danesh et al., 2000; Seman et al., 1999; Sniderman et al., 2003). In addition,

since plant sterols may have an anti-inflammatory action (Bouic, 2001), the present study

aimed to examine the effect of plant sterols on C-reactive protein (CRP), an

inflammatory mediator that predicts coronary heart events (Rifai and Ridker, 2001).

The primary hypothesis was that the effect of the new preparation of plant sterol on blood

lipid levels, (i.e. plant sterols combined with or esterified to fatty acids from fish

oil),would be the same as traditional forms of plant sterols, (i.e. free plant sterols and plant sterols esterified to fatty acid from vegetable oils). In addition, we hypothesized that the esterification of plant sterols with fatty acids from fish oil would not affect the action 37 of plant sterols in terms of plant sterol incorporation into micelles to compete with cholesterol absorption. Thus, we expected plant sterols to have a similar effect on blood lipids when they are either combined with, or esterified to, fatty acids from fish oil. All plant sterol preparations were given as a single dose at breakfast to ensure compliance.

3.3 METHODS:

3.3.1 Subjects

Thirty eight male and postmenopausal female subjects were recruited through the distribution of flyers and newspaper advertisements in Montreal and surrounding areas.

Criteria for being considered for the study included the following: age range of 40-85 y; body mass index between 22 and 34 kg/m2; LDL- cholesterol > 2.6 mmol/L (100 mg/dl); non-smoker; free from cardiovascular, kidney or liver disease; non diabetic; not consuming lipid- or glucose - lowering medications and normotensive or hypertensive,

and controlled by medications; normotensive; or hypertensive and controlled medications within the last 3 months. Two women who were on hormone replacement therapy maintained their current regimen for the study duration. Subjects provided a medical history and underwent a physical examination by the study physician. Eight subjects

dropped out for personal reasons including lack of time and difficulties in reaching the

research clinic. The experimental protocol was approved by the Institutional Research

Ethics Board for the School of Medicine at McGill University, Montreal, Canada and

Tufts University- New England Medical Center, Boston, US. All volunteers gave their

written informed consent to participate in the trial prior to the commencement of the

study. Baseline characteristics of the study subjects are presented in Table 3.1. 38

3.3.2 Protocol and diet Study subjects consumed each of the 5 experimental diets for periods of 29 d using a randomized, single-blind, crossover design in which every subject completed every treatment. Both food and beverages were provided in amounts to maintain a stable body weight as estimated using the Mifflin equation (Mifflin et al., 1990). At days 1 and 2, and then again at days 26, 27 and 28 of each diet phase, blood samples were obtained after a

12 h fast. Each diet phase was followed by a 2-4 week washout period during which the subjects consumed their habitual diets. Diets were designed to contain 55% of energy from carbohydrates, 15% from protein and 30% from fat (Table 3.2). Foods were identical for all diet phases with the exception of the experimental component.

Subjects consumed their breakfast each morning at the clinical unit under supervision of the staff. The 2 remaining meals were prepared and packed for consumption at work or at home. The experimental components of the diet were administrated as a single daily dose provided with the margarine component of the breakfast meal. Subjects were instructed to consume only meals prepared by the clinical kitchen and not to consume any other food or drink, including alcohol or other beverages. Body weights were monitored daily throughout the intervention phases and maintained at baseline ± 1 kg of baseline weight by adjusting the amount of food provided, if necessary. Body weights at the end of treatment periods were 79.0 ±16.3 kg, 78.2±16.4 kg, 78.3±16.5 kg, 78.6±16.6 kg and

78.9±16.7 kg for control, sterol, sterol and long chain fatty acids offish oil, sterol ester of long chain fatty acids from fish oil, and sterol ester of sunflower oil phases, respectively. 39

Subjects were randomly assigned to each of the following 5 treatments using a Latin

square design: control margarine (Unilever, Inc, USA); free form of plant sterols given at

a dose of 22 mg • kg body weight"1 »d_1 (Forbes Medi-Tech Inc, Canada); plant sterols

esterified to fatty acids from sunflower oil provided in margarine and given at a dose that provide 22 mg plant sterol • kg body weight"1 "d"1 (Unilever, Inc, USA); plant sterols

esterified to long chain n-3 polyunsaturated fatty acids (n-3 PUFA) from fish oil given at a dose of 35.2 mg • kg body weight"1 'd'1 (equivalent to 22 mg plant sterols) (Forbes

Medi-Tech Inc, Canada); and free form of plant sterols (Forbes Medi-Tech Inc, Canada) given at a dose of 22 mg • kg body weight"1 'd'1 in combination with long chain n-3

PUFA from fish oil (Roche Lipid Technologies, Switzerland) given at a dose of 13.2 mg • kg body weight"1 •d"1. All plant sterols used in the study were derived from soybeans

(Table 3.3). The fatty acid composition of the fish oil and the sterol fish oil ester was the

same and consisted of 41.1 % EPA and 19.8 % DHA. To standardize the dose across

subjects who ranged considerably in body mass, the plant sterol dose was administrated according to body weight. The mean of the dose of plant sterols was 1.7g/d with a range of 1.0 -1.8 g/d. The mean intake of long chain n-3 PUFA was 1.1 g/d, ranging from 0.7-

2.1g/d.

3.3.3 Analyses

Fasting blood samples were centrifuged for 20 min at 520 x g at 4°C and aliquots were

stored at -80°C until further analyses. Plasma total cholesterol, LDL-, and HDL-

cholesterol, triacylglycerol, apo A-I and apo B, Lp(a) and CRP were analyzed as previously described (Lichtenstein et al., 2003; Lichtenstein et al., 2002). 40

Plasma plant sterols, campesterol and P-sitosterol, were analyzed in duplicate by gas- liquid chromatography, as reported previously (Ntanios and Jones, 1998). Briefly, plasma samples were saponified with methanolic KOH solution and extracted twice with petroleum ether. 5-a cholestane was used as the internal standard. Samples were analyzed by a gas-liquid chromatograph equipped with a flame ionization detector (HP 5890 Series

II, Hewlett Packard, Palo Alto, CA) and a 30 m capillary column (SAC-5, Supelco,

Bellefonte, PA). Detector and injector temperatures were 300 °C. Sterols peaks were identified by comparison with standards (Supelco). Sitosterol and campesterol levels were analyzed in order to verify the bioavailability of plant sterols from the new formulations.

3.3.4 Statistical analyses

Data are expressed as mean ± standard deviation of the mean ± SD. A sample size of 26 subjects was calculated as sufficient for detecting a change of 0.5 mmol/L at a (2-sided) =

0.05 and power = 0.80 and using a value for the standard deviation of the change of 0.5 mmol/L as obtained from previous studies. Bonferroni correction was applied due to the comparison of more than two groups. ANOVA was used to determine statistical significance. When treatment effects were identified as significant, Tukey's test was used to identify significant effects between treatments. Student's paired t tests were used to compare start and endpoint values within each treatment period. Normal distribution was tested with Shapiro-Wilks test. If a variable was not normally distributed, data were logarithmically transformed prior to analysis (Hallikainen et al., 2000b). Genders also were evaluated separately by ANOVA. Differences were considered significant if P <

0.05. The data were analyzed using Proc-General Linear Model SAS (version 8.0; SAS

Institute). 41

3.4 RESULTS

The addition of free sterol, free sterol plus fish oil, sterol esterified to fish oil or sterol esterified to sunflower oil to the diet for 4 weeks did not affect percent changes (data not shown) or end point plasma concentration of total cholesterol, LDL- cholesterol or triacylglycerol compared to control (Table 3.4). When genders were analyzed separately, results were similar to those of the whole group (data not shown). Plasma HDL- cholesterol levels were higher (P < 0.05) after subjects consumed each of the diets containing the sterols and relative to the period of control diet. Although the trend was similar in men and women, the magnitude of difference was greater in the latter. The increase in HDL cholesterol resulted in a lower (P < 0.05) total cholesterol:HDL cholesterol ratio at the end of each sterol-supplemented diet period, relative to the control diet period, although the differences were significant for all treatments only in women (P

< 0.05) subjects only. The difference in HDL-cholesterol levels was not reflected in the plasma apo A-I concentrations that were not affected by the treatments. The treatments also did not affect apo B, Lp (a) or CRP concentrations.

Relative to the control, plasma campesterol levels were higher (P < 0.05) only after subjects consumed the diet of plant sterol esterified to sunflower oil supplemented (Table

3.4). The pattern for plasma P-sitosterol levels was different than that of campesterol.

The levels of |3-sitosterol rose (P < 0.05) after the subjects consumed the different plant sterols formulations relative to the control diet. However, the increase in P-sitosterol was lower (P < 0.05) after subjects consumed sterols esterified to fatty acids from sunflower oil than when they consumed the other sterol preparations. Over the 4-week controlled diet period, plasma campesterol levels rose (P < 0.05) by 42% for the sterols esterified to 42 fatty acids from sunflower oil, and P-sitosterol levels rose by (P < 0.05), 24%, 49%,

39%, and 26%, for the free sterols, free sterols plus fish oil, sterols esterified to fish oil and sterol esterified to sunflower oil treatments, respectively.

Although the sterol preparations did not affect plasma lipid, lipoprotein, apolipoprotein and CRP levels, with the exception of HDL cholesterol, among diet groups, there were effects of the diet interventions on LDL- and HDL-cholesterol, and campesterol and |3-

sitosterol levels attributable to shifting individuals from their habitual diet to the

controlled diets. Over the 4-week controlled diet period, plasma LDL- cholesterol levels

declined from baseline (P < 0.05) by 8%, 11%, 7%, 3% and 11%, for control, free

sterols, free sterols plus fish oil, sterols esterified to fish oil and sterols esterified to

sunflower oil periods, respectively. Triacylglycerol declined from baseline(P < 0.05) by

12%, 23%o, 23%, 12% and 13%, for control, free sterols, free sterols plus fish oil, sterols

esterified to fish oil and sterols esterified to sunflower oil periods, respectively. In

contrast, HDL- cholesterol declined from baseline (P < 0.05) by 3, 5% and 6% for

control, sterols esterified to fish oil and sterols esterified to sunflower oil periods,

respectively.

3.5 DISCUSSION

The aim of this study was to determine effects of free sterols and sterols esterified to

different fatty acids and mixed with very long chain n-3 fatty acids on plasma lipid and

lipoprotein levels, CRP and plasma plant sterols. Somewhat unexpectedly, a single daily

dose of plant sterols, regardless of their physical form, taken with the breakfast meal, did

not significantly lower plasma cholesterol concentrations compared with the control 43 treatment, but was associated with an increase in HDL cholesterol. Although some previous studies reported that the lack of efficacy of plant sterols as cholesterol-lowering agents, this could be attributed to poor solubility of plant sterols in the formulations tested

(Denke, 1995; Jones et al., 2003), the lack of plant sterol efficacy in our study appeared not to be related to the form consumed or their bioavailability, because the data were similar for those administrated as free plant sterols or as plant sterols esterified to fatty acids from either sunflower or fish oil. Plant sterol preparations examined in our study included preparations previously shown to be bioavailable and to lower blood cholesterol, namely free plant sterols blended with a spread (Jones et al., 1999; Vanstone et al., 2002) and plant sterol esters of fatty acids from vegetable oil (Hallikainen and Uusitupa, 1999;

Jones et al., 2000; Maki et al., 2001; Neil et al., 2001; Nestel et al., 2001). Thus, even if plant sterols from the new formulations are not bioavailable, this does not explain why previously tested plant sterol preparations did not reduce blood cholesterol levels in this

study.

The increase in HDL-cholesterol concentrations observed after plant sterol-supplemented

diet phases has been reported in a few (Gylling and Miettinen, 1994; Mussner et al.,

2002) but not majority (Jones et al, 1999; Maki et al., 2001; Mensink et al., 2002;

Vanstone et al., 2002; Weststrate and Meijer, 1998) of previous studies. Regardless of the

significant HDL cholesterol-raising effect of the plant sterol preparations tested, no

reciprocal effect on triacylglycerol concentrations was seen. The increase in HDL-

cholesterol concentrations observed in this study may be due to chance. Furthermore,

supplementing the diet with fish oil, either mixed with plant sterols or esterified to the plant sterols, did not exert any effect on triacylglycerol concentrations. The lack of an 44 effect of fish oil on triacylglycerol levels is likely due to the quantity of fish oil fed and the absence of hypertriacylglycerolemia, by design, in the individuals studied.

Differences in increases of plasma sitosterol and campesterol between the different study formulations may correspond to the differences in plant sterol content of the formulations.

As discussed previously, the lack of efficacy of different plant sterols in the present study is unlikely to be related to improper solubility of plant sterol formulations examined here.

In addition, it is unlikely that the lack of efficacy of plant sterols was due to inadequate power as other plant sterols studies have been performed with a similar number or even fewer subjects. In addition sample size calculation was corrected for the multiple comparisons. The lack of efficacy of different plant sterols in this study was likely due to the fact that plant sterols were administrated as a single morning dose, to ensure compliance with consuming the breakfast meal under supervision. Therefore, poor

compliance was not responsible for the lack of effects. Previous studies that have shown

cholesterol-lowering efficacy distributed the plant sterol treatment in 2 (Hallikainen et al.,

2000b; Maki et al., 2001; Ntanios et al., 2002; Weststrate and Meijer, 1998) or 3 (Jones et

al., 2000; Jones et al., 1999) (Mensink et al., 2002; Plat and Mensink, 2000; Vanstone et

al., 2002) doses per day. However, a single dose of plant sterols has also been shown to

lower cholesterol levels (Matvienko et al., 2002; Plat et al., 2000). In a crossover study,

2.5 g/d of plant stanol esters, incorporated into margarine or shortening, and consumed

either once at lunch or 3 times/d for 4 weeks, LDL- cholesterol concentrations were

reduced by 9% and 10%, respectively (Plat et al., 2000). In another study, a 10% reduction in LDL- cholesterol occurred in subjects given a 2.7 g dose of plant sterols

solely at lunch for 4 weeks (Matvienko et al., 2002). Moreover, the diets were moderate in fat and cholesterol and should not have contributed to the absence of an effect of plant 45 sterols on circulating cholesterol concentrations. The single dose of plant sterols used in this study was provided with the morning meal that contained similar cholesterol content to the other meals. Each of the 3 meals supplied about 83 mg, 86 mg, and 79 mg of cholesterol, respectively. Even at low cholesterol intake, plant sterols have been shown to reduce blood cholesterol concentrations (de Graaf et al., 2002; Hallikainen et al., 2000b;

Hallikainen and Uusitupa, 1999; Homma et al., 2003; Maki et al., 2001; Nestel et al.,

2001).

Plat et al. (Plat et al., 2000) hypothesized that plant stanols remain in the intestinal lumen

for extended periods, suggesting that it is not necessary to consume plant stanol ester

products with every meal (Plat et al., 2000). Our results do not support this hypothesis. A

single morning dose of plant sterols reduced plasma cholesterol in some individuals, but

overall, there was no significant reduction in blood cholesterol compared with the control

diet phase. One of the major differences between the studies of Plat et al. (Plat et al.,

2000) and Matvienko et al. (Matvienko et al., 2002), and our study is the size of the single

dose of plant sterols. Compared with the 1.0-1.7 g/d dose of the present study, the single

dose sizes for Plat et al.(Plat et al., 2000) and Matvienko et al. (Matvienko et al., 2002)

were 2.5 and 2.7 g/d, respectively. It is likely that a higher dose of plant sterols is needed

when administrated as a single dose. Plant sterols were given on a body weight basis to

standardize the dose. In one of our previous studies (Jones et al., 1999) we also gave plant

sterols at a dose of 22 mg« kg body weight"1 'd'1 and observed a 15.5% reduction in LDL-

cholesterol levels compared with the control. However, in that study plant sterol dose

was distributed throughout the day. 46

Another major difference is that the single dose was consumed at lunch in the Plat et al.

(Plat et al., 2000) and Matvienko et al. (Matvienko et al., 2002) studies, whereas in the present study plant sterol treatments were consumed at breakfast. The efficacy of plant sterols as a cholesterol-lowering agent may demonstrate a time-of-day variation, possibly coinciding with the circadian rhythm of cholesterol metabolism. Circadian rhythm in cholesterol synthesis has been shown in animals (Edwards et al., 1972; Hamprech et al.,

1969; Ho, 1975; Jurevics et al., 2000) and in humans (Cella et al., 1995; Jones et al.,

1992; Jones and Schoeller, 1990), whereas the circadian variation in cholesterol absorption has not been studied. In rats and hamsters, maximum cholesterol synthesis occurs at midnight, whereas minimum synthesis occurs at noon (Edwards et al., 1972;

Hamprech et al., 1969; Ho, 1975; Jurevics et al., 2000). However, in these rodents, which normally eat at night, circadian rhythm of cholesterol biosynthesis is dependent on time of food intake rather than on the light cycle (Edwards et al., 1972; Ho, 1979). Similar to the data from animals, the circadian rhythm of cholesterol biosynthesis in humans is affected by food intake. Delaying the meal time by a 6.5 h resulted in a 8.6 h and 6.5 h delay in maximum and minimum cholesterol synthesis rate, respectively (Cella et al.,

1995). Because cholesterol synthesis and absorption are inversely related, one can speculate that cholesterol absorption is low early in the morning and increases during the daytime period. Thus, further lowering of cholesterol absorption by plant sterols may not lead to a decrease in blood cholesterol. It is possible that a single dose of plant sterols taken in the morning may not lead to optimal cholesterol reduction.

In conclusion, because traditional forms of plant sterols did not reduce blood cholesterol levels, we cannot confirm the efficacy of the new formulation of plant sterols 47 administrated as single morning dose. Future studies are needed to address whether a higher dose of plant sterols is needed when taken as a single dose or whether intake should be distributed throughout the day. Moreover, studies are needed to investigate whether the time of day affects the efficacy of a single dose of plant sterols, given that recent recommendations (Cleeman et al., 2001) encourage their use in reducing the risk of

CVD. In addition, promoting plant sterol-enriched products for consumption once a day should be based on efficacy data from well controlled studies. Recently, a number of proteins that regulate cholesterol absorption have been identified. Niemann-pick CI Like

1 (NPC1L1) plays a critical role in the uptake of cholesterol across the intestinal enterocytes (Altmann et al., 2004). In contrast, ATP-binding cassette transporter (ABC)

G5 and G8 proteins pump the sterols from the enterocytes back into the intestinal lumen

(Schmitz et al., 2001). The study of the level and/or gene expression of these proteins, presents a promising approach for investigating the interplay among circadian rhythm, plant sterols and cholesterol metabolism.

3.6 ACKNOWLEDGEMENT:

We thank Susan Jalbert from Tufts University for analyzing blood lipid concentrations and Dr. William Parsons for monitoring the participants during the study. We also thank

Mary Emily Clinical Nutrition Research Unit staff for preparing and serving the meals for the study. Supported by grant HL 54727 from the National Institutes of Health,

Bethesda, MD and the U.S. Department of Agriculture, under agreement 58-

1950-4-401. S.S.A.M. was supported by a student scholarship from the Hashemite

University, Jordan. 48

Table 3.1. Baseline characteristics of subjects.'

~~ Variable All (n=30)

Age, yr 59 ±10

Weight, kg 79 ±17

BMI, kg/m2 28 ± 5

Lipids, mmol/L

Total cholesterol 5.9 ±0.8

Low density lipoprotein 3.8 ±0.8

High density lipoprotein 1.4 ±0.4

Triacylglycerol 1.6 ±0.9

Values are expressed as mean ± SD. 49

Table 3.2. Macronutrient composition of the study diet /12552 Kj (3000

Kcal).

Nutrient Composition

Protein,% energy 15%

Carbohydrate, % energy 56%

Total fat, % energy 31 %

Saturated fatty acids, % energy 8%

Monounsaturated fatty acids, % energy 12%

Polyunsaturated fatty acids, % energy 8%

Cholesterol, mg 248

Fiber, g 37 50

Table 3.3. Major plant sterol concentration of the study treatments (% w/w).

Free plant sterol Sterol ester of Plant sterol-fish

sunflower oil oil ester

"^Sitosterol 702 403 57.9

Campesterol 9.7 22.7 7.5

Stigmasterol 0.2 18.1 0.2

Other sterols 19.9 18.9 34.4 Table 3.4. Plasma Lipid, lipoprotein, apolipoprotein, CRP and sterol concentrations in subjects at the end of each dietary intervention.1 Parameter Control Free sterol Sterol+fish oil Sterol ester of Sterol ester of P-value fish oil sunflower oil

Total cholesterol, 5.74±1.09 5.67±1.11 5.78±1.11 5.80±1.11 5.70±1.14 0.562 mmol/L LDL-choIesteroI, 3.65±0.93 3.60±1.01 3.70±0.98 3.76±1.01 3.60±1.06 0.391 mmol/L HDL-cholesterol, 1.27±0.28b 1.37±0.34a 1.37±0.34a 1.35±0.34a 1.35±0.34a 0.0001 mmol/L Triacylglycerol2, 1.5U0.63 1.44±0.66 1.39±0.63 1.48±0.71 1.56±0.88 0.198 mmol/L Total 4.72±1.25a 4.34±1.23b 4.39±1.24a,b 4.54±1.25a'b 4.49±1.36a'b 0.0275 cholesterol/HDL Apo B (g/L) 0.98±0.21 0.97±0.21 0.98±0.20 1.00±0.21 0.98±0.21 0.444 Apo A-I, g/L 1.25±0.17 1.28±0.17 1.29±0.19 1.27±0.18 1.27±0.18 0.0845 Lp(a), jimol/L 0.96±0.71 1.00±0.75 1.00±0.71 1.00±0.75 1.00±0.71 0.5746 CRP, mg/L 4.8±7.7 2.5±21 2.8±3.2 3.7±4.9 5.5±10.1 0.106 Campesterol,2 18.6±8.3a 19.4±9.1a 19.3±8.5a 19.5±7.6a 24.1±12.6b O.0001 jimol/L P-Sitosterol,2 jimol/L 7.0±3.7C 9.9±5.6a 10.8±5.9a 10.2±4.7a 8.7±5.5b 0.0002

i - 2 Values are expressed as mean ± SD. Means within a row with different superscript letters are significantly different, P<0.05. Data log transformed prior to analysis. 52

BRIDGE 2.

The primary objective of Manuscript 2 was to determine effects of novel preparations of plant sterol on plasma lipid and lipoprotein levels, however, traditional forms of plant sterol preparations fed under full compliance situations for an appropriate duration were found not to reduce circulating cholesterol levels compared to control. As explained in

Manuscript 2; the lack of efficacy of different plant sterols in that study could be attributed to the consumption of plant sterols as a single morning dose. Plant sterols were given in the morning to ensure compliance as the subjects consumed only the breakfast meal under supervision. The majority of studies that have shown cholesterol-lowering efficacy distributed plant sterol treatments across 2 or 3 doses per day. A single dose of plant sterols given at lunch has also been shown to lower cholesterol levels and it was hypothesized that plant sterols remain in the intestinal tract for an extended period of time.

However, results from Manuscript 2 do not support that hypothesis. Results of Manuscript

2 suggest that the dosing frequency of plant sterols or the time of intake of single dose of plant sterols could affect their cholesterol-lowering action. Therefore, the primary objective of Manuscript 3 was to examine the cholesterol- lowering efficacy of plant sterols consumed throughout the day or once a day in the morning. 53

CHAPTER 4. Manuscript 3 (Study # 2). In preparation for submitting to the

European Journal of Clinical Nutrition.

EFFECT OF PLANT STEROL CONSUMPTION FREQUENCY ON PLASMA

LIPID LEVELS AND CHOLESTEROL KINETICS IN HUMANS

Suhad S. AbuMweis1, Catherine A. Vanstone1, Alice H. Lichtenstein2,

Peter J.H. Jones1,3

Author affiliation:

'School of Dietetics and Human Nutrition

McGill University, Ste-Anne-de-Bellevue, Montreal, Quebec, Canada

Cardiovascular Nutrition Laboratory, Jean Mayer USDA Human Nutrition Research

Center on Aging, Tufts University

Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba,

Canada

Correspondence and reprint requests:

Peter J.H. Jones, Ph.D,

Richardson Centre for Functional Foods and Nutraceuticals

University of Manitoba, Smartpark

196 Innovation Drive, Winnipeg, Manitoba, R3T 6C5

Phone: (204) 474-8883, Fax: (204) 474-7552

Email: [email protected] 54

4.1 ABSTRACT

The objective of this study was to compare the efficacy of single verses multiple doses of

plant sterols on circulating lipid levels, and cholesterol kinetics. A randomized, single- blind, placebo-controlled, 3 phase (6 days/phase) crossover, supervised feeding trial was

conducted in 19 subjects (16 males and 3 females) with mean age of 55 ± 8 yr. Subjects

were provided control margarine with each meal; plant sterol-enriched margarine (1.8 g/d

plant sterols) with breakfast (BF) (single-BF) and control margarine with lunch and

dinner, or plant sterol-enriched margarine divided equally at each of the 3 daily meals (3

times/d). Relative to control, endpoint plasma low density lipoprotein (LDL) cholesterol

concentrations were significantly lower after consuming plant sterols 3 times/day (P<

0.05) but not significantly different when consumed once per day (3.43 ± 0.62, 3.22 ±

0.58 and 3.30 ± 0.65 mmol/L, control, 3 times/d and single-BF, respectively).

Cholesterol fractional synthesis rate was highest (P <0.05) after the 3 times/d phase

(0.083 ± 0.028, 0.083 ± 0.025 and 0.091 ± 0.022 pool/d, control, single-BF and 3 times/d,

respectively). Cholesterol absorption decreased (P < 0.05) after both the 3 times/d and

single-BF phase, relative to control. The change in LDL-cholesterol relative to the control

phase tended to be directly associated with change in cholesterol absorption efficiency for

the 3 times/d (r =0.43, P =0.0623) but not for the single-BF phase (P =0.4858). These

data suggest that for an equivalent amount of plant sterol, consuming it as a divided dose

3 times/d was more efficacious than when consumed as a single dose.

Key words: Plant sterols, single dose, LDL-cholesterol, diet, lipoproteins 55

4.2 INTRODUCTION

In addition to lifestyle change, the National Cholesterol Education Panel and the

American Heart Association have suggested the use of plant sterols as an approach to control circulating levels of low density lipoprotein (LDL) cholesterol (Cleeman et al.,

2001; Fletcher et al., 2005). Plant sterols and stanols, the hydrogenated form of sterols, have been reported to reduce cholesterol absorption consequently increasing cholesterol synthesis, with a net effect of reducing LDL-cholesterol concentrations about 10 percent

(Gylling and Miettinen, 1994; Jones et al., 2000; Vanstone et al., 2002).

The majority of studies have shown efficacy of plant sterol supplementation when distributed in two (Hallikainen et al., 2000a; Maki et al., 2001; Ntanios, 2001; Weststrate and Meijer, 1998) or three (Jones et al., 2000; Mensink et al., 2002; Plat and Mensink,

2000; Vanstone et al., 2002) doses per day. There is discordant data on the effect of a single dose of plant sterols on plasma cholesterol concentrations. A single dose of plant sterol also lowers cholesterol levels to a similar extent as multiple doses of the same amount (Matvienko et al., 2002; Plat et al., 2000). Plat et al. (Plat et al., 2000) hypothesized that plant stanols remain in the intestinal lumen for extended periods where they decrease cholesterol absorption, thus it is not necessary to consume plant stanol ester products throughout the day. It has also been reported that a single dose of plant sterol failed to lower LDL-cholesterol concentrations when administered as a single morning dose (AbuMweis et al., 2006b). Nonetheless, current marketing of plant sterol-containing products is largely directed towards consumption once a day with breakfast meals. There have been no studies examining the effects of plant sterol consumption frequency on lipid profile, and cholesterol kinetics as part of a controlled low-fat, low-cholesterol diet. 56

The objective of this study was to compare under controlled and supervised conditions the effect of plant sterols given once in the morning or three times throughout the day on plasma lipids and cholesterol kinetics.

4.3 SUBJECTS AND METHODS

4.3.1 Subjects

Males and postmenopausal female subjects 40-80 yr with a BMI between 22 and 32 kg/m2 were recruited from a database of subjects who had participated in previous studies at the Mary Emily Clinical Nutrition Research Unit (MECNRU) of McGill University.

Respondents underwent a thorough historical and physical examination to exclude those with thyroid disease, diabetes mellitus, kidney disease, liver disease, smokers or people consuming an excessive amount of alcohol (>14 drinks/wk). Volunteers taking medication known to affect lipid metabolism, high dose dietary supplements, fish oil capsules or plant sterol for 3 months or more prior to the start of the study were excluded from participation. At the first visit volunteers were screened for LDL-cholesterol > 3.0 mmol/L. Eligible volunteers were asked to return for a second screening to measure complete blood counts, serum biochemistry and lipid profiles that included total cholesterol, triacylglycerol, LDL-cholesterol and high density lipoprotein (HDL) cholesterol levels.

4.3.2 Study design and protocol

A randomized, single-blind crossover placebo-controlled clinical trial was carried out at the MECNRU. The study consisted of 3 phases of 6 days each. During day 1 through to day 5 subjects consumed a precisely controlled weight-maintaining diet. At the end of 57 each phase, i.e. day 6, blood samples were collected but no diets were provided. The 3 phases of treatments included control phase: no plant sterols were consumed (control), single morning dose phase: plant sterol dose was given only at breakfast (single-BF) and three times dose phase: plant sterol dose was divided equally among 3 daily meals, breakfast, lunch and dinner (3 times/d). Each phase was separated by a washout period of

2 weeks during which time the subjects consumed their habitual diets. Subjects were randomly assigned to 1 of 6 predetermined sequences using a Latin square design.

Diets were prepared in the metabolic kitchen of the MECNRU where foods were weighed to the nearest 0.5 g. Three isocaloric meals were prepared each day for the 5 study days for every subject during each phase. Total caloric intake was matched to each subject's energy requirements. Diets were designed to contain 55% of energy (E) from carbohydrates, 15% E from protein and 30% E from fat, 80 mg/1000 Kcal of cholesterol and 12 g/1000 Kcal of fibers. During the study period subjects weighed themselves every morning. Subjects consumed breakfasts and the dinners at the MECNRU under supervision. The lunch meals were packed to be taken out. Plant sterol enriched margarine (Take Control-Unilever Inc. Netherlands) was used to provide a dose of 1.8 g/d of plant sterols and the same margarine without plant sterols was used as the control margarine (Table 4.1). For the single-BF phase, the plant sterol enriched margarine was incorporated in the breakfast meal and the control margarine was incorporated in the lunch and dinner meals. During the divided phase, plant sterol enriched margarine was divided equally among the breakfast, lunch and dinner meals. 58

Subjects consumed a controlled diet including the assigned study treatment from day 1 to day 5. On the second day (24h) of each phase, subjects ingested 75 mg [3, 4- C] cholesterol (CDN Isotopes, Montreal) to measure cholesterol absorption over the following 72 h (Vanstone et al., 2002). The 13C-cholesterol was dissolved in 5 g of warmed margarine and consumed on a slice of toast. On day 5 of each feeding period, approximately 25 ml of deuterium oxide was given orally to each subject before breakfast to measure endogenous cholesterol synthesis. Fasting blood samples were taken at baseline prior to isotope administration (24h), as well as on days 3, 4, 5 and 6 to monitor enrichment levels of both isotopes.

4.3.3 Analyses

4.3.3.1 Plasma lipid profile

Fasting blood samples collected on day 5 and 6 were used to measure circulating lipid concentrations. Blood was centrifuged for 20 min at 520 * g and 4°C to separate plasma from RBCs. Plasma total and HDL cholesterol, and triacylglycerol concentrations were measured using standardized procedures as previously described (Lichtenstein et al.,

2006). The Friedewald equation was used to calculate LDL-cholesterol concentrations

(Friedewald et al., 1972).

4.3.3.2 Cholesterol absorption determination

Cholesterol absorption was assessed using the stable isotope single tracer method as previously described (Ostlund et al., 2002; Ostlund et al., 1999; Shin et al., 2005;

Spilburg et al., 2003; Varady and Jones, 2006). The area under the 13C-enrichment curve or the average C -enrichment correlates with the cholesterol absorption rate as measured 59 by the dual stable isotope ratio method (Wang et al., 2004). Compared to the dual stable isotope ratio method, the single stable isotope single tracer method is less invasive.

Free cholesterol extracted from red blood cells (RBCs) was used to determine 13C- cholesterol enrichments using on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Lipids were extracted from RBCs in duplicate using a modified Folch extraction procedure (Folch et al., 1957). Briefly, 2 g of RBCs from each timepoint was combined with methanol and heated. Hexane-chloroform 4:1 (v/v) and was added and the mixture was shaken for 20 min. Water was added, the sample was shaken again, and the upper layer solvent layer was removed after centrifugation. The extraction procedure was then repeated and solvent layers were combined. Lipid extracts dissolved in hexane were injected into a gas chromatograph (Agilent 6890N) interfaced with a

Finnigan Delta V Plus isotope ratio mass spectrometer (Bremen, Germany) through a

Finnigan combustion interface (Combustion Interface III, Bremen, Germany). Isotope abundance, expressed in delta (5) per mil (%o), was calculated using CO2 as a reference gas. The delta values were further expressed relative to the international reference standard, Pee Dee Belemnite (PDB) limestone, using a calibration curve of working standards that were analyzed previously using a conventional IRMS (SIRA 12; Isomass,

Cheshire, United Kingdom). From 24 h to 96 h (72 h post 13C cholesterol ingestion), two kinetic parameters were calculated using NCSS (version 2000; NCSS Statistical

Software, Kaysville, UT) software: area under the 13C-enrichment curve versus time curve [AUC (24-96 h)] and maximum 13C -cholesterol enrichment (E-max). The AUC 60

(24—96 h) corrected for baseline values was calculated with the use of the trapezoidal rale.

4.3.3.3 Endogenous cholesterol synthesis determination

Cholesterol synthesis rates were assessed after 24 h of deuterium water administration, using the deuterium incorporation approach (Jones et al., 1993). This method measures

cholesterol synthesis as the rate of deuterium incorporation from body water into RBC

membrane free cholesterol over a 24-h period. The deuterium incorporation method has

been validated against the sterol balance method (Jones et al., 1998a). Deuterium

enrichment was measured in both RBC free cholesterol and in plasma water. The

measurement of plasma water deuterium enrichment was performed as described

previously (Vanstone et al., 2002). Enrichments were expressed relative to standard

mean ocean water (SMOW) and a series of standards of known enrichment.

The measurement of free cholesterol deuterium enrichment was performed using on-line

gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/JJRMS) as described

above. Lipids were extracted from RBCs as described above for cholesterol absorption

determination. Isotope abundance, expressed in 5 %o, was calculated using H_2 as

reference gas. The delta values were further expressed relative to SMOW using a

calibration curve of working standards.

Fractional synthesis rate (FSR) is taken to represent RBC free cholesterol deuterium

enrichment values relative to the corresponding mean plasma water sample enrichment 61 after correcting for the free cholesterol pool. FSR represents that fraction of the cholesterol central pool or Mi that is replaced daily by newly synthesized cholesterol.

This pool consists of cholesterol that equilibrates rapidly with plasma cholesterol and includes that in the RBCs, liver, intestine and other viscera. The initial 1 -2 day period after deuterium intake is considered to give the most interpretable synthesis data. FSR is calculated as per the equation (Jones et al., 1993):

FSR (pools per day) = del cholesterol/(Je/ plasma * 0.478) where del for deuterium is the enrichment difference between free cholesterol and plasma water between 72 h (day 5) and 96 h (day 6) in parts per thousand relative to a SMOW standard. The factor 0.478 accounts for the ratio of labelled H atoms replaced by deuterium (22/46) during in vivo biosynthesis. Cholesterol absolute synthesis rates were calculated by multiplication of FSR values by the measured Mi pool size and a factor of

0.33.

4.3.3.4 Statistics

All data are expressed as means ± SDs. Statistical analysis was carried out using SAS

(version 8.0; SAS Institute INC, Cary, NC). The principal statistical model used was the

ANOVA taking into account subject as a random effect, study treatment effect, period effect, sequence effect and carryover effect. Residuals of every outcome were checked for normality using Shapiro-Wilk test. If the normality assumption was violated for a variable, then log transformation was performed. In the event of a statistically significant treatment effect (P-value < 0.05) pair wise comparisons were performed using Tukey's test. Tests for associations between variables were performed using Pearson Correlation

Coefficient analyses. 62

4.4 RESULTS

Characteristics of the study subjects are shown in Table 4.2. There were no significant

weight changes across any of the study phases. Concentrations of plasma lipids and

lipoproteins at the end of each treatment phase are shown in Table 4.3. Relative to the

control, endpoint plasma LDL-cholesterol level was 0.21±0.27 mmol/L (6%) lower at the

end of the 3 times/d phase (P< 0.05) and not significantly different at the end of the

single-BF phase compared to control. Total cholesterol tended to be lowest during the 3

times/d phase (P= 0.0639), while plasma HDL and triacylglycerol concentrations were

not different among the study treatments. The total cholesterol :HDL ratio was lower (P <

0.05) at the end of the 3 times/d phase by 4.4%, relative to control phase.

Cholesterol absorption AUC (24-96 h) values of RBC cholesterol during the single-BF

phase and 3 times/d phase were lower by 39% and 36%, respectively, compared to the

control phase (Table 4.4). Similarly, E-max values were lower (P <0.0001) during the

single-BF phase and 3 times/d phase than the control phase. When subjects were

analyzed by treatment grouping, the change in LDL relative to the control phase tended to be associated with the change in AUC (24-96 h) relative to the control phase for the 3

times/d phase (r =0.43, P =0.0623), but not for the single-BF phase CPM3.4858) (Figure

1 "X

4.1). Maximum values for C-enrichment were attained at around 48 h (Figure 4.2).

Cholesterol FSR and ASR values were highest (P < 0.05) during the plant sterol 3 times/d

phase (Table 4.4). 63

4.5 DISCUSSION

The major findings of this study are that consumption of a single morning dose of plant

sterols was not as efficacious in lowering LDL concentrations as the 3 times per day

dosing pattern and that the significant reduction in plasma LDL concentration was

achieved after a relatively short period. Previous studies have reported a reduction in

cholesterol concentrations after one day (Miettinen et al., 2000), 8 days (Hallikainen et

al., 2002) and 10 days (Jones et al., 1998b) of plant sterol/stanol consumption. However,

the single day study by Miettinen et al. (Miettinen et al., 2000) carried out in

colectomized individuals whose cholesterol metabolism differed from that of healthy

individuals. The 8-day study of Hallikainen et al. (Hallikainen et al., 2002) lacked a

control group and the reduction in serum cholesterol concentrations by stanol ester

consumption was relative to day 0 thus a time effect could have contributed to the

reduction in cholesterol concentrations. Although the duration of the present study was

relatively short, plant sterol ester lowered LDL-cholesterol concentrations by 0.21 mmol/L (6%). The magnitude of reduction in LDL-cholesterol concentrations observed

in the current study is consistent with that typically observed after 3-6 week intervention

periods (Colgan et al., 2004; Hendriks et al, 1999; Jakulj et al, 2005; Kratz et al., 2007;

Sierksma et al., 1999) or even for 52 weeks (Hendriks et al., 2003).

Limited data are available on plant sterol daily frequency of intake and outcomes. An

earlier study demonstrated that 2.5 g/d of plant stanol consumed for 4 weeks once per day

at lunch or divided among 3 meals (0.42 g at breakfast, 0.84 g at lunch and 1.25 g at

dinner), lowered LDL-cholesterol concentrations to a similar extent, about 10% (Plat et

al., 2000). A more recent study reported that consuming a single dose of plant sterols 64 provided in a yoghurt drink with lunch resulted in a larger decrease in LDL-cholesterol concentrations than consuming the same dose with breakfast (Doornbos et al., 2006).

In addition to monitoring changes in plasma cholesterol concentration the mechanism(s) accounting for these changes were also assessed. In accordance with previous work,

administration of plant sterols at a dose of 1.8 g/d was shown to significantly decrease

intestinal cholesterol absorption efficiency. Notably, cholesterol FSR was highest during

the 3 times/day phase, likely in response to the decrease in cholesterol absorption

efficiency. On the contrary, no reciprocal increase in cholesterol synthesis in response to

the decrease in absorption during the single-BF phase was observed. Changes in LDL-

cholesterol concentrations tended to be associated with changes in area under the 13C-

cholesterol enrichment curve during the 3 times/d phase, but not during the single-BF

phase. Previous studies utilizing different experimental approaches have shown that

consumption of plant sterols/stanols reduced cholesterol absorption and consequently

increased cholesterol synthesis, with a net reduction in LDL-cholesterol concentrations in

hypercholesterolemic individuals (Gylling and Miettinen, 1994; Jones et al., 2000;

Vanstone et al., 2002). The regulatory process behind this phenomenon is not clearly

understood yet.

Plant sterols consumed during the single-BF phase reduced cholesterol absorption

efficiency, as measured by the stable isotope single tracer method, but did not reduce

LDL-cholesterol concentrations compared to control. The discrepancy may be due to the

method used to assess cholesterol absorption efficiency. The single stable isotope single

tracer method detects only the relative efficiency of cholesterol absorption rather than the 65 absolute amount that depends on endogenous biliary cholesterol secretion (Ostlund et al.,

2002). Whether, the frequency of intake of plant sterols affects the absolute amount of cholesterol absorption and/or bile acid excretion was not assessed. Additionally, the observed reduction in efficiency of dietary cholesterol absorption may not always reflect the magnitude of reduction in circulating cholesterol. This explanation is supported by a finding from a previous work. Jones et al. (Jones et al., 2000) reported no difference in the cholesterol absorption coefficient between plant sterol and plant stanol-containing diets although the decline in LDL-cholesterol concentrations was greater at the end of plant sterol than plant stanol phase. Gylling et al. (Gylling et al., 1997) have shown that the sitostanol-induced change in LDL-cholesterol was significantly associated with the change in cholesterol absorption efficiency (r =0.443). Likewise, Jones et al. (Jones et al.,

2000) reported an r =0.530 between plasma total cholesterol concentrations and cholesterol absorption coefficient with plant sterol treatment. In the present study the changes in LDL-cholesterol concentration tended to be associated with changes in AUC of 13C cholesterol when plant sterols were consumed 3 times a day. The proportion of the total variation in circulating total and LDL-cholesterol concentrations that is explained by cholesterol absorption efficiency in the present study and prior studies (Gylling et al.,

1997; Jones et al., 2000) ranges from 18-28%. These findings suggest that either the methods used to measure absorption are insensitive to changes in cholesterol absorption efficiency or plant sterols/stanols reduce circulating levels by other means.

Nevertheless, the results of this study have implications for the mechanism of action of plant sterols as cholesterol-lowering agents. In contrast to the hypothesis that plant sterols/stanols are retained in the enterocyte, it does not appear that a single dose 66 consumed early in the morning is as efficacious as divided doses. Recent work suggests that the AUC (24-96 h) of RBCs "C-cholesterol and E -max values when single dose of plant sterols was consumed with dinner were similar to control values (unpublished data,

AbuMweis S et al.). Cholesterol absorption is a gradual process as labeled cholesterol usually peaks after 48 h of administration (Bosner et al., 1993), and the ingested dietary cholesterol is secreted by the small intestine in chylomicrons into the circulation during >

3 subsequent postprandial periods in human (Beaumier-Gallon et al., 2001). Thus, it appears that plant sterols are not retained in the intestinal lumen or enterocytes and do not prevent the absorption of pre-taken dietary cholesterol during subsequent periods.

In summary, results from this study indicate that dosing frequency of plant sterols may affect their action as cholesterol-lowering agents. Consumption of 1.8 g/d of plant sterols distributed over the day was more effective in lowering LDL-cholesterol than the same dose once a day with breakfast, in spite of a reduction in cholesterol absorption efficiency. Moreover, cholesterol FSR increased when plant sterols were consumed in divided doses which may be attributed to feedback up-regulation of cholesterol FSR in response to reduced sterol absorption.

4.6 ACKNOWLEDGEMENTS

We wish to thank Unilever Research for providing the sterol-enriched and control margarines in-kind. We also thank the study subjects for their enthusiastic participation. 4.7 FIGURE LEGENDS

Figure 4.1. Correlation between changes in the area under the curve (AUC (per mil x h))

1 T of C-enrichment in RBCs- cholesterol and the changes in LDL-cholesterol concentrations (mmol/L) relative to control, at the end plant sterol single-BF (A) and 3 times/day phases (B). Data were analyzed using a Pearson correlation.

Figure 4.2. Enrichment of 13C in RBCs- cholesterol at the end of control phase, and plant sterol single-BF and 3 times/d phases. Values are expressed as mean ± standard error.

Areas under the curve with different superscripts are significantly different (P < 0.05). 68

Table 4.1. Nutrient and plant sterol composition of control and sterol-enriched margarine }

Component (per 100 g) Control Sterol-enriched

margarine margarine

Energy (Kcal) 327 327

Total fat (g) 35 35

Saturated fatty acids (g) 8.2 8.2

Polyunsaturated fatty acids (g) 18.0 18.0

Monounsaturated fatty acids•(g ) 8.3 8.3

Trans fatty acids (g) 0.5 0.5

Protein (g) 0.1 0.1

Carbohydrate (g) 3.2 3.2

Fiber (g) 0.3 0.3

Total plant sterols (g) 0.01 8.0

Plant sterol composition (% w/w)

Brassicasterol - 2.9

Campesterol - 22.2

Campestanol - 0.6

Stigmasterol - 9.6

p-Sitosterol - 58.1

P-Sitostanol - 3.6

D5-Avenasterol - 0.9

Other - 1.9

As provided by the margarine supplier. 69

Table 4.2. Characteristics of the subjects at the time of screening.1

Variable

Age, yr 55 ± 8

Weight, kg 80 ±15

BMI, kg/m2 26 ± 4

Lipids, mmol/L

Total cholesterol 5.10 ±0.52

LDL- cholesterol 3.44 ± 0.41

HDL- cholesterol 1.11 ± 0.23

Triacylglycerol 1.16 ± 0.45

All values are mean ± SD, n=19 (16 males and 3 females) 70

Table 4.3. Lipid and lipoprotein concentrations at the end of each experimental diet

phase.1

Parameter Control Single-BF 3 times/d P-value Total Cholesterol (mmol/L) 5.29 ±0.72 5.14 ±0.68 5.05 ±0.71 0.0637

HDL- Cholesterol (mmol/L) 1.25±0.21 1.24 ±0.21 1.25 ±0.22 0.9145 LDL-Cholesterol (mmol/L)2 3.43±0.62a 3.30±0.65a,b 3.22±0.58b 0.0452

Total Cholesterol/ HDL3 4.32±0.80a 4.22 ±0.77a'b 4.13±0.79b 0.0380 Triacylglycerol (mmol/L) 1.32 ±0.66 1.30 ±0.64 1.26 ±0.61 0.5024 All values are mean ± SD, n= 19; weight change was entered as a covariant. Single-BF,

plant sterols were consumed only with breakfast; 3 times/d, plant sterol enriched

margarine was divided equally among the 3 meals, breakfast, lunch and dinner. Within a

row, values with different superscripts are significantly different P-values for pairwise

comparisons adjusted by Tukey's test are as follows: P= 0.2616 control vs. single-BF;

P= 0.0371 control vs. 3 times/d; P= 0.5860 single-BF vs. 3 times/d3 P-values for

pairwise comparisons adjusted by Tukey's test are as follows: P= 0.3729 control vs.

single-BF; P= 0.0292 control vs. 3 times/d; P= 0.3874 single-BF vs. 3 times/d 71

Table 4.4. Cholesterol kinetics as measured from free cholesterol from RBCs in

study subjects during each of dietary phase.1 __

Parameter Control Single-BF 3 times/d value

Cholesterol absorption

a b b AUC24-96h(per 398±79 242±61 253±58 <0.0001 mil x h)

E-max (per mil) 6.3±1.3a 3.9±1.0b 4.1 ± 1.0b <0.0001

Cholesterol synthesis

Fractional 0.083 ±0.028a 0.083 ± 0.025 a,b 0.091 ± 0.022b 0.0219 synthesis rate

(pool/d)2'3

Absolute 0.749 ±0.316a 0.733 ±0.219a,b 0.810 ± 0.226b 0.0266 synthesis rate (g/d) 2'4

All values are mean ± SD, n=19. Single-BF, plant sterols were consumed only

with breakfast; 3 times/d, plant sterol enriched margarine was divided equally

among the 3 meals, breakfast, lunch and dinner; AUC 24-96h, area under the curve of

13C enrichment of RBCs cholesterol during a 24-96 h period following the oral dose

of 13C -cholesterol; E-max; maximum 13C -cholesterol enrichment in RBCs. Within

a row, values with different superscripts are significantly different. 2 All values

were log-transformed prior to statistical analysis; arithmetic means are reported.

Weight change was entered as a covariant 3 P-values for pairwise comparisons

adjusted by Tukey's test are as follows: P= 0.9097 control vs. single-BF; P= 0.0281 72 control vs. 3 times/d; P= 0.0764 single-BF vs. 3 times/d 4 F-values for pairwise comparisons adjusted by Tukey's test are as follows: P= 0.9109 control vs. single-

BF; P= 0.0266 control vs. 3 times/d; P= 0.0718 single-BF vs. 3 times/d 73

Figure 4.1.

A) Single-BF phase y = 0.001x+0.0335 R2 = 0.029 1.00 0.50 .E ell 0.00 » -S ""- S -0.50 C 0 =J O -1.00

B) 3 times/d phase y = 0.0024x+0.1475 R2 = 0.1898 2 o 1.00 2 a>

$ iti v 0.50 TO

ho i 0 o L_ "tp- 0.00 i Q | c "o 8-0.50 E

l( m -1.00 c a> ro x: _>0 O -1.50 -250 -200 -150 -100 -50 0 Change in AUC (per mil x h) ofr 13,C-enrichment , relative to control 74

Figure 4.2.

Control

6.0 - ^^^^<^lpT3»«~____^ o "L-^-"' £ 5.0 - i a 4 f| 3.'°0" - - J 2.0 - . / 1.0

0.0 z_.) . 24 48 72 96 ( Time (h)

Single-BF phase

7.0

6.0

E 5.0

«• 4.0

| 3-0 H B ^ 2.0 1.0

0.0 J^ 24 48 72 96 Time (h)

3 times/d phase

7.0

6.0

96 75

BRIDGE 3.

The findings of Manuscript 3 indicate that dosing frequency of plant sterols may be a critical factor that determines plant sterol efficacy as cholesterol- lowering agents. About

1.8g/ d of plant sterols distributed equally between breakfast, lunch and dinner reduced

LDL-cholesterol levels compared to control. However, the same dose size consumed as a single dose with breakfast did not reduce LDL-cholesterol levels. The importance of plant sterols dosing frequency was demonstrated through: (i) reduction in LDL-cholesterol when plant sterols were consumed at each meal but not as a single morning dose; relative to control, and (ii) an increase in cholesterol synthesis when plant sterols were consumed with each meal, which could be attributable to feedback up-regulation of cholesterol synthesis in response to reduced cholesterol absorption. Since, diurnal variation is known to exist in human cholesterol metabolism; the time of intake may also affect the efficacy of single dose of plant sterols. Given that plant sterols are not only incorporated in vegetable oil spread but also in a wide variety of products which include breakfast products such as milk, yoghurt and cereal bars, it is possible that a single dose of plant sterols taken in the morning may not lead to optimal cholesterol reduction. Therefore,

Manuscript 4 was undertaken to compare efficacy of new breakfast plant sterol enriched yoghurt consumed with the morning or the evening meals, in lowering blood cholesterol of hypercholesterolemic individuals. 76

CHAPTER 5. Manuscript 4 (Study # 3).

EFFICACY OF PLANT STEROL - CONTAINING YOGHURT CONSUMED

ONCE A DAY WITH BREAKFAST OR DINNER IN MANAGEMENT OF

HYPERCHOLESTEROLEMIA IN HUMANS

1 1 9 Suhad S. AbuMweis , Iwona Rudkowska , Peter J.H. Jones ,

Author affiliation:

'School of Dietetics and Human Nutrition

McGill University, Ste-Anne-de-Bellevue, Montreal, Quebec, Canada

2 Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba,

Winnipeg, Manitoba, Canada

Corresponding author:

Peter JH Jones, PhD

Richardson Centre for Functional Foods and Nutraceuticals

University of Manitoba, Smartpark

196 Innovation Drive, Winnipeg, Manitoba, R3T 6C5

Phone: (204) 474-8883, Fax: (204) 474-7552

Email: [email protected] 77

5.1 ABSTRACT

Studies on the efficacy of single dose of plant sterols have been inconsistent in terms of magnitude of reduction in LDL levels. Thus, the objective of this study was to evaluate

effects of new plant sterol-containing yoghurt on plasma LDL-cholesterol levels when

consumed with meal as a single dose in the morning or in the evening. In a randomized,

placebo-controlled, crossover, feeding trial, 26 subjects received two bottles each of 100

ml of yoghurt for 4 weeks during each of the following 3 interventions including: (i)

placebo yoghurt at breakfast and dinner (control); (ii) active yoghurt at breakfast and

placebo yoghurt at dinner (single morning dose); or (iii) placebo yoghurt at breakfast and

active yoghurt at dinner (single evening dose). The active yoghurt contained 1.6 g of plant

sterol/100 ml as sterol ester. Overall, there was no significant difference in LDL-

cholesterol levels across interventions at endpoints (3.51± 0.66, 3.39 ± 0.77, 3.43 ± 0.59

mrnol/L, control, single morning and single evening, respectively). However, in the

group of subjects with a low basal cholesterol absorption efficiency, the LDL-cholesterol

levels decreased (P = 0.0008) by 8.5 % and 6.8 %, relative to control, due to the

consumption of single morning and single evening dose of plant sterols, respectively. In

conclusion, the consumption of low-fat yoghurt containing 1.6 g/d plant sterol did not

lower LDL-cholesterol when it was consumed once a day in the morning or the evening,

but there was wide interindividual variation in LDL responses to the intervention. A

substantial reduction in LDL-cholesterol was only observed in subjects with low

cholesterol absorption efficiency irrespective of the time of intake suggesting that some

subjects may benefit from the consumption of single dose of plant sterols.

Keywords: cholesterol, plant sterol ester, yoghurt, single dose 78

5.2 INTRODUCTION

Diet therapy is a cornerstone strategy for lowering low density lipoprotein (LDL) cholesterol levels (Cleeman et al., 2001). Plant sterols and stanols, the hydrogenated forms of plant sterols, are among natural health ingredients that are suggested by many experts to reduce circulating levels of cholesterol (Cleeman et al., 2001; Fletcher et al.,

2005). Plant sterols/stanols have been incorporated into a variety of food products such as margarine (Gylling and Miettinen, 1994; Jones et al., 2000; Mussner et al., 2002; Ntanios,

2001; Plat and Mensink, 2000), milk (Noakes et al., 2005; Thomsen et al., 2004), and yoghurt (Doornbos et al., 2006; Hyun et al., 2005; Mensink et al., 2002; Noakes et al.,

2005; Volpe et al., 2001).

Plant sterols/stanols reduce cholesterol levels by reducing cholesterol absorption (Gylling

and Miettinen, 1994; Jones et al., 2000; Normen et al, 2000; Vanstone et al., 2002). The

exact mechanism by which plant sterol reduce cholesterol absorption has not been fully

elucidated. The conventional proposed mechanism of action involves plant sterols

competing with cholesterol for incorporation into the micelles (Heinemann et al., 1991).

This suggests that plant sterols should be consumed at each meal to achieve full potential

cholesterol-lowering effect (Plat et al., 2000). Actually, the majority of studies

demonstrating the cholesterol- lowering action of plant sterols/stanols gave two

(Hallikainen et al., 2000a; Maki et al., 2001; Ntanios, 2001; Weststrate and Meijer, 1998)

or three (Jones et al., 2000; Mensink et al., 2002; Plat and Mensink, 2000; Vanstone et al.,

2002) servings per day of plant sterol/stanol-containing products.

Nevertheless, it has been suggested that the consumption of single dose of plant sterols

will increase consumers' compliance to various products (Plat et al., 2000). Therefore, a 79 number of studies have been carried supplementing plant sterols as single daily dose. The results have been inconsistent with the placebo adjusted reduction in LDL levels ranged

from 0.05 to 0.62 mmol/L (AbuMweis et al., 2006b; Doornbos et al., 2006; Hyun et al.,

2005; Matvienko et al., 2002; Pineda et al., 2005; Plat et al., 2000). This wide variation

could be explained by differences in type of administrated plant sterols and food matrix,

time of day of intake due to diurnal variation, known to exist in both cholesterol (Jones

and Schoeller, 1990) and bile acid synthesis (Galman et al., 2005), consumption of single

dose of plant sterols with and without a meal, and subjects' genetic makeup and their

basal cholesterol absorption efficiency. For example, plant sterol enriched margarine

reduced cholesterol levels when was consumed as single dose with lunch (Plat et al.,

2000). However, when plant sterol enriched margarine was consumed with breakfast, no

reduction in cholesterol levels was observed (AbuMweis et al., 2006b). In another study,

intake of the single dose of plant sterols provided in yoghurt drinks with lunch resulted in

a larger decrease in LDL-cholesterol levels than the intake of same dose of plant sterols

30 min before breakfast. This lower efficacy was attributed to the fact that morning

yoghurt was ingested in a fasted state (Doornbos et al., 2006). On the other hand, the

efficacy of plant sterols consumed in the morning may be influenced by diurnal variation

in cholesterol metabolism. The efficacy of novel plant sterol enriched yoghurt consumed

as single dose with meal and at different time of the day remains to be examined.

Thus, the objective of this study was to evaluate the effects of a yoghurt drink

supplemented with plant sterols on plasma LDL-cholesterol levels when consumed with

breakfast or dinner meal. The null hypothesis of the present study was that the timing of

intake of single dose of plant sterols consumed with meal does not affect the magnitude

of reduction in circulating LDL-cholesterol levels. 80

5.3 SUBJECTS AND METHODS

5.3.1 Subject selection

Thirty males and postmenopausal females subjects aged 40-80 years were recruited using flyers and newspaper advertisements in the West Island of Montreal. The study consisted of three sequential stages: first screening (day -28); second screening (day -14) and intervention stage consisted of 3 phases. Subjects were selected based on the following inclusion criteria: LDL-cholesterol level > 3.0 mmol/L, Plasma concentrations of P-

sitosterol and campesterol <10,000 ng/mL, body mass index between 22 to 32 kg/m2, no use of medications likely to affect lipid metabolism for at least the previous 3 months.

Exclusion criteria were presence of thyroid disease, diabetes mellitus, kidney disease, liver disease, or vascular disease, high blood pressure (95140 mmHg and/or

4590 mmHg), triacylglycerols > 2.8 mmol/L, history of metabolic or gastrointestinal disease with the exception of appendicectomy, consumption of large amounts of alcohol, receiving systemic treatment likely to interfere with evaluation of the study parameters, presenting allergy or hypersensitivity to milk proteins, or refusing to consume them, female subjects likely to change her hormonal substitutive treatment and currently in an exclusion period following participation in another clinical trial and smokers. The study was approved by the Institutional Research Ethics Board for the

School of Medicine at McGill University, Montreal, Canada. All subjects signed a consent form, prior to the commencement of the study.

5.3.2 Study design and protocol

A randomized, crossover, placebo-controlled clinical study was carried out at Mary Emily

Clinical Nutrition Research Unit (MECNRU) at the Macdonald Campus of McGill 81

University. After screening, all eligible subjects were enrolled into the study. The study consisted of 3 phases of 30 days during which subjects consumed a fixed composition precisely controlled weight-maintaining diet. Diets were planned to contain 55% of energy from carbohydrates, 15% from protein and 30% from fat. At the end of each phase, a washout period of 4 weeks was followed during which the subjects consumed their habitual diets.

The study product was a commercially available yoghurt drink (Table 5.1). Subjects received 2 bottles of 100 ml of yoghurt per day for each of the following treatments in a random order (i) placebo yoghurt provided at breakfast and dinner (control phase), (ii)

active yoghurt provided with breakfast and placebo yoghurt provided at dinner (single morning dose phase), and (iii) placebo yoghurt provided at breakfast and active yoghurt

provided at dinner (single evening dose phase). Active yoghurt contained 1.6 g of plant

sterols/100 ml. Plant sterols were extracted from tall oil and esterified to rapeseed oil.

Subjects consumed a prepared breakfast meal as part of the control diet as well as meals

containing the plant sterol rich yoghurt in the MECNRU under supervision. The two other

meals were prepared and packed for consumption at work or at home. Subjects were

instructed to consume the yoghurts during the latter half of the meal and to consume only

the prepared meals and not to consume alcohol or caffeinated beverages. Subjects were

instructed to shake yoghurt bottles before consumption. In addition, subjects were

encouraged to keep their lifestyle, such as level of physical activity, constant throughout

the study. Subjects weighed themselves every day. 82

During the study subjects were asked to report any important deviation in lifestyle as well as current illnesses and use of concomitant medication. All reported or observed clinical adverse effects and clinically significant laboratory adverse effects were recorded. At the start and end of each treatment phase, fasting blood samples were taken for measurement of lipid profile.

5.3.3 Analyses

Fasting blood samples were centrifuged for 20 min at 520 X g and the separated aliquots were kept frozen until analysis. Samples obtained from second screening were used to analyze plasma plant sterols. Briefly, plasma samples were saponified with methanolic

KOH solution. Sterols were then extracted twice with petroleum ether. Extracted sterols were derivatized using tri-methylsylation procedures (Lutjohann et al., 1993).

Plasma samples from days 1, 29 and 30 were analyzed for total cholesterol, high density lipoprotein (HDL) cholesterol and triacylglycerol levels using an automated analyzer

(Dade Behring Inc.). Low-density lipoprotein cholesterol was calculated from the total and HDL- cholesterol and triacylglycerol measurements using the Friedewald equation

(Friedewald et al., 1972). All samples from the same subjects were analyzed in the same batch. The daily coefficients of variation for the individual lipids were all < 6%.

The sample size calculation was determined to detect an anticipated difference in LDL- cholesterol levels due to plant sterol treatment of 12% (0.54 mmol/L) using an standard deviation (SD) of 0.732 mmol/L (Nigon et al., 2001). The alpha and power were 0.05 and

0.7, respectively. A sample size of 28 subjects was calculated, with a target of 24 83

subjects, taking into account the block size and the estimated premature withdrawal rate of subjects of 10%.

Statistical analysis was carried out using SAS (version 8.0; SAS Institute INC, Cary, NC)

and was conducted on the per-protocol population, defined as subjects who completed the

3 phases of the study. Data are expressed as mean ± SD. The principle statistical model that was used is the analysis of variance taking into account subject as a random effect,

study treatment effect, period effect and carryover effect (period-treatment interaction).

The residuals of every outcome were checked for normality using Shapiro-Wilk test. If the normality assumption was violated for a variable, then a non-parametric analysis was used. In the event of statistical significant treatment effect (P-value < 0.05) pairwise comparisons were performed using Tukey's test.

5.4 RESULTS

Thirty subjects commenced the study with 26 subjects, 16 men and 10 women, completing the 3 phases of the study. Three subjects dropped out for personal reasons and one subject dropped out for an adverse event that was not related to the study product.

Baseline characteristics of the subjects as obtained from the second screening (day -14) are shown in Table 5.2. At the study entry subjects mean age was 63.4 ± 6.6 yr, and mean

BMI was 26.0 ± 2.4 kg/m . According to the Adult Treatment Panel III, subjects mean

LDL-cholesterol level was classified as high level (Cleeman et al, 2001). There were no significant mean group weight changes across any of the 3 treatment phases. 84

Mean plasma lipid levels are shown in Table 5.3. There was no significant difference in endpoint levels of total and LDL-cholesterol among study phases. Total cholesterol levels at endpoint were 5.53 ± 0.93, 5.32 ± 0.95 and 5.37 ± 0.79 mmol/L with control, single morning dose of plant sterols, and single evening dose of plant sterols treatments, respectively. Endpoint LDL-cholesterol levels after control, single morning dose of plant sterols, and single evening dose of plant sterol consumption were 3.51 ± 0.66, 3.39 ± 0.77 and 3.43 ± 0.59 mmol/L, respectively. Endpoint HDL-cholesterol and triacylglycerol levels were also not significantly different between study treatments. Consequently, endpoint ratios of total cholesterol to HDL-cholesterol were not different among study treatments. A periodic effect on some parameters was detected (Table 5.3) and a carryover effect, i.e. period by treatment interaction, was only detected on triacylglycerol start values, change from start and % change from start values.

Although overall there was no significant reduction in plasma LDL-cholesterol concentrations compared to the control diet phase; a single dose of plant sterols reduced blood cholesterol for some individuals (Figure 5.1). The plant sterol treatment resulted in large individual variation in responsiveness of LDL-cholesterol levels. The differences in

LDL-cholesterol between the plant sterol treatment and placebo treatment ranged from -

1.49 tol.06 mmol/L, and from -1.33 to 1.17 mmol/L for the single morning and single evening phases, respectively. Thus, subgroup analyses were conducted to test the effect of baseline capacity of cholesterol absorption on LDL-cholesterol responsiveness to treatment with plant sterols. We used screening values of campesterol as an indicator of cholesterol absorption efficiency. Previously it was shown that circulating levels of campesterol correlates positively with the fractional absorption of cholesterol (Tilvis and 85

Miettinen, 1986). Subjects were divided into 2 groups according to basal campesterol levels: one group with highest levels (n=13; range 10.7-26.0 umol/L) and the other group with lowest levels (n=13; range 6.0-10.6 umol/L). Subgroup analysis according to subjects' baseline characteristics have been used elsewhere with sample size close to ours or even smaller (Hallikainen et al., 2000b; Jones et al., 1998b; Thuluva et al., 2005).

Thus, the sample size per group in this study may be enough to detect a statistically significant effect. In the group with the low levels of basal campesterol, the LDL- cholesterol levels decreased by 8.5 % and 6.8 % after the consumption of single morning and single evening dose of plant sterols relative to control, respectively (Table 5.4).

However, in the group with high levels of basal campesterol, LDL-cholesterol levels did not differ between study phases. Notably, 8/10 and 9/12 of non responders during single morning and single evening phases, respectively, were classified in the group with high basal cholesterol absorption efficiency. Additionally, 8 subjects were classified as non responders to both phases of plant sterol intervention.

5.5 DISCUSSION

This is the first study to look into the cholesterol- lowering effect of plant sterol- containing yoghurt consumed as single daily dose with meal and at different point of time. The major finding was that consumption of low-fat yoghurt containing 1.6 g/d of plant sterols once a day with breakfast or with dinner for 4 weeks did not lower LDL levels. The absence of lowering effect of single dose of plant sterols consumed at different times of the day suggests that the frequency of intake and not the time of intake may be the influential factor for the cholesterol- lowering efficacy of plant sterols. 86

Nevertheless, the consumption of single dose of plant sterols was particularly beneficial in subjects with low baseline cholesterol absorption efficiency suggesting that the plant sterols provided in the yoghurt drink were bioavailable to compete with cholesterol for absorption. As a result, the absence of overall cholesterol- lowering action of plant

sterols in the present study could not be attributed to the food matrix used.

Overall, the lack of effect found in this study with a single-dose of plant sterols is

inconsistent with some recent studies. The discrepancy between the present study and past findings may be a result of different approaches used to present the data as well as of

different study protocols implemented.

For example, some previous studies did not directly compare LDL levels between the

active and the placebo groups and only reported the changes from baseline (Hyun et al.,

2005; Pineda et al., 2005), and therefore we can not compare directly our results to these

two studies. Another potential explanation could be related to the dose size of plant

sterols consumed. In a study that used a larger dose size of plant sterols than here, plant

sterol ester in yoghurt drink lowered LDL-cholesterol levels by 6% and 9.4% when taken

as single dose before breakfast or with lunch (Doornbos et al., 2006). In the study by

Doornnbos et al. (Doornbos et al., 2006) the yoghurt contained 3 g of plant sterols while

in this study the yoghurt contained 1.6 g of plant sterols, i.e. half the dose given by

Doornnbos et al. (Doornbos et al., 2006). Intake of single dose of plant sterols with lunch

resulted in a larger decrease in LDL-cholesterol levels than the intake of same dose of

plant sterols before breakfast which was contributed to the fact that morning yoghurt was

ingested in a fasted state (Doornbos et al., 2006). Although in the present study the

morning dose of plant sterols was consumed with the second half of breakfast, no 87 reduction in LDL-cholesterol levels was achieved. Other studies have used a dose size of plant sterols similar to the one used in the present study or a smaller one incorporated into yoghurt has been reported to reduce LDL-cholesterol levels relative to control (Korpela et

al., 2006) or from baseline (Volpe et al., 2001); however, the frequency and or time of intake of plant sterol were not reported. The gastric emptying rate of the liquid

component of a meal is faster than the solid component, with this rate being slowed by

simultaneous ingestion of solids (Fisher et al., 1982). Therefore, it is possible that plant

sterols provided in liquid foods have less time to mix with other gastrointestinal contents

including micelles in the small intestine (Jones et al., 2003), and thus a large dose size

may be indicated. Most of the dose response studies were carried out on plant sterols and

stanols incorporated into some form of fat spread (Christiansen et al., 2001; Hallikainen

et al., 2000b; Hendriks et al., 1999; Maki et al., 2001) and consumed throughout the day

and thus extrapolation of these results to plant sterols incorporated into liquid matrix and

consumed once a day is not valid. There is one study that looked at the effect of plant

sterols in a liquid matrix in a dose-dependent manner; however in that study plant sterols

were consumed in 2 servings (Thomsen et al., 2004). It should be noted that the yoghurt

tested in this study is a commercially available product in Europe and USA that is being

promoted to be consumed once a day. Apparently, the dose size of plant sterol may be a

critical issue when plant sterol-containing yoghurt to be consumed once a day which calls

for dose response studies.

In the present study, there was a large individual variation in responsiveness of plasma

LDL-cholesterol to plant sterols. Indeed, 30% and 38% of the subjects were not responders to plant sterols consumed as a single dose in the morning or the evening, 88 respectively. Non responsiveness to plant sterols treatment, defined as no change or an increase in LDL-cholesterol levels after plant sterol treatment comparing to control, has been reported previously. Sierksma et al. (Sierksma et al., 1999) found that 37% of subjects who consumed 0.8g/d of plant sterols in margarine for 3 weeks did not show a reduction in LDL-cholesterol levels. Likewise, Jakulj et al. (Jakulj et al., 2005) demonstrated that after 4 weeks of plant sterol-enriched margarine, corresponding to 2 g/d of plant sterols, 33% of the subjects did not show reduced LDL-cholesterol levels with plant sterol intervention. In another study, 20% of subjects did not show a reduction in LDL- cholesterol levels after consumption of 1.2 g/d or 1.6 g/d of plant sterols in milk

(Thomsen et al., 2004). The plant sterol dose was divided in two servings consumed immediately after breakfast and lunch (Thomsen et al., 2004). Thus, it appears that factors including dose size, food matrix, and time of intake of plant sterols contribute to higher level of non-responsiveness of LDL-cholesterol levels to plant sterol treatments. Non- responsiveness of LDL-cholesterol levels to plant sterol consumption may be related to the aforementioned factors as well as genetic determinants of such a non- responsiveness which should be investigated in the future.

Although the examination of treatment effects in subgroup analysis is burdened with potential limitations, and the modest sample size limits statistical power and interpretation, the pattern of non-responsiveness observed here is worth further investigation. Thus, subgroup analysis offers an opportunity to evaluate trends in the data, which may further improve our understanding of the efficacy of single dose of plant sterols consumed daily and the causes of non-responsiveness. Potential determinant of such non-responsiveness is basal cholesterol absorption capacity of the individuals. 89

Previous studies have shown that individuals with high baseline absorption of cholesterol benefit more from plant sterol/stanol treatment than individuals with a lower baseline absorption (Gylling and Miettinen, 2002; Mussner et al., 2002). In opposition, subjects with high baseline synthesis of cholesterol benefit more from statin treatment than subjects with a lower baseline synthesis (O'Neill et al., 2001). Contrary to the observations in other studies, in the present study a single dose of plant sterols reduced

LDL-cholesterol levels in subjects with lower efficiency in baseline cholesterol absorption as measured indirectly by basal campesterol levels. A major dissimilarity between previous studies and the present study is the consumption frequency of plant sterols/stanols. Unlike the studies by Mussner et al. (Mussner et al., 2002) and Gyllling and Miettinen (Gylling and Miettinen, 2002) with plant sterols/stanols consumed in 2-3 times/d, plant sterols were consumed once/d in the present study. It may be taken from these results that individual with low cholesterol absorption, and therefore with high cholesterol synthesis, may benefit from a single dose of plant sterols. Thus, subjects with low efficiency of cholesterol absorption and therefore with high cholesterol synthesis may also be candidates for a combined treatment with statin and plant sterols, giving that the best dose regime is applied. Further investigation is needed to determine why a single dose of plant sterols was effective in subjects with low cholesterol absorption efficiency.

Alternatively, it is likely that single dose of plant sterols is not enough to reduce LDL- cholesterol levels in subjects with high cholesterol absorption efficiency. 90

In conclusion, there was no overall reduction in LDL-cholesterol levels when a 1.6 g/d of single dose of plant sterols provided in yoghurt was consumed with breakfast or dinner.

The efficacy of single dose of plant sterols in yoghurt drink could be improved by increasing the dose size or the frequency of intake. Nevertheless, a reduction in LDL- cholesterol was observed in subjects with low cholesterol absorption efficiency irrespective of time of intake. Since single dose of plant sterols reduced LDL-cholesterol levels in some subjects, the results from this investigation indicate that some individuals may benefit from consumption of single dose of plant sterols.

5.6 ACKNOWLEDGEMENT

We wish to thank Kristen Rindress, the clinical study coordinator, and Catherine

Vanstone for overseeing the study. We also thank Adrielle Houwelling for analyzing the plasma plant sterols levels and Dr. William Parsons for monitoring the participants during the study. We deeply thank the participants as well as MECNRU staff for preparing and serving the meals for the study. The study was supported by Danone Vitapole. 91

5.7 FIGURE LEGEND

Figure 5.1. Individual differences in LDL-cholesterol levels between the end of the 4- week treatment with plant sterols enriched yoghurt consumed in the morning or in the evening and at the end of the 4-week control phase 92

Table 5.1. Nutritional composition of the placebo and plant sterol-containing

yoghurt.

Component (per 100 ml) Placebo yoghurt Active yoghurt

Energy (Kcal) 65 65

Carbohydrates (%) 10 10

Protein (%) 3.2 3.2

Lipids (ro)1 1.4 1.4

Sterols (as free sterols)(g) - 1.6

P-sitosterol 75

(%wt/wt of sterols)

Campesterol 8.4

(%wt/wt of sterols)

Including rapeseed oil 93

Table 5.2. Baseline characteristics of the subjects.

Variable All Men Women

(n=26) (n=16) (n=10)

Age (yr) 58.6 ±9.4 55.6 ±9.8 63.4 ±6.6

Weight (kg) 79.2 ± 14.2 84.8 ±13.8 70.4 ±10.2

BMI (kg/m2) 26.6 ± 2.7 26.9 ±2.8 26.0 ±2.4

Blood pressure (mm Hg)

Systolic 130 ±14 130 ±12 129 ±19

Diastolic 81±7 83 ±6 79 ±9

Heart rate (beats/min) 71 ±7 72 ±7 71±7

Lipids (mmol/L)

Total cholesterol 6.23 ± 0.82 6.02 ± 0.88 6.57 ±0.59

LDL-cholesterol 4.20 ±0.69 4.18 ±0.77 4.24 ± 0.57

HDL-cholesterol 1.25 ±0.43 1.03 ±0.18 1.61 ±0.49

Triacylglycerol 1.71 ± 0.65 1.78 ± 0.58 1.60 ± 0.76

Plasma plant sterols (umol/L)

Campesterol 12.5 ±5.3 12.1 ±5.6 13.1 ±5.1

P-Sitosterol 6.8 ±2.8 6.6 ±2.8 7.2 ±2.9

Values are mean ±SD 94

Table 5.3. Effect of study treatments on lipid profile.

Parameter (mmol/L) Control Single Morning Single Evening P-value

Total Cholesterol Start 5.74±0.85 5.79±0.97 5.82±1.04 0.9329 End1 5.53±0.93 5.32±0.95 5.37±0.79 0.1129 Change from start -0.22±0.67 -0.47±0.65 -0.45±0.56 0.2588 % Change from start -3.41±13.09 -7.79±11.46 -6.74±9.93 0.3357

LDL- Cholesterol Start 3.61±0.76 3.67±0.73 3.67±0.79 0.8885 End1'2 3.51±0.66 3.39±0.77 3.43±0.59 0.3077 Change from start -0.10±0.59 -0.28±0.55 -0.23±0.36 0.3795 % Change from start -0.87±18.52 -7.21±15.52 -5.02±10.18 0.2964

HDL-Cholesterol Start1 1.37±0.44 1.41±0.44 1.41±0.51 0.2171 End 1.27±0.40 1.23±0.36 1.23±0.36 0.0802 Change from start -0.11±0.19 -0.19±0.18 -0.18±0.22 0.0813 % Change from start1 -6.75±13.14 -12.13±10.75 -10.39±13.43 0.1424

Triacylglycerol Start2'3 1.87±1.09 1.60±0.58 1.67±0.85 0.2735 End 1.66±0.57 1.58±0.62 1.56±0.55 0.7672 Change from start' -0.21±0.97 -0.02±0.41 -0.10±0.78 0.5905 % Change from start 1.57±34.49 0.12±24.76 4.34±35.92 0.6173

TotahHDL Cholesterol Start1 4.44±1.02 4.31±0.93 4.39±1.05 0.2859 End 4.59±1.02 4.52±1.00 4.56±0.97 0.9431 Change from start 0.16±0.48 0.22±0.48 0.17±0.36 0.8132 % Change from start 4.24±10.89 5.56±11.37 5.00±9.30 0.8182 Values are mean ± SD (n=26) 1 Significant period effect, 2 Analyzed by non-parametric test,3 Significant period*treatment effect 95

Table 5.4. LDL-cholesterol levels after 4 weeks of study treatments classified

according to basal cholesterol absorption efficiency measured with basal

campesterol levels.

Single Control Single Morning Evening P-value1

Low 3.65±0.52a 3.34±0.78b 3.40±0.66b 0.0008 (n=13)

High 3.38 ±0.78 3.44 ±0.79 3.47 ±0.53 0.9822 (n=13)

Values are mean ± SD (mmol/L).

1 Analyzed by non-parametric test. Means within a row with different superscript letters are significantly different. Difference in LDL- c cholesterol relative to control (mmol/L)

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BRIDGE 4.

The previous manuscript found that the consumption of 1.6 g of plant sterol in yoghurt once a day with breakfast or dinner did not affect LDL-cholesterol levels. However, a large individual variation in LDL-cholesterol levels responsiveness to a single dose was observed. The lack of efficacy of plant sterols has also been reported in previous studies.

Moreover, the degree of placebo-adjusted reduction in LDL-cholesterol levels caused by plant sterols differed widely between studies, ranging from 5% to 15%. The two review articles included in this thesis show that clinical investigators have used a variety of food as carriers for plant sterols/stanols; differing concentrations of plant sterols/stanols consumed at differing frequency and time of the day; and subjects with different baseline characteristics. The next chapter of this thesis attempted to combine the results of multiple studies with different sample size to enhance the precision of the estimates of the effect of plant sterols on changes in LDL-cholesterol levels and to explore potential causes of heterogeneity in effect size between studies. Thus, the next chapter presents a systematic meta-analysis that quantifies the magnitude of the effect of plant sterols on

LDL levels and that answers questions concerning the effect of differences in studies on the efficacy of plant sterols as cholesterol-lowering agents. 98

CHAPTER 6. Manuscript 5. In preparation for submitting to the British Medical

Journal

PLANT STEROLS/STANOLS AS CHOLESTEROL- LOWERING AGENTS: A

META-ANALYSIS OF RANDOMIZED CONTROLLED TRIALS

Suhad S. AbuMweis1, Roula Barake *and Peter J.H. Jones1'2

Author affiliation:

School of Dietetics and Human Nutrition

McGill University, Ste-Anne-de-Bellevue, Montreal, Quebec, Canada

2 Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba,

Canada

Correspondence and reprint requests:

Peter J.H. Jones, Ph.D,

Richardson Centre for Functional Foods and Nutraceuticals

University of Manitoba, Smartpark

196 Innovation Drive, Winnipeg, Manitoba, R3T 6C5

Phone: (204) 474-8883, Fax: (204) 474-7552

Email: [email protected] 99

6.1 ABSTRACT

The consumption of plant sterols/stanols has been reported to reduce low density lipoprotein (LDL) cholesterol concentrations by 5 to 15%. Thus, this systematic meta­ analysis was conducted to more precisely quantify the effect of plant sterols on LDL- cholesterol concentrations in humans. Fifty nine eligible randomized clinical trials published from 1992 to 2006 were identified from five databases. All studies evaluated the hypocholesterolemic effects of 0.3 to 8.6 g plant sterols 16 incorporated into different food products and consumed by individuals with ranging LDL-cholesterol levels for greater than two weeks. Weighted mean effect sizes were calculated for net differences in

LDL-cholesterol concentrations using random effect model (RevMan 4.2). In the overall pooled estimate, plant sterol-containing products decreased LDL levels by 0.31 mmol/L

(95% CI, -0.35 to -0.27, P= <0.0001) compared with placebo. Between trial heterogeneity was evident (Chi test, P= <0.0001). The reductions in LDL levels were larger in individuals with high baseline LDL levels compared with those with normal to borderline baseline LDL levels. The reduction in LDL was greater when plant sterols were incorporated into fat spreads, mayonnaise and salad dressing, milk and yoghurt comparing with other food products. Plant sterols consumed a single morning dose did not reduce LDL levels. This is the first systematic meta-analysis to resolve the controversy seen to exist concerning the influences of food matrix and frequency and time of intake of plant sterols, as well as subjects' baseline characteristics, on efficacy of plant sterols. In conclusion, this meta-analysis shows that the reduction in LDL was greater in studies conducted on individuals with high baseline LDL levels, when plant sterols were incorporated into fat spreads, mayonnaise and salad dressing, milk and yoghurt comparing with other food products such as beverages and bakery products, and 100 when plant sterols were consumed in 2-3 portions/d or as single dose with lunch or main meal but not with breakfast. 101

6.2 INTRODUCTION

Incorporation of plant sterols/stanols in the diet has been suggested for blood cholesterol reduction (Fletcher et al., 2005; National Cholesterol Education Program Expert Panel,

2002). The consumption of plant sterols/stanols has been reported to reduce low density

lipoprotein (LDL) cholesterol levels by 5 to 15% (Berger et al., 2004). Reasons for such

large variations include different dosages and consumption frequency of plant

sterols/stanols, different formulations of plant sterols/stanols, and different types of

subjects.

A number of meta-analyses have been conducted to evaluate the efficacy of plant

sterols/stanols as cholesterol-lowering agent. The first meta-analysis was conducted in

2000 (Law, 2000) and looked at the cholesterol-lowering action of plant sterols/stanols

added to fat spreads mostly margarine. Another meta-analysis in 2003 (Katan et al., 2003) looked at the efficacy and safety of plant sterol/stanols enriched products. In addition, since 2003 a number of clinical trials have examined the efficacy of low fat foods containing plant sterols/stanols and observed substantially weaker effects. A recent meta­

analysis was published in 2006 (Moruisi et al., 2006), however, the objective of that meta-analysis was to investigate specifically the efficacy of plant sterols/stanols in lowering total and LDL-cholesterol levels of familial hypercholesterolemia subjects. The two main previous meta-analyses that were conducted on plant sterols were non-

systematic reviews (Katan et al., 2003; Law, 2000) and therefore did not describe how the reviewers searched, selected and evaluated the quality of studies. While systematic meta­ analysis includes a comprehensive search of the primary studies on a specific clinical question, selection of studies using clear eligibility criteria, critical evaluation of the 102 quality of studies, and generating results according to pre-specified method (Pai et al.,

2004). Meta-analysis is an unbiased tool to assess an intervention and may lead to resolution of controversy. Therefore, a systematic meta-analysis could be used to resolve controversy seen to exist regarding the efficacy of plant sterols as cholesterol-lowering agents. Results of research conducted thus far lead to conflicting conclusions concerning the influences of food matrix, frequency and time of intake, and subjects' baseline characteristics on cholesterol- lowering action of plant sterols.

Earlier studies that have tested the efficacy of plant sterols as cholesterol- lowering agents incorporated plant sterols/stanols into either regular or low-fat spreads. Since it appears counterintuitive to use a high-fat food product to deliver a cholesterol- lowering agent; clinical trials have been conducted to test the efficacy of plant sterols incorporated into low-fat products (St-Onge and Jones, 2003). A number of clinical trials have tested the efficacy of plant sterols incorporated into low-fat foods including low-fat milk (Noakes et al., 2005; Thomsen et al., 2004), low-fat yoghurt (Doornbos et al., 2006; Hyun et al.,

2005; Mensink et al., 2002; Noakes et al., 2005; Volpe et al., 2001), bakery products

(Quilez et al., 2003), orange juice (Devaraj et al., 2006; Devaraj et al., 2004), cereal bars

(Yoshida et al., 2006) and low- and nonfat beverages (Jones et al., 2003; Shin et al., 2005;

Spilburg et al., 2003). However, plant sterols incorporated into low-fat food did not always reduce blood cholesterol (Davidson et al., 2001; Jones et al., 2003; Yoshida et al.,

2006) and the same food matrix tested in different trials did not always result in same magnitude in LDL-cholesterol reduction. Plant sterol/stanol -enriched yoghurt and milk drinks have resulted in LDL-cholesterol reduction in the range of 5% to 14% in various clinical trials (AbuMweis et al., 2006a). One study compared side by side effect of plant 103 stanol consumed as an ester in a number of food matrices including bread, breakfast cereal, milk and yoghurt on plasma lipids (Clifton et al., 2004). The results of the study by Clifton et al. (Clifton et al., 2004) showed that plant stanol esters in low-fat milk were almost three times more effective than in bread and cereal in lowering plasma cholesterol levels. Whether all plant sterols/stanols enriched low-fat food matrices are efficacious as plant sterols/stanols enriched spread matrix in lowering blood cholesterol has not been studied thoroughly. It remains to be determined which food matrices are viable carriers to deliver an effective dose of plant sterols/stanols.

The optimal number of servings per day of plant sterol/stanol-containing products was addressed in only one study. Plat et al. (Plat et al., 2000) showed that 2.5 g of plant stanol consumed for 4 weeks once per day at lunch or divided over 3 meals, lowered LDL- cholesterol levels to a similar extent, about 10%. The intake of single dose of plant sterols/stanols enriched products is thought to increase consumers' compliance.

Thereafter, studies that have been conducted using a single dose of plant sterols/stanols consumed either at breakfast (AbuMweis et al., 2006b; Doornbos et al., 2006; Hyun et al.,

2005), or with lunch or the principal meal (Doornbos et al., 2006; Matvienko et al., 2002;

Pineda et al., 2005; Plat et al., 2000) yielded conflicting results. For example, plant stanol-enriched margarine reduced cholesterol levels when consumed throughout the day or as single dose with lunch (Plat et al., 2000). However, when plant sterol-enriched margarine was consumed with breakfast, no reduction in cholesterol levels was observed

(AbuMweis et al., 2006b). In another study, intake of the single dose of plant sterols provided in yoghurt drinks with lunch resulted in a larger decrease in LDL-cholesterol levels than the intake of same dose of plant sterols 30 min before breakfast (Doornbos et 104 al., 2006). Since plant sterols/stanols product are being marketed for consumption once a day, it remains to be investigated whether the effect of single dose of plant sterols/stanols consumed at different time of the day is comparable to that consumed throughout the day.

Several potential effect modifiers for the effect of plant sterols/stanols supplementation on reduction of LDL levels were studied in some trials, including age and gender, LDL levels as baseline, and genetic profile. Again, results from various studies are inconsistent. For example, baseline LDL levels have been shown to modify the effect of plant sterols/stanols in some (Mussner et al., 2002; Naumann et al., 2003), but not other studies (Maki et al., 2001; Ntanios et al., 2002; Weststrate and Meijer, 1998).

Identification of which effect modifiers play a role in the cholesterol-lowering action of plant sterols/stanols will help targeting the individuals who may benefit more from such an intervention.

Accordingly, instead of running more randomized clinical trials to resolve disagreement that seen to exist concerning influences of food matrix, frequency and time of intake, and subjects' baseline characteristics on cholesterol-lowering action of plant sterols, it was considered that an appropriate meta-analysis could be used as an alternative novel approach.

An updated meta-analysis that encompasses recently published trials and looks into what factors may affect the efficacy of plant sterols as cholesterol-lowering agents is warranted as plant sterols enriched products are marketed in many countries. Thus, the primary objective of this meta-analysis was to evaluate systematically the effect of plant sterol 105 enriched products on LDL-cholesterol levels by pooling the results from randomized controlled trials. The secondary objective was to explore the potential factors affecting the efficacy of plant sterols as cholesterol-lowering agents such as the matrix, frequency and time of intake.

6.3 METHODS

6.3.1 Data collection

6.3.1.1 Search strategy

Studies that examined the efficacy of plant sterols as cholesterol-lowering agents in humans where identified by searching five databases (from 1900 to 2006) PubMed,

Embase, Medline, Cochrane Library and Web of Science using the terms "plant sterol", plant stanol" "phytosterol" and "phytostanol" as words in the title, abstract or keywords.

When available, the search was restricted to clinical trials. In addition, a manual search using reference lists of review articles (Berger et al., 2004; Jones et al., 1997; Katan et al.,

2003; Law, 2000) was performed. For non-English-language literature, if existing, the abstract written in English was used to extract the required information; otherwise the trial was not included in the analysis. All citations were exported into reference manager software (EndNote version 8.0.2) and studies on plant sterols and cholesterol metabolism were identified.

6.3.1.2 Selection of trials

Randomized placebo controlled studies conducted to test the efficacy of plant sterols incorporated into a food matrix on circulating cholesterol levels in adults were included in this meta-analysis. Therefore, studies were first excluded from the meta-analysis for not 106

measuring circulating cholesterol levels as a primary or secondary outcome, for having

duration of intervention of < 2 weeks, for conducting in children or in adults who were

homo- or heterozygote for sitosterolemia or with history of cardiovascular disease.

Studies were also excluded for having a co-intervention that could not be separated from

plant sterol treatment, for incorporating plant sterols in the form of capsules or tablets, or

for not having a control group or an appropriate control/placebo. In addition, studies were

excluded if lipid profile was done on non-fasting blood samples or if lipid profile data

were published elsewhere. A total of 84 clinical trials met the first inclusion criteria and

were then were screened for the quality criteria (Figure 6.1).

6.3.1.3 Study quality assessment

Randomized controlled studies were assessed for methodological quality with the Jadad

score as described in Table 6.1 (Jadad et al., 1996). A Jadad score of 3 or above, out of a maximum of 5, was used to indicate that a study is of reasonable quality to be included in the meta-analysis (Whelan et al., 2006).

6.3.2 Data extraction

For studies that met the inclusion criteria and that possessed a Jadad score of > 3, data

were extracted for parameters related to (i) trial design: parallel vs. crossover, (ii) type of

plant sterols: sterol vs. stanols and free vs. esterified, (iii) dose (g/d) and duration of plant

sterol treatment, (iv) frequency and time of intake of plant sterol enriched products, (v)

food matrix to which plant sterols were incorporate, (vi) characteristics of the study population, including mean age; proportion of males; subjects baseline LDL-cholesterol levels, (vii) the mean values and the standard deviations (SD) of LDL-cholesterol levels, 107 and (viii) sample size. Two reviewers (SA and RB) independently assessed articles for inclusion, assessed quality and extracted data.

For studies that reported multiple time points for the same subjects, only endpoints for the longest duration of the intervention were used. For studies in which the outcomes were presented as percentage change from baseline, and no endpoint data were available (Maki et al., 2001; Seki et al., 2003; Simons, 2002), we imputed endpoint data using the baseline values and percentage change from baseline and the SD of the baseline data for the endpoint SD. Where studies reported absolute change from baseline and no endpoint data were available (Homma et al., 2003; Miettinen and Vanhanen, 1994; Spilburg et al.,

2003), we imputed endpoints using baseline plus change for the mean and using the SD of the baseline data for the endpoint SD.

6.3.3 Statistical analysis

The primary outcome for this meta-analysis was the difference in LDL-cholesterol levels due to plant sterols/stanols treatment. In this meta-analysis LDL-cholesterol levels are reported in mmol/L. For parallel trials, endpoint LDL-cholesterol in the treatment group was subtracted from endpoint LDL-cholesterol in the control group (Deeks et al., 2005).

For crossover trials, the LDL-cholesterol value at the end of the treatment period was subtracted from the LDL-cholesterol value at the end of the control period (Deeks et al.,

2005). Within-individual changes were used when presented; otherwise, group means were used. Standard deviations were extracted from the studies or, if not reported, derived from standard errors of mean, confidence intervals, paired t-value or P-value

(Deeks et al., 2005). If different treatments were tested within the same trial, they were 108

evaluated as separate strata and this is described by a, b, c and d suffixes in tables and

figures. To obtain the pooled treatment effect size, the effect size estimates and standards

error were entered into RevMan 4.2 under the "generic inverse variance" outcome.

Heterogeneity between trial results was tested for using a standard chi-squared test. A P- value < 0.1 was used to indicate that significant heterogeneity was present (Deeks et al.,

2005). Calculations used in this meta-analysis are presented in Appendix 3. Estimates of the pooled treatment effect size of plant sterol-containing food on LDL-cholesterol levels

and 95 CIs were calculated by using both fixed-effect and random effect models. If the test for heterogeneity was significant, we presented the results of the random effect models. Otherwise, estimated results based on a fixed effect model are presented.

6.4 RESULTS

Fifty nine studies comprising 95 relevant strata proved to be eligible for meta-analysis with > 4500 subjects. The language of reports was English for all trial except for one trial.

A summary of trials design and characteristics is presented in Table 6.2 and Table 6.3.

Twenty nine were crossover design trials, and 30 were parallel design trials. Sample sizes ranged from 8 to 185 subjects. Baseline BMI was reported for the majority of the studies.

Subjects in 30 strata had a normal weight, 48 strata contained overweight subjects and 5

strata contained obese subjects. Most of the studies recruited males and females. In 7

studies, > 95% of the subjects were males. In 48 studies the background diets were

subjects' habitual diets, while 9 studies provided controlled diets. The diet background was not reported in 2 studies. Only 2 out of the 59 studies provided data on the ethnicity of the subjects. Studies were carried out in USA (n= 12), Canada (n=7), Europe (n=30),

Australia (n= 2), New Zealand (n=l), Japan, (n=l), Korea (n=l), and Brazil (n=l). The 109 majority of the studies reported the source of funding. Thirty six studies were funded or partially funded by the industry.

Individual trial results and the pooled effect size for all trials are presented in Figure 6.2.

In the overall pooled estimate, plant sterol/stanol decreased LDL-cholesterol levels by

0.31 mmol/L (95% CI, -0.35 to -0.27, P= <0.0001) compared with placebo. Between trial heterogeneity was evident (Chi 2 test, P= <0.0001). Thus, we performed subgroup analysis by subject characteristics and study design features as summarized in Table 6.4.

The placebo-adjusted reduction in LDL levels produced by consumption of plant sterols/stanols was the same across all age groups (Figure 6.3). Subjects with high to very high levels of baseline LDL-cholesterol (study n=22) had a greater decrease in LDL levels than did subjects with optimal to borderline high levels of baseline LDL- cholesterol (study n=33) (Figure 6.4). The LDL-cholesterol levels of the former decreased by 0.37 mmol/L (95% CI: -0.42, -0.31) and those of the latter decreased by

0.28 mmol/L (95% CI: -0.31, -0.25).

There was evidence of a dose-response effect (Figure 6.5). The minimum (-0.25 mmol/L;

95%o CI: -0.32, -0.18) and the maximum (-0.42 mmol/L; 95% CI: -0.46, -0.39) reductions in LDL levels were achieved by the intake of < 1.5g/d (study n=8) and > 2.5 g/d (study n=13) of sterols/stanols, respectively. Between-trial heterogeneity was evident with intake of < 1.5g/d and 1.5-2.0 g/d (study n=35), whereas test for heterogeneity was not significant with intakes of > 2.1 g/d. 110

The favorable effect of plant sterols on LDL-cholesterol is influenced by the matrix to which plant sterols/stanols are incorporated (Figure 6.6). Plant sterols incorporated into fat spreads, mayonnaise and salad dressing or milk and yoghurt reduced LDL-cholesterol levels to a greater extent than plant sterols incorporated into other food products.

Compared to control, LDL levels were reduced by 0.33 (95% CI, -0.38 to -0.28), 0.32

(95% CI, -0.40 to -0.25), 0.34 (95% CI, -0.40 to -0.28) and 0.20 (95% CI, -0.28 to -

0.11) mmol/L in the fat spreads (study n=38), mayonnaise and salad dressing (study n=6)

, milk and yoghurt (study n=7) and other food products (study n=l 1), respectively. Other food product subgroups included studies testing the efficacy of plant sterols incorporated into chocolate (de Graaf et al., 2002; Polagruto et al., 2006), orange juice (Devaraj et al.,

2006; Devaraj et al., 2004), cheese (Jauhiainen et al., 2006), non-fat beverage (Jones et al., 2003; Spilburg et al., 2003), meat (Matvienko et al., 2002), croissants and muffins

(Quilez et al., 2003), oil in bread (Seki et al., 2003), and cereal bars (Yoshida et al.,

2006).

The favorable effect of plant sterols on LDL-cholesterol levels is also influenced by the frequency and time of intake of plant sterols (Figure 6.7). For instance, plant sterols consumed 2-3 times/d (study n=38) reduced LDL-cholesterol levels by 0.34 mmol/L

(95%o CI: -0.38, -0.18) while plant sterols consumed once per day (study n=4) in the morning did not result in a significant reduction in LDL levels. On the other hand, plant sterols consumed once/d with lunch or the principal meal (study n=3) reduced LDL levels by 0.30 mmol/L (95%: -0.39, -0.21). Ill

6.5 DISCUSSION

Since meta-analyses of Law (Law, 2000) and Katan et al (Katan et al., 2003) examining plant sterol/stanol effects on circulating cholesterol levels, several studies have been conducted examining the action of various plant sterol-containing products using different study designs. The present meta-analysis is the first systematic quantitative review of randomized clinical trials yielding information on factors that might affect efficacy of plant sterols as cholesterol-lowering agents. The present work shows that the intake of plant sterol/stanol- containing food products was associated with a significant decrease in

LDL-cholesterol (-0.31 mmol/L). However, the substantial heterogeneity among individual trials indicates that the effects of plant sterols/stanols on LDL-cholesterol levels are not uniform.

There was a larger reduction in LDL-cholesterol levels in subjects with high to very high baseline levels of LDL than in those with optimal to borderline high baseline levels.

Some previous (Mussner et al., 2002; Naumann et al., 2003), but not other studies (Maki et al., 2001; Ntanios et al., 2002; Weststrate and Meijer, 1998), have reported that the higher the baseline levels of LDL-cholesterol the more the reduction in LDL due to plant sterols consumption. The present meta-analysis has confirmed that baseline LDL- cholesterol levels affect magnitude of reduction in LDL after plant sterol consumption which could explain the wide variation seen in previous studies. Nevertheless, plant sterols do reduce LDL levels in individuals with normal to high baseline LDL levels as well as in adults across different age groups. Therefore, everyone, excluding individuals with P-sitosterolemia and heterozygotes for the disease, can benefit from the consumption of plant sterols. 112

A positive dose-response relationship was apparent with the greatest reduction in LDL levels obtained with intakes of 2.5 g/d of plant sterols/stanols. It should be noted that studies included in the subgroups with intakes > 2.1 g/d incorporated plant sterols/stanols mainly in fat spreads while the other subgroups included a variety of food products. This could explain why heterogeneity was absent with intakes of > 2.1 g/d.

Plant sterols/stanols reduce circulating levels of cholesterol through interfering with cholesterol absorption (Gylling et al., 1997; Jones et al., 2000; Normen et al., 2000;

Vanstone et al., 2002). Because of their inert crystalline structure, pure plant sterols are not consistently effective in lowering cholesterol absorption (Ostlund 2004). Thus, plant sterols should be adequately formulated before use. The most accepted method that has been used to optimize the effect of plant sterols on cholesterol absorption is esterification of plant sterols to fatty acids and dissolving plant sterols in food fats (Ostlund, 2004).

Some studies have shown that free plant sterols/stanols when mixed with fat spread are also effectual in reducing LDL-cholesterol levels (Jones et al., 1999; Vanstone et al.,

2002). Earlier efforts to improve the effect of plant sterols on cholesterol absorption were esterification of plant sterols to fatty acids and dissolving plant sterols in food fats. Later on, plant sterols/stanols were added to low and non-fat food products. The results presented here show that compared with plant sterol-containing fat spreads, mayonnaise and salad dressing, and milk and yoghurt, other plant sterol-containing food products, including chocolate (de Graaf et al., 2002; Polagruto et al., 2006), orange juice (Devaraj et al., 2006; Devaraj et al., 2004), cheese (Jauhiainen et al., 2006), non-fat beverage

(Jones et al., 2003; Spilburg et al., 2003), meat (Matvienko et al., 2002), Croissants & muffins (Quilez et al., 2003), oil in bread (Seki et al., 2003), cereal bars (Yoshida et al., 113

2006) demonstrated a lower LDL-reduction efficacy. This finding highlights the

importance of food matrix and proper formulation of plant sterols. Although milk and

yoghurts drink contain much less fat than fat spreads and mayonnaise, they demonstrated

similar efficacy as of products with higher fat content. Thus, the food matrix to which

plant sterols are added does not have to contain a high fat content to be an effective

means of release of plant sterols to compete with cholesterol absorption, giving that a

proper plant sterols formulation is applied. Unfortunately, the exact methods used to

formulate plant sterols/stanols in the milk and yoghurt studies were not described in

detail. Studies only reported if they used free (Thomsen et al., 2004; Volpe et al., 2001) or

esterified (Doornbos et al., 2006; Hyun et al., 2005; Mensink et al., 2002; Noakes et al.,

2005; Pineda et al., 2005) sterols or stanols. It is also possible that plant sterols in milk may be more readily incorporated into milk globule membranes thus accessible for

competing with cholesterol for transfer into the micelles, while in the other low-fat foods plant sterols may be trapped in the centre of the lipid droplets and not available until the

fat is digested (Clifton et al., 2004). Future work is needed to identify proper formulation of plant sterols to improve their efficacy in food products other than those with high fat

content, i.e. vegetable and dairy spreads and mayonnaise, or milk and yoghurts.

In a previous study from our group, consumption of a single dose of different preparations of plant sterols in the morning failed to lower LDL levels (AbuMweis et al.,

2006b). Some studies showed that consumption of single dose of plant sterols with lunch lowered LDL levels (Matvienko et al., 2002; Plat et al., 2000). One study has tested the efficacy of plant stanol consumed at different frequency. In the study by Plat et al. (Plat et al., 2000) subjects consumed the plant stanol enriched margarine at breakfast and at lunch 114

and ate a cake or cookie containing plant stanol-enriched shortening within 1 h after

dinner. The higher portion of plant stanol during the 3 times/d phase was given using a

different food matrix and was consumed without a meal in comparison to the single dose

phase, thus, the study comparison had multiple factors that might contributed to the

differences between the study phases. Additionally, the availability of plant stanol in the

cakes and cookies might be affected by baking conditions. To what extent this affected

the cholesterol- lowering action of 3 times/d phase of plant stanol intake is unknown.

Hereby, we conducted a subgroup analysis by the frequency and time of intake of plant

sterols. The results of this meta-analysis show that the time of intake of a single dose of

plant sterols/stanols may affect their cholesterol-lowering action as consumption of single

dose with lunch or main meal, but not with a breakfast meal, lowered LDL levels. The

results of the subgroup analyses by time of intake of plant sterols should be interpreted

with caution. The number of subjects included in the individual subgroups was small and

many of the included studies did not report data on time of intake, resulting in the potential to be misled by bias. The exact mechanisms responsible for the effects of plant

sterols/stanols on LDL levels are still being investigated. Based on current knowledge, plant sterols/stanols reduce solubilization of cholesterol in the micelles and also may have

effects on the absorption site and during intra-cellular trafficking of cholesterol (Rozner

and Garti, 2006). Therefore, until mechanisms have been elucidated by which plant

sterols and in particular single dose of plant sterols reduce LDL levels, and until there are

more studies on consumption of plant sterols as single dose; plant sterols should be

consumed in two to three portions per day. 115

This is the first meta-analysis to resolve the controversy concerning the influence of food matrix and frequency and time of intake of plant sterols as well as subjects' baseline characteristics on efficacy of plant sterols. In conclusion, this meta-analysis shows that the reduction in LDL was greater in studies conducted on individuals with high baseline

LDL levels, when plant sterols were incorporated into fat spreads, mayonnaise and salad dressing, milk and yoghurt comparing with other food products such as beverages and bakery products, and when plant sterols were consumed in 2-3 portions/d or as single

dose with lunch or main meal but not with breakfast.

6.6 Acknowledgment

We would like to thank Mrs. Mary Cheang, a statistical consultant at the Department of

Community Health Sciences, University of Manitoba, for reviewing the method section

and providing statistical advice. 116

6.7 FIGURE LEGENDS

Figure 6.1. Selection of randomized placebo-controlled studies for meta- analysis of plant sterols and circulating cholesterol levels

Figure 6.2. Effect size and 95% CI in LDL-cholesterol levels associated with consumption of plant sterol/stanol-containing food products

Figure 6.3. Effect size and 95% CI in LDL-cholesterol levels associated with consumption of plant sterol/stanol-containing food products, analysis based on subjects' age

Figure 6.4. Effect size and 95% CI in LDL-cholesterol levels associated with consumption of plant sterol/stanol-containing food products, analysis based on subjects' baseline LDL-cholesterol levels

Figure 6.5. Effect size and 95% CI in LDL-cholesterol levels associated with

consumption of plant sterol/stanol-containing food products, analysis based on plant

sterols dose

Figure 6.6. Effect size and 95% CI in LDL-cholesterol levels associated with

consumption of plant sterol/stanol-containing food products, analysis based on carrier matrix

Figure 6.7. Effect size and 95% CI in LDL-cholesterol levels associated with

consumption of plant sterol/stanol-containing food products, analysis based on frequency

of intake and time of intake of plant sterols 117

Table 6.1. Calculation of Jadad score to assess study quality1

Criterion Score

If study was described as randomized (this includes words such as randomly, random, and randomization) 0/1

If the method used to generate the sequence of randomization was described and was appropriate (table of random numbers, computer- generated, etc) 0/1

Deduct one point if the method used to generate the sequence of randomization was described and it was inappropriate (patients were allocated alternately, or according to date of birth, hospital number, etc). 0/-1

If the study was described as double blind 0/1

If the method of double blinding was described and was appropriate (identical placebo, active placebo, dummy, etc) 0/1

Deduct one point if the study was described as double blind but the method of blinding was inappropriate (e.g., comparison of tablet vs. injection with no double dummy). 0/-1

If there was a description of withdrawals and dropouts 0/1 l Adapted from Jadad et al. (Jadad et al., 1996) Table 6.2. Design and subject characteristics of randomized controlled studies of plant sterols/stanols

BMI4 Study ID Reference Design' Duration n Subjects2 Sex3 Age wk yr kg/m2 (AbuMweis et AbuMweis et al. 2006 a al, 2006b) 2 4 30 3 NR 59 2 (AbuMweis et AbuMweis et al. 2006 b al, 2006b) 2 4 30 3 NR 59 2 (Pineda et al, Algorta Pineda et al. 2005 2005) 1 3 32 4 4 42 2 (Alhassan et 53Tx Alhassan et al. 2006 al, 2006) 1 5 26 2 2 52Co 2 (Andersson et Andersson et al. 1999 al, 1999) 1 8 40 4 2 55 2 (Ayesh et al, Ayeshetal. 1999 1999) 1 3&4 21 1 2 36 1 (Cater et al, Cater et al. 2005 a 2005) 2 6 8 NR 4 58 2 (Cater et al, Cater et al. 2005 b 2005) 2 6 8 NR 4 58 2 (Cater et al, Cater et al. 2005 c 2005) 2 6 8 NR 4 58 2 (Cater et al, Cater et al. 2005 d 2005) 2 8 10 2 5 66 2 (Christiansen Christiansen et al. 2001 a etal, 2001) 1 26 92 4 NR 51 1 (Christiansen Christiansen et al. 2001 b et al, 2001) 1 26 89 4 NR 51 2 (Cleghorn et Cleghorn et al. 2003 al, 2003) 2 4 50 3 2 47 2 )

Table 6.2 (Continued) Study ID Reference Design1 Duration n Subjects2 Sex3 Age wk yr kg/m2 (Davidson et Davidson et al. 2001 a al., 2001) I 8 42 3 4 46 NR (Davidson et Davidson et al. 2001 b al., 2001) 1 8 40 3 4 46 NR (Davidson et Davidson et al. 2001 c al., 2001) L 8 44 3 4 46 NR (deGraafet 56 Tx DeGraafetal. 2002 al., 2002) I 4 62 4 4 58 Co 2 (Devaraj et 44 Tx Deavarj et al. 2006 al., 2006) [ 8 72 3 2 48 Co 1 (Devaraj et 41 Tx Devaraj et al. 2004 al., 2004) ]I 8 72 3 2 44 Co 2 (Doombos et Doombos et al. 2006 a al., 2006) 1 4 72 3 2 57 2 (Doombos et Doorabos et al. 2006 b al, 2006) 1 4 71 3 2 57 2 (Doombos et Doombos et al. 2006 c al., 2006) 1 4 69 3 2 57 2 (Doombos et Doombos et al. 2006 d al., 2006) 1 4 71 3 2 57 2 (Gylling and Miettinen, Gylling etal. 1994 1994) 2 6 11 NR 5 58 2 (Gylling and Miettinen, Gylling etal. 1999 1999) 2 5 21 3 1 53 2

^o Table 6.2 (Continued) _^ , RIVTT4 Study ID Reference Design1 Duration n Subjects2 Sex3 Age wk yr kg/m2 (Hallikainen and Uusitupa, 41 Tx 1TX2 Hallikainen et al. 1999 a 1999) 1 8 37 4 2 46 Co Co (Hallikainen and Uusitupa, 43 Tx Hallikainen et al. 1999 b 1999) 1 8 35 4 2 46 Co 2 (Hallikainen Hallikainen et al. 2000 a et al., 2000a) 2 4 34 4 NR 49 1 (Hallikainen Hallikainen et al. 2000 b et al., 2000a) 2 4 34 4 NR 49 1 (Hendriks et Hendriks et al. 1999 a al., 1999) 2 3.5 80 2 2 37 1 (Hendriks et Hendriks et al. 1999 b al., 1999) 2 3.5 80 2 2 37 1 (Hendriks et Hendriks et al. 1999 c al., 1999) 2 3.5 80 2 2 37 1 (Hendriks et Hendriks et al. 2003 al., 2003) 1 52 185 3 2 48 1 (Hyun et al., Hyun et al. 2005 2005) 1 4 51 2 4 29 1 (Jakulj et al., Jakulj et al. 2005 2005) 2 4 39 5 4 56 2 (Jones et al., Jones et al. 1999 1999) 1 4.3 32 4&5 5 NR NR (Jones et al., Jones et al. 2000 a 2000) 2 3 15 4 5 NR NR Table 6.2 {Continued) BMI4 Study ID Reference Design' Duration n Subjects2 Sex3 Age 2 wk yr kg/m (Jones et al., Jones et al. 2000 b 2000) 2 3 15 4 5 NR NR (Jones et al., Jones et al. 2003 a 2003) 2 3 15 3 4 NR NR (Jones et al., Jones et al. 2003 b 2003) 2 3 15 3 4 NR NR (Judd et al, Judd et al. 2002 2002) 2 3 53 3 2 47 2 (Jauhiainen et Jauhianen et al. 2006 al., 2006) 1 5 67 3 2 43 NR (Lau et al., Lau et al. 2005 a 2005) 2 3 15 3 2 55 2 (Lau et al., Lau et al. 2005 b 2005) 2 3 14 8 2 55 3 (Lee et al., 60 TX Lee et al. 2003 2003) 1 12 81 9 2 62 Co 2 (Lottenberg et Lottenberg et al. 2003 al., 2003) 2 4 60 5 2 NR NR (Maki et al., 59 Tx Makietal.2001a 2001) 1 5 158 3 2 58 Co 2 (Maki et al., 60 Tx Makietal. 2001b 2001) 1 5 118 3 2 58 Co 2 (Matvienko et 22 Tx Matvienko et al. 2002 al., 2002) 1 4 34 3 5 22 Co 2 (Mensink et Mensink et al. 2002 al., 2002) 1 4 60 2 2 36 1 Table 6.2 (Continued) BMI4 Study ID Reference Design' Duration n Subjects2 Sex3 Age wk yr kg/m2 (Miettinen and Miettinen and Vanhanen Vanhanen, 1994 a 1994) 1 9 17 NR 4 45 2 (Miettinen and Miettinen and Vanhanen Vanhanen, 1994 b 1994) 1 9 15 NR 4 45 2 (Miettinen and Miettinen and Vanhanen Vanhanen, 1994 c 1994) 1 9 15 NR 4 45 2 (Mussner et Mussner et al. 2002 al., 2002) 2 3 63 3 2 42 1 (Naumann et 32 w Naumann et al. 2003 a al,2003) 2 3 42 2 2 37 m 1

(Naumann et 32 w Naumann et al. 2003 b al., 2003) 2 3 42 2 2 37 m 1 (Neil et al., 53 Tx Neil etal. 2001 2001) 2 8 29 5 2 50 Co 2 (Nguyen et Nguyen et al. 1999 a al., 1999) 1 8 159 3 2 53 2 (Nguyen et Nguyen etal. 1999 b al., 1999) 1 8 157 3 2 53 2 (Nguyen et Nguyen etal. 1999 c al., 1999) 1 8 162 3 2 53 2 (Nigon et al., Nigon etal. 2001 2001) 2 8 53 3&4 2 58 1 )

Table 6.2 (Continued) BIVIT4 Study ID Reference Design' Duration n Subjects2 Sex3 Age wk yr kg/m2 (Noakes et al., 58 w Noakes et al. 2002 a 2002) 2 3 46 4 2 55 m 2 (Noakes et al., 58 w Noakes et al. 2002 b 2002) 2__ 3 46 4 2 55 m 2 (Noakes et al., 56 w Noakes et al. 2002 c 2002) 2 3 35 4 4 58 m 2 (Noakes et al., Noakes et al. 2005 a 2005) 2 3 40 4 2 60 2 (Noakes et al., Noakes et al. 2005 b 2005) 2 3 40 4 2 60 2 (Ntanios et al., Ntanios et al. 2002 2002) 2 3 53 2 2 45 (Plat and Plat and Mensink et al. 2000 Mensink, a 2000) 1 8 78 2 2 33 (Plat and Plat and Mensink et al. 2000 Mensink, b 2000) 1 8 76 2 2 33 (Plat et al., Plat et al. 2000 a 2000) 2 4 39 1 2 31 (Plat et al., Plat et al. 2000 b 2000) 2 4 39 1 2 31 (Polagruto et 49 Tx Polagruto et al. 2006 al., 2006) 1 6 67 4 2 56 Co 2 (Quilez et al., Quilez et al. 2003 2003) 1 8 57 1 2 31 (Saito et al., 38 Tx Saito et al. 2006 a 2006) 1 4 33 3 5 39 Co Table 6.2 (Continued) BMI4 Study ID Reference Design' Duration n Subjects2 Sex3 Age wk yr kg/m2 (Saito et al., Saito et al. 2006 b 2006) 1 4 33 3 5 39 1 (Saito et al., 38 Tx Saito et al. 2006 c 2006) 1 4 34 3 5 39 Co 1 (Seki et al., Seki etal.2003 2003) 1 12 60 3 5 39 1 (Sierksma et Sierksma et al. 1999 al., 1999) 2 3 75 NR 4 44 1 (Simons, 58 Tx Simons et al. 2002 2002) 1 4 77 5 4 60 Co 2 (Spilburg et Spilburg et al. 2003 al.,2003) 1 4 24 3 4 51 2 (Temme et al., Temme et al. 2002 2002) 2 4 42 4 4 55 2 (Thomsen et Thomsen et al. 2004 a al., 2004) 2 4 69 4 2 60 2 (Thomsen et Thomsen et al. 2004 b al., 2004) 2 4 69 4 2 60 2 (Vanhanen et 48 Tx Vanhanen et al. 1993 al., 1993) 1 6 67 3 4 43 Co 2 (Vanhanen, Vanhanen et al. 1994 1994) 1 6 14 3 2 55 2 (Vanstone et Vanstone et al. 2002 a al., 2002) 2 3 15 4 4 48 3 (Vanstone et Vanstone et al. 2002 b al., 2002) 2 3 15 4 4 48 3 (Vanstone et Vanstone et al. 2002 c al., 2002) 2 3 15 4 4 48 3 Table 6.2 (Continued) BMI4 Study ID Reference Design 1 Duration n Subjects2 Sex3 Age wk yr kg/m2 (Vissers et al, Vissers et al. 2000 2000) 2 3 60 NR 2 NR NR (Volpe et al., Volpeetal. 2001 2001) 2 4 30 4 4 NR 1 (Weststrate and Meijer, Weststrate et al. 1998 a 1998) 2 3.5 95 3 3 45 1 (Weststrate and Meijer, Weststrate et al. 1998 b 1998) 2 3.5 95 3 3 45 1 (Yoshida et Yoshida et al.2006 a al., 2006) 2 3 16 4 2 55 2 (Yoshida et Yoshida et al.2006 b al., 2006) 2 3 13 8 2 57 3

NR= not reported, Tx= treatment; Co= control; w= women; m^men 1 Study design: 1= parallel; 2= crossover 2 Subjects were classified according to total or cholesterol baseline levels reported in baseline characteristic. Classification based on ATPIII (Cleeman et al., 2001): 1= optimal; 2= near or above optimal; 3= borderline high; 4= high; 5= very high 3 Predominant sex: 1= < 5% males; 2= 5%-50% males; 3= 50% males; 4= 50%- 95% males; 5= > 95% males; 6= not clear 4 Body Mass Index kg/m2: 1= < 24.9; 2= 25-29.9; 3= 30=34.9; 4= > 35 >

Table 6.3. Features of plant sterol intervention of randomized controlled studies of plant sterols/stanols

Study ID Reference Plant sterols/stanols Dose Matrix1 Type2 g/d as Frequency Time3 free (AbuMweis et AbuMweis et al. 2006 a al., 2006b) 1 1 1.7 1 2 (AbuMweis et AbuMweis et al. 2006 b al, 2006b) I 3 1.7 1 2 (Pineda et al., Algorta Pineda et al. 2005 2005) :5 4 2.0 1 6 (Alhassan et al., Alhassan et al. 2006 2006) I 4 NR NR NR (Andersson et Andersson et al. 1999 al., 1999) L 4 1.9 NR NR (Ayesh et al., Ayeshetal. 1999 1999) I 3 8.6 2 9 (Cater et al., Cater et al. 2005 a 2005) 1[ 4 2.0 3 8 (Cater et al., Cater et al. 2005 b 2005) ][ 4 3.0 3 8 (Cater et al, Cater et al. 2005 c 2005) 1 4 4.0 3 8 (Cater et al., Cater et al. 2005 d 2005) 1 4 3.0 3 8 (Christiansen et Christiansen et al. 2001 a al, 2001) 1 1 1.5 at least 2 NR (Christiansen et Christiansen et al. 2001 b al., 2001) 1 1 3.0 at least 2 NR Table 6.3 (Continued) Study ID Reference Plant sterols/stanols Dose Matrix1 Type2 g/das Frequency Time3 free (Cleghorn et al, Cleghorn et al. 2003 2003) 1 3 2.0 NR NR (Davidson et Davidson et al. 2001 a al., 2001) 1 3 3.0 NR NR (Davidson et Davidson et al. 2001 b al., 2001) 9 3 6.0 NR NR (Davidson et Davidson et al. 2001 c al., 2001) 13 3 9.0 NR NR (deGraafet al., DeGraafetal. 2002 2002) 17 1 1.8 3 8 (Devaraj et al., Deavarj et al. 2006 2006) 4 1 2.0 2 9 (Devaraj et al., Devaraj et al. 2004 2004) 4 1 2.0 2 NR (Doornbos et Doorabos et al. 2006 a al., 2006) 3 3 3.2 1 2 (Doornbos et Doornbos et al. 2006 b al, 2006) 3 3 3.2 1 3 (Doornbos et Doombos et al. 2006 c al., 2006) 3 3 2.8 1 2 (Doornbos et Doornbos et al. 2006 d al, 2006) 2.8 Table 6.3 (Continued) Study ID Reference Plant sterols/stanols Dose 1 Matrix TyPe2 g/d as Frequency Time3 free (Gylling and Miettinen, Gylling et al. 1994 1994) 1 4 3.0 3 1 (Gylling and Miettinen, Gylling etal. 1999 1999) K 4 2.5 NR NR (Hallikainen and Uusitupa, Hallikainen et al. 1999 a 1999) 1 4 2.2 NR NR (Hallikainen and Uusitupa, Hallikainen et al. 1999 b 1999) 1 4 2.3 NR NR (Hallikainen et Hallikainen et al. 2000 a al., 2000a) 1 3 2.1 2 to 3 NR (Hallikainen et Hallikainen et al. 2000 b al., 2000a) 1 4 2.0 2 to 3 NR (Hendriks et al., Hendriks et al. 1999 a 1999) 1 3 0.8 2 5 (Hendriks et al., Hendriks et al. 1999 b 1999) 1 3 1.6 2 5 (Hendriks et al., Hendriks et al. 1999 c 1999) 1 3 3.2 2 5 (Hendriks et al., Hendriks et al. 2003 2003) 3 1.6 2 10 Table 6.3 (Continued) Study ID Reference Plant sterols/stanols Dose Matrix Type g/d as Frequency Time free (Hyun et al., Hyun et al. 2005 2005) 3 4 2.0 1 2 (Jakulj et al., Jakulj et al. 2005 2005) 1 3 2.0 NR NR (Jones et al., Jones et al. 1999 1999) 1 1 1.7 3 (Jones et al., Jones et al. 2000 a 2000) 1 3 1.8 3 (Jones et al., Jones et al. 2000 b 2000) 1 4 1.8 3 (Jones et al, Jones et al. 2003 a 2003) 4 1 1.8 3 (Jones et al., Jones et al. 2003 b 2003) 4 1 1.8 3 (Judd et al., Judd et al. 2002 2002) 9 3 2.2 2 5 (Jauhiainen et Jauhianen et al. 2006 al., 2006) 5 4 2 lor 2 3 or 6 (Lau et al., Lau et al. 2005 a 2005) 1 1 1.8 1 2 (Lau et al., Lau et al. 2005 b 2005) 1 2 1.8 1 2 (Lee et al., Lee et al. 2003 2003) 1 3 1.6 2 9 Table 6.3 (Continued) Study ID Reference Plant sterols/stanols Dose Matrix1 Type2 g/d as Frequency Time3 free (Lottenberg et Lottenberg et al. 2003 al., 2003) 1 3 1.7 3 1 (Maki et al, Makietal. 2001a 2001) 1 3 1.1 2 NR (Maki et al, Maki et al. 2001 b 2001) 1 3 2.2 2 NR (Matvienko et Matvienko et al. 2002 al., 2002) 18 3 2.7 1 3 (Mensink et al., Mensink et al. 2002 2002) 3 4 3.0 2 or 3 8 or 9 (Miettinen and Miettinen and Vanhanen Vanhanen, 1994 a 1994) 11 1 0.7 NR NR (Miettinen and Miettinen and Vanhanen Vanhanen, 1994 b 1994) 11 2 0.7 NR NR (Miettinen and Miettinen and Vanhanen Vanhanen, 1994 c 1994) 11 4 0.8 NR NR (Mussner et al, Mussner et al. 2002 2002) 1 3 1.8 2 9 (Naumann et Naumann et al. 2003 a al., 2003) 1 7 2.0 NR NR Table 6.3 (Continued) Study ID Reference Plant sterols/stanols Dose Matrix1 Type2 g/d as Frequency Time3 free (Naumann et Naumann et al. 2003 b al, 2003) L 7 2.0 NR NR (Neil et al., Neil et al. 2001 2001) 1I 3 2.5 NR NR (Nguyen et al., Nguyen etal. 1999 a 1999) I 4 3.0 3 NR (Nguyen et al., Nguyen etal. 1999 b 1999) ]I 4 3.0 3 NR (Nguyen et al., Nguyen etal. 1999 c 1999) 1[ 4 2.0 3 NR (Nigon et al., Nigon et al. 2001 2001) 1i 3 1.6 3 1 (Noakes et al., Noakes et al. 2002 a 2002) ][ 3 2.3 3 1 (Noakes et al., Noakes et al. 2002 b 2002) ][ 4 2.5 3 1 (Noakes et al., Noakes et al. 2002 c 2002) 1 3 2.0 3 1 (Noakes et al., Noakes et al. 2005 a 2005) 3 3 1.8 2 NR (Noakes et al., Noakes et al. 2005 b 2005) 3 4 1.7 2 NR (Ntanios et al., Ntanios et al. 2002 2002) 1 3 1.8 2 10 ) ^

Table 6.3 (Continued) Study ID Reference Plant sterols/stanols Dose Matrix1 Type2 g/d as Frequency Time3 free Plat and Mensink et al. 2000 (Plat and a Mensink, 2000) 1 4 3.8 3 1 Plat and Mensink et al. 2000 (Plat and b Mensink, 2000) 1 4 4.0 3 1 (Plat et al., Plat et al. 2000 a 2000) 1 4 2.5 1 3 (Plat et al., Plat et al. 2000 b 2000) 19 4 2.5 3 1 (Polagruto et Polagruto et al. 2006 al., 2006) 17 3 1.5 2 11 (Quilez et al., Quilez et al. 2003 2003) 16 3 3.2 2 NR (Saito et al., Saito et al. 2006 a 2006) 11 3 0.3 1 NR (Saito et al., Saito et al. 2006 b 2006) 11 3 0.4 1 NR (Saito et al., Saito et al. 2006 c 2006) 11 3 0.5 1 NR (Seki et al., Seki et al. 2003 2003) 15 3 0.5 3 NR (Sierksma et al., Sierksma et al. 1999 1999) 0.8 NR NR Table 6.3 (Continued) Study ID Reference Plant sterols/stanols Dose Matrix1 Type2 g/d as Frequency Time3 free Simons et al. 2002 (Simons, 2002) 1 3 2.0 2 NR (Spilburg et al., Spilburg et al. 2003 2003) 4 6 1.9 3 1 (Temme et al, Temme et al. 2002 2002) 1 3 2.0 3 1 (Thomsen et al., Thomsen et al. 2004 a ' 2004) 2 1 1.2 2 7 (Thomsen et al., Thomsen et al. 2004 b 2004) 2 1 1.6 2 7 (Vanhanen et Vanhanen et al. 1993 al,1993) 11 4 3.4 NR NR (Vanhanen, Vanhanen et al. 1994 1994) 11 4 1.5 NR NR (Vanstone et al., Vanstone et al. 2002 a 2002) 10 1 1.8 3 1 (Vanstone et al., Vanstone et al. 2002 b 2002) 10 2 1.8 3 1 (Vanstone et al, Vanstone et al. 2002 c 2002) 10 5 1.8 3 1 (Vissers et al., Vissers et al. 2000 2000) 1 1 2.1 NR NR (Volpe et al., Volpe et al. 2001 2001) 3 1 1.0 1 NR Table 6.3 {Continued) Study ID Reference Plant sterols/stanols Dose Matrix1 Type2 g/d as Frequency Time3 free (Weststrate and Weststrate et al. 1998 a Meijer, 1998) 1 3 3.2 2 5 (Weststrate and Weststrate et al. 1998 b Meijer, 1998) 1 4 2.7 2 5 (Yoshida et al., Yoshida et al.2006 a 2006) 8 1 1.8 3 11 (Yoshida et al., Yoshida et al.2006 b 2006) 8 1 1.8 3 11

NR= not reported, !Food matrix to which plant sterols/stanols were added: 1= fat spread (margarine); 2= milk; 3= yoghurt; 4= juice or beverage; 5= soft cheese; 6= hard cheese; 7= bread; 8= cereals or cereals bars; 9= salad dressing; 10= butter or dairy spread; 11= mayonnaise; 12= spread + cereals+ bread; 13= spread + salad dressing; 14= chips; 15= vegetable oil; 16= croissants and muffins; 17= chocolate bar; 18= meat; 19= margarine + shortening in cakes and cookies 2Type of plant sterols/ stands: 1= free plant sterols (>50%); 2= free plant stands (>50); 3= plant sterol esters (>50%); 4= plant stanol esters (>50%); 5= mixture of free plant sterols and stanols; 6= stanol lecithin; 7= mixture of plant sterol and stanol esters 3Time of consumption of plant sterol/stanol enriched products: 1= at breakfast + lunch + dinner; 2= at breakfast; 3= at lunch; 4= at dinner; 5= at lunch and dinner; 6- with the main meal; 7= at breakfast + lunch; 8= with each meal; 9= at breakfast + lunch or dinner; 11= between meals (snack) Table 6.4. Pooled estimates of treatment effect on LDL-cholesterol in subgroups of trials

defined by subject characteristics and study design features

No. of Effect size (95% Test of Variables trials CI) P heterogeneity n mmol/L P

Age (y) 20-39 10 -0.29 (-0.35, -0.23) < 0.0001 0.16 40-49 15 -0.32 (-0.41,-0.24) < 0.0001 < 0.0001 50-60 21 -0.30 (-0.37, -0.23) < 0.0001 < 0.0001

Baseline LDL-cholesterol levels Optimal to border line high 33 -0.28 (-0.31,-0.25) O.0001 0.38 High to very high 22 -0.37 (-0.42,-0.31) O.0001 0.01

Plant sterol dose (g/d) <1.5 8 -0.25 (-0.32,-0.18) O.0001 0.05 1.5-2.0 35 -0.29 (-0.34, -0.24) O.0001 0.0003 2.1-2.5 9 -0.32 (-0.36, -0.28) O.0001 0.12 >2.5 13 -0.42 (-0.46, -0.39) O.0001 0.57 Matrix Fat spreads 38 -0.33 (-0.38, -0.28) 0.0001 0.0001 Mayonnaise and salad dressing 6 -0.32 (-0.40, -0.25) 0.0001 0.3 Milk and yoghurt 7 -0.34 (-0.40, -0.28) 0.0001 0.18 Other than fat spreads, mayonnaise, salad dressing and milk and 11 yoghurt -0.20 (-0.28,-0.11) 0.0001 0.21

Frequency of intake and time of intake 2-3 times/d 38 -0.34 (-0.38, -0.30) 0.0001 0.0001 Once/ d in the morning 4 -0.14 (-0.29, 0.00) 0.05 0.60 Once/ d in the afternoon or with main meal 3 -0.30 (-0.39, -0.21) 0.0001 0.82 Figure 6.1.

Studies of plant sterols supplementation in humans and cholesterol metabolism (n=188) Studies excluded for the following reasons: Subjects were children (n=14) Study did not measure total cholesterol and LDL-cholesterol (n=19) Plant sterols/stanols were not incorporated into a food matrix (n=11) The duration of intervention was < 2 weeks(n=6) The study had a co-intervention component and can not separate effect of plant sterols/stanols (n=18) The lipid data were published elsewhere (n=18) The study did not have a control group or an appropriate placebo (n=8) The subjects were homo- or heterozygote for sitosterolemia (n=3) Duration of control and treatment interventions was not the same (n=1) The study language is not English (n=4) i r Subjects had previous cardiovascular Potential studies for quality assessment disease event (n=2) (n=83) Used non fasting blood samples (n=1)

Studies of insufficient quality or insufficient information: Jadad score < 3 (n=19) Insufficient information (n=5) >' Studies included in meta-analysis (n=59) 137

Figure 6.2.

Revfcyv". Hani-sterols 2007 Cafiparfson: 05Alitudle* Outcomes 01 LW.*h

Sto* Effect sizetranaom). VMeight Effect.sb* (random)

or auto-Category; 85*0 '•:* *$%ci 01 Relevant comiwifSiona (most studies v^ plant sterols andan doses) AbuMwels 2008b — 1.77 -0.05 •0.27, Al^orta Pineda 2005 4 • 0.51 -0.62 •1.13. AtomsssanSOOe •— 0.40 -0.26 •0.85. Anoersson1999 i • 0.68 -0.68 •1.11, Ayesh"t999 4 • 0.73 -0.66 •1.07, Cater2005c 4 « 0.5» -0.S7 •1.04, ChrisBsnsen 2001b • 1.33 -0.4S 0.72, deghom2003 -»- 2.83 -0.Z7 •0.39. Davidson 2001b » 0.63 -COS •0.54, DeOre*12002 • 1.46 -0.48 •0.73, Deavw)2006 —: »> 0.79 -0.16 •0.55. Devaraj2004 •:• . • •• 1.21 -0.34 •0.63, Doornbos 2006a •*« 1.11 -0.31 •0.62, <3ving1994 —*-— 1.95 -0.37 •0.57, ©yino;1999 - • .. 1.35 -0.45 •0.65, Heftakwi 1899a ,.- •••»•—- 0.68 -0.37 -0.80, HaKsahen 2000a -if 2.83 -0.45 •0.57, Hen*k» 1B99C -** 2.83 -0.30 •0.42. Hen#»te2003 •• •.' » 1.77 -0.26 -0.48, Hyun2005 . *• 1.21 -0.1S •0.44, Jakuj2O05 ••«." 1.77 23 •0.4*, JauNaNsn2006 0.73 38 -0.79, >Jonesi999 4 19 •1.99, Jones 2000a 56 •0.75. Jones 3003a •0.41, Judd2002 32 -0.40, Lau 2005a -0.30 -0.55, lee 2003 0.12 -0.19, lottenberg 2003 3.07 -0.30 -0.40, MaW 2001b 1.61 .38 •0.62, MaMer*0 2O02 0.S9 .40 97, MensWcJSXK 0.73 .24 55, Wetthen 1994a < 0.24 .51 29, Mussner2002 2.IS .26 44, Neumann 2003a 2.IS .17 35, Neil 2001 2.15 -0.34 52. Nguyen 1999b 2.IS -0.42 60, NlgoniOOt 1.77 -0.23 45, Noaltes20Q2a 2.15 -0.33 51. Noake* 2002s 1.77 -0.40 -D.62, Noakes 2005a 3.07 -0.27 -0.37, l*ar*>*20O2 3.07 -0.28 -0.38, flat SMsrvsink 2000a 1.11 39 -0.70, Rat 2000a 3.07 29 -0,39, Pota8ruto2D08 1.02 06 -0.39, Qi*sz20D3 1.02 26 -0.S9, Sato 2008c 1.11 58 -0.89, SeM2003 1.02 02 -0.31, S'Bfksma1999 3.66 19 -0.23, Simons 2002 < O.SS 0.58 -1.07, Spjfcrs20O3 2.15 0.04 -0.22, Ternme2002 3.07 0.41 -0.51, Thomsen 2004b' 3.07 0.44 -0.54, Vanhanen1993 0.94 0.30 -0.65, Vanhanen1994 0.30 0.20 -0.49, Vanstone 2002a 1.61 0.41 -0.65, Vfc*ers20O0 3.07 0.20 -0.30, Vo)pe2001 2.37 0.34 -O.SO, Wsststcate 1993a * 3.66 0.44 -0.48, Yosr**a2006a , —:~M 1.61 0.24 -0.48, Subtotal (95% CT) f 100.00 0.31 -0.34, test (of heterogenetv: CW- 17241,'.*.«3B*(J>•«"ffiijjoootj;M »f».7* T«s((orovi!fa»e««ci;Z«tS»(et*.OjBIiaMj

Total (95% O) > 100.BO -0.53.1 1-B.M, -^,27»- Test.for beteroojenely: CW »172.11, df« S9(P « 0 J0001), P » 65 7% Test faroyerel affect Z«1S.6«(P«c 0.00001) -1 -0.S 0 0.5 1 Favour* treatment Favours control Figure 6.3

Review: Plant sterols 2007 Comparison30 Age 20-39 yr Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

Ayesh 1999 f- 2.23 -0.6S [-1.07, -0.25] Hendriks 1999c 27.32 [-0.42, -0.18) Hyun 2005 4.37 t-0.44, 0.14] Matvienko 2002 1.71 [-0.87, 0.07] Mensink 2002 2.23 [-0.6S, 0.17] Naumann 2003a 12.14 t-0.35, 0.01] Plat & Mensink 2000b 3.40 -0.36 [-0.69, -0.03] Plat 2000b 39.34 -0.31 [-0.41, -0.21] Saito 2006c 3.84 -0.58 [-0.89, -0.27] Seki 2003 3 .40 0.02 [-0.31, 0.35]

Total (95% CI) + 100.00 •0.29 [-0.35, -0.23] Test for heterogeneity: Chi2 = 12.98, iJf = 9 (P = 0.16), I2 = 30.7% Test for overall effect: Z = 9.31 (P < (.00001)

-1 -0.5 0 0.5 1 Favours treatment Favours control

Review: Plant sterols 2007 Comparisons Age 40-49 yr (dose equal or > 1.5 j Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (random) Weight Effect size (random) or sub-category 95% CI % 95% CI

Algorta Pineda 2005 f- 1.40 -0.62 [-1.13, -0.11] Davidson 2001a 1.50 0.12 [-0.37, 0.61] Deavarj 2006 2.26 -0.16 [-0.55, 0.23] Devaraj 2004 3.73 -0.34 [-0.63, -0.05] Hallikainen 1999a 1.90 -0.37 [-0.80, 0.06] Hallikainen 2000a 12.31 -0.45 [-0.57, -0.33] Hendriks 2003 6.05 -0.26 [-0.48, -0.04] Jauhiainen 2006 4.17 -0.38 [-0.65, -0.11] Judd 2002 16.28 -0.32 [-0.40, -0.24] Mussner 2002 7.95 -0.26 [-0.44, -0.08] Ntanios 2002 14.22 -0.28 [-0.38, -0.18] Vanhanen 1993 2.73 -0.30 [-0.65, 0.05] Vanstone 2002a 5.31 -0.41 [-0.65, -0.17] Weststrate 1998a 20.19 -0.44 [-0.48, -0.40]

Total (95% CI) + 100.00 -0.35 [-0.41, -0.29] Test for heterogeneity: Chi2 = 25.27, df 13 (P = 0.02), I2 = 48.6% Test for overall effect: Z = 10.97 (P < 0 00001) -1 -0.5 0 0.5 1 Favours treatment Favours control Review: Plant sterols 2007 Comparison29 Age 50-60 yr Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (random) Weight Effect size (random) or sub-category 95% CI % 95% CI

AbuMweis 2006b 5.26 -0.05 0.27, 0.17] Andersson 1999 <—*— 2.08 -0.68 1.11, -0.25] Cater 2005c 4 •- 1.81 -0.57 1.04, -0.10] Christiansen 2001a —• 4.00 -0.48 0.75, -0.21] De Graaf 2002 —• 4.38 -0.48 0.73, -0.23] Doornbos 2006a — 3.36 -0.31 0.62, 0.00] Gylling1994 5.77 -0.37 •0.57, -0.17] Jakulj 2005 26 -0.23 •0.45, -0.01] Lau 2005a 38 -0.30 0.55, -0.05] Lee 2003 36 0.12 0.19, 0.43] Maki 2001b — 80 -0.38 •0.62, -0.14] Neil 2001 32 -0.34 •0.52, 16] Nguyen 1999a 32 -0.34 •0.52, 16] Nigon 2001 26 -0.23 •0.45, ,01] Noakes 2002a 6.32 -0.33 •0.51, .15] Noakes 2005a 8.81 -0.27 -0.37, -0.17] Simons 2002 4- 1.69 -0.58 -1.07, -0.09] Spilburg 2003 6.32 -0.04 -0.22, 0.14] Thomsen 2004b 8.81 -0.44 -0.54, -0.34] -0.49, Vanhanen 1994 0.93 0.20 0.89] -0.48, Yoshida 2006a 4.80 -0.24 0.00]

Total (95% CI) • 100.00 -0.30 [-0.37, -0.23] Test for heterogeneity: Chi2 = 42.06 df = 20 (P = 0.003), I2 = 52.4% Test for overall effect: Z = 8.45 (P < 0.00001) 1 -1 -0.5 0.5 Favours treatment Favours control Figure 6.4

Review: Plant sterols 2007 Comparison:! 5 Subject baseline LDL- optimal to borderline high Outcome: 01 LDL-cholesterol

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

01 Relevant comparisions (most studfes with plant sterols and all doses ) AbuMweis 2006b 2..3 1 -0.05 -0.27, Alhassan 2006 - 0..3 1 -0.26 •0.85, Ayesh 1999 i * 0..6 3 -0.66 -1.07, Cleghorn 2003 7..7 5 -0.27 -0.39, Davidson 2001b — 0..5 3 -0.09 -0.54, Deavarj 2006 .70 -0.16 -0.55, Devaraj 2004 • 1 .24 -0.34 -0.63, Doornbos 2006a 1 1 .09 -0.31 -0.62, Gylling 1999 —*- 2 .79 -0.45 -0.65, Hendriks 1999c -i 7 .75 -0.30 -0.42, Hendriks 2003 — 2 .31 -0.26 -0.48, Hyun 2005 — 1 .24 -0.15 -0.44, Jauhiainen 2006 »- 0 .63 -0.38 -0.79, Jones 2003a - 0 .97 -0.08 -0.41, Judd 2002 -« 17 .44 -0.32 -0.40, Lau 2005a —s 1 .65 -0.30 -0.55, Maki 2001b —*- 1 .94 -0.38 -0.62, Matvienko 2002 •- 0 .48 -0.40 -0.87, Mensink 2002 0 .63 -0.24 -0.65, Mussner 2002 — 3 .45 -0.26 -0.44, Naumann 2003a 3 .45 -0.17 -0.35, Nguyen 1999b —*- 3 .45 -0.42 -0.60, Ntanios 2002 11 .16 -0.28 -0.38, Plat & Mensink 2000a •- 1 .09 -0.39 -0.70, Plat 2000a H 11 .16 -0.29 -0.39, Quilez 2003 0 .97 -0.26 -0.59, Saito 2006a — 1 .09 -0.22 -0.53, Seki 2003 0 .97 0.02 -0.31, Spilburg 2003 3 .45 -0.04 -0.22, Vanhanen 1993 1 0 .86 -0.30 -0.65, .23 -0.49, Vanhanen1994 — 0.20 .36 -0.50, Volpe 2001 -i 4 -0.34 .94 -0.48, Yoshida 2006a — 1 -0.24 .00 -0.31, Subtotal (95% CI) 100 -0.28 • 2 = 5.2% Test for heterogeneity: Chi2 = 33.74, If = 32 (P = 0.38), I Test for overall effect: Z = 16.73 (P < 0.00001)

Total (95% CI) • 100.00 •0.28 [-0.31, -0.25] Test for heterogeneity: Chi2 = 33.74, >if = 32 (P = 0.38), I2 = 5.2% Test for overall effect: Z = 16.73 (P < 0.00001)

-1 -0.5 0 0.5 1 Favours treatment Favours control Review: Plant sterols 2007 Comparison:!6 Subject baseline LDL-high to very high Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (random) Weight Effect size (random) or sub-category 95% CI % 95% CI

01 Relevant comparisions (most studies with plant sterols and all doses) Algorta Pineda 2005 4 • l .10 -0.62 -1.13, -0.11] Andersson 1999 4—i 1 49 -0.68 -1.11, -0.25] Christiansen 2001b —• 3 ,12 -0.45 -0.72, -0.18] De Graaf 2002 —•— 3 .48 -0.48 -0.73, -0.23] Hallikainen 1999a • 1 ,49 -0.37 -0.80, 0.06] Hallikainen 2000a -f- ,01 -0.45 -0.57, -0.33] Jakulj 2005 —•— .37 -0.23 -0.45, -0.01] Jones 1999 4 ,47 -1.19 -1.99, -0.39] Jones 2000a —•— ,93 -0.56 -0.76, -0.36] Lee 2003 — .54 0.12 -0.19, 0.43] Lottenberg 2003 -*- .98 -0.30 -0.40, -0.20] Neil 2001 —•— .56 -0.34 -0.52, -0.16] Noakes 2002a —»— ,56 -0.33 -0.51, -0.15] Noakes 2002c —*— .37 -0.40 -0.62, -0.18] Noakes 2005a -*- ,98 -0.27 -0.37, -0.17] Polagruto 2006 « ,31 -0.06 -0.39, 0.27] Simons 2002 4 • .19 -0.58 -1.07, -0.09] Temme 2002 -t- .98 -0.41 -0.51, -0.31] Thomsen 2004b •*- .98 -0.44 -0.54, -0.34] Vanstone 2002a —•— 3 .90 -0.41 -0.65, -0.17] Volpe 2001 -§- 6 .29 -0.34 -0.50, -0.18] Yoshida 2006a —•— 3 .90 -0.24 -0.48, 0.00] Subtotal (95% CI) • 100 .00 -0.37 -0.42, -0.31] Test for heterogeneity: Chi2 = 38.69, Jf = 21 (P = 0.01), I2= 45.7% Test for overall effect: Z = 12.81 (P < 0.00001)

Total (95% CI) • 100.00 -0.37 [-0.42, -0.31] Test for heterogeneity: Chi2 = 38.69, jf = 21(P = 0.01), I2 = 45.7% Test for overall effect: Z = 12.81 (P < 0.00001) -1 -0.5 0 0.5 1 Favours treatment Favours control Figure 6.5

Review: Plant sterols 2007 Comparison: 38 Dose < 1.5 g/d Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (random) Weight Effect size (random) or sub-category 95% CI % 95% CI

Hendriks 1999a 17.30 -0.19 [-0 31 -0.07] Maki 2001a 10.94 -0.31 [-0 49 -0.13] Miettinen 1994a 0.82 -0.51 [-1 29 0.27] Saito 2006a 4.50 -0.22 [-0 53 0.09] Seki 2003 4.05 0.02 [-0 31 0.35] Sierksma 1999 29.52 -0.19 [-0 23 -0.15] Thomsen 2004a 20.17 -0.34 [-0 44 -0.24] Volpe 2001 12.70 -0.34 [-0 50 -0.18]

Total (95% CI) • -0.25 [-0.32, -0.18] Test for heterogeneity: Chi2 = 13.99, df = 7 (P 0.05), I2 = 50.0% Test for overall effect: Z = 6.78 (P < 0.00001)

-1 -0.5 0 0.5 1 Favours treatment Favours control

Review: Plant sterols 2007 Comparison: 37 Dose 1.5-2.0 g/d Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (random) Weight Effect size (random) or sub-category 95% CI % 95% CI

01 Relevant comparisions AbuMweis 2006b 3.00 05 -0.27, 0.17] Algorta Pineda 2005 0.82 62 -1.13, -0.11] Andersson 1999 68 -1.11, -0.25] Cater 2005a 52 -0.95, -0.09] Christiansen 2001a 48 -0.75, -0.21] Cleghorn 2003 27 -0.39, -0.15] De Graaf 2002 48 -0.73, -0.23] Deavarj 2006 16 -0.55, 0.23] Devaraj 2004 34 -0.63, -0.05] Hendriks 1999b 28 -0.40, -0.16] Hendriks 2003 26 -0.48, -0.04] Hyun 2005 2.01 15 -0.44, 0.14] Jakulj 2005 3.00 23 -0.45, -0.01] Jauhiainen 2006 1.19 38 -0.79, 0.03] Jones 1999 0.36 19 -1.99, -0.39] Jones 2000a 3.33 56 -0.76, -0.36] Jones 2003a 08 -0.41, 0.25] Lau 2005a 30 -0.55, -0.05] Lee 2003 12 -0.19, 0.43) Lottenberg 2003 30 -0.40, -0.20] Mussner 2002 26 -0.44, -0.08] Naumann 2003a 17 -0.35, 0.01] Nguyen 1999c 21 -0.37, -0.05] Nigon 2001 23 -0.45, -0.01] Noakes 2002c 40 -0.62, -0.18] Noakes 2005a 27 -0.37, -0.17] Ntanios 2002 28 -0.38, -0.18] Polagruto 2006 .06 -0.39, 0.27] Simons 2002 .58 -1.07, -0.09] Spilburg 2003 70 .04 -0.22, 0.14] Temme 2002 51 .41 -0.51, -0.31] Thomsen 2004b 51 .44 -0.54, -0.34] Vanhanen 1994 0.48 .20 -0.49, 0.89] Vanstone 2002a 2.70 .41 -0.65, -0.17] Yoshida 2006a 2.70 .24 -0.48, 0.00] Subtotal (95% CI) • 100.00 .29 -0.34, -0.24] Test for heterogeneity: Chf 58, df = 34 (P f 0.0003), Is = 51.1 % Test for overall effect: Z = 11.61 (P < 0.00001)

Total (95% CI) • -0.29 [-0.34, -0.24] Test for heterogeneity: Chi2 = 69.58, df = 34 (P10.0003), I2 = 51.1 % Test for overall effect: Z = 11.61 (P < 0.00001)

-1 -0.5 0 0.5 1 Favours treatment Favours control Review: Plant sterols 2007 Comparison:35 Dose 2.1-2.5 g/d Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

01 Relevant comparisions Gylling 1999 —•— 4.67 -0.45 [-0.65, -0.25] 0.97 -0.37 [-0.80, 0.06] Hallikainen 2000a -*- 12.98 -0.45 [-0.57, -0.33] Judd 2002 + 29.21 -0.32 [-0.40, -0.24] 3.25 -0.38 [-0.62, -0.14] Neil 2001 —*— 5.77 -0.34 [-0.52, -0.16] Noakes 2002a —•— 5.77 -0.33 [-0.51, -0.15] Plat 2000a -*- 18.69 -0.29 [-0.39, -0.19] Vissers 2000 -*- 18.69 -0.20 [-0.30, -0.10] Subtotal (95% CI) • 100.00 -0.32 [-0.36, -0.28] Test for heterogeneity: Chi2 = 12.87, c F=8(P = 0.12), I2 = 37.8% Test for overall effect: Z = 14.76 (P < i 1.00001)

Total (95% CI) • 100.00 -0.32 [-0.36, -0.28] Test for heterogeneity: Chi2 = 12.87, c f = 8(P = 0.12), I2 = 37.8% Test for overall effect: Z = 14.76 (P < I 1.00001) 1 1 h- h -1 -0.5 0 0.5 1 Favours treatment Favours control

Review: Plant sterols 2007 Comparison: 36 Dose > 2.5 g/d Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

01 Relevant comparisions Ayesh 1999 i • 0.70 -0.66 0.25] Cater 2005c 4 • 0.53 -0.57 0.10] Cater 2005d —»— 3.79 -0.44 -0.62 0.26] Christiansen 2001b » 1.56 -0.45 -0.72 0.18] Davidson 2001b 0.58 -0.54 .36] Doornbos 2006b »— 0.85 -0.75 0.01] Gylling 1994 —•— 3.07 -0.37 -0.57 0.17] Hendriks 1999c -*- 8.52 -0.30 -0.42 0.18] Matvienko 2002 « 0.53 -0.40 -0.87 .07] Plat & Mensink 2000a •— 1.20 -0.39 -0.70 0.08] Quilez 2003 »- 1.06 -0.26 -0.59 .07] Vanhanen 1993 •- 0.95 -0.30 -0.65 .05] Weststrate 1998a • 76.67 -0.44 -0.48 0.40] Subtotal (95% CI) • 100.00 -0.42 -0.46 0.39] Test for heterogeneity: Chi2 = 10.51, df = 12 (Pi = 0.57), I2 = 0% Test for overall effect: Z = 24.08 (P < 0.00001)

Total (95% CI) • -0.42 [-0.46, -0.39] Test for heterogeneity: Chi2 = 10.51, df = 12 (F : 0.57), I2 = 0% Test for overall effect: Z = 24.08 (P < 0.00001)

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Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

Davidson 2001b 60 -0.09 [-0.54 0.36] Judd 2002 82 -0.32 [-0.40 -0.24] Miettinen 1994a 86 -0.51 [-1.29 0.27] Saito 2006c 36 -0.58 [-0.89 -0.27] Vanhanen1993 24 -0.30 [-0.65 0.05] Vanhanen1994 12 0.20 [-0.49 0.89]

Total (95% CI) • 100.00 3.32 [-0.40, -0.25] 2 Test for heterogeneity: Chi = 6.08, df (P = 0.30), I2 = 17.8% Test for overall effect: Z = 8.71 (P < 0.00001

-1 -0.5 0 0.5 1 Favours treatment Favours control

Review: Plant sterols 2007 Comparison09 Milk and yogurt Outcome: 01 LDL-cholesterol

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

01 Milk and yogurt (most studies with plant sterols and dose >1g/d) Algorta Pineda 2005< • 1.37 -0.62 [-1.13, -0.11] Doornbos 2006a • 3.63 -0.31 [-0.62, 0.00] Hyun2005 • 4.13 -0.15 [-0.44, 0.14] Mensink 2002 •—\— 2.11 -0.24 [-0.65, 0.17] Noakes 2005a 37.13 -0.27 [-0.37, -0.17] Thomsen 2004b 37.13 -0.44 [-0.54, -0.34] Volpe 2001 14.50 -0.34 [-0.50, -0.18] Subtotal (95% CI) 100.00 -0.34 [-0.40, -0.28] Test for heterogeneity: Chi2 = 8.97, jf = 6 (P = 0.18), I2 = 33.1% Test for overall effect: Z = 11.29 (P 0.00001)

Total (95% CI) • 100.00 0.34 -0.40, -0.28] Test for heterogeneity: Chi2 = 8.97, {)f=6(P = 0.18), I2 = 33.1% Test for overall effect: Z = 11.29 (P 0.00001)

-1 -0.5 0 0.5 1 Favours treatment Favours control Review: Plant sterols 2007 Comparison24 Other than fat spread, mayonnaise, salad dressing and milk and yogurt Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

01 Others (most studies with plant stefols and all doses) De Graaf 2002 n. 84 -0.48 [-0.73, -0.23] Deavarj 2006 — 5. 00 -0.16 [-0.55, 0.23] Devaraj 2004 8. 89 -0.34 [-0.63, -0.05] Jauhiainen 2006 — 4. 54 -0.38 [-0.79, 0.03] Jones 2003a — 6. 92 -0.08 [-0.41, 0.25] Matvienko 2002 — 3. 47 -0.40 [-0.87, 0.07] Polagruto 2006 6. 92 -0.06 [-0.39, 0.27] Quilez 2003 6. 92 -0.26 [-0.59, 0.07] Seki 2003 6. 92 0.02 [-0.31, 0.35] Spilburg 2003 24. 69 -0.04 [-0.22, 0.14] Yoshida 2006a 13. 89 -0.24 [-0.48, 0.00] Subtotal (95% CI) • 100. 00 -0.20 [-0.28, -0.11] Test for heterogeneity: Chi2 = 13.22, c|f = 10(P = 0.21), l2 = 24.3% Test for overall effect: Z = 4.38 (P < 0. 0001)

Total (95% CI) • 100. 00 -0. 20 -0.28 -0.11] Test for heterogeneity: Chi2 = 13.22, c = 10(P :0.21), l2 = 24.3% Test for overall effect: Z = 4.38 (P < 0 0001) -1 -0.5 0 0.5 Favours treatment Favours control )

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Q. O cn CD V CQ^ Review: Plant sterols 2007 Comparisont4 Once/d in the afternoon or with main meal Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

01 Studies with plant sterol frequency of intake once/d in the afternoon Doornbos 2006b 1— 6.22 -0.38 [-0.75, -0.01] Matvienko 2002 1— 3.90 -0.40 [-0.87, 0.07] Plat 2000a # 89.88 -0.29 [-0.39, -0.19] Subtotal (95% CI) • 100.00 -0.30 [-0.39, -0.21] Test for heterogeneity: Chi2 = 0.39, df = 2(P = 0.82), l2 = 0% Test for overall effect: Z = 6.33 (P • 0.00001)

Total (95% CI) • 100.00 -0.30 [-0.39, -0.21] 2 2 Test for heterogeneity: Chi = 0.39; df = 2 (P = 0.82), I = 0% Test for overall effect: Z = 6.33 (P • 0.00001)

-1 -0.5 0 0.5 1 Favours treatment Favours control

Review: Plant sterols 2007 Comparisont3 Once/d in the morning Outcome: 01 LDL-cholesterol mmol/L

Study Effect size (fixed) Weight Effect size (fixed) or sub-category 95% CI % 95% CI

01 Studies with plant sterol frequerjcy of intake once/d in the morning AbuMweis 2006b — 43.77 -0.05 -0.27, 0.17] Doornbos 2006a *- 20.69 -0.31 -0.62, 0.00] Hyun 2005 1 — 23.54 -0.15 -0.44, 0.14] Lau 2005b 1 — 12.01 -0.19 -0.60, 0.22] Subtotal (95% CI) 4 100.00 -0.14 -0.29, 0.00] Test for heterogeneity: Chi2 = 1.86, df = 3 (P = 0.60), I2 = 0% Test for overall effect: Z = 1.98 (P = 0.05)

Total (95% CI) 4P 100.00 -0.14 [-0.29, 0.00] Test for heterogeneity: Chi2 = 1.86, df = 3 (P = 0.60), I2 = 0% Test for overall effect: Z = 1.98 (P : 0.05)

-1 -0.5 0 0.5 1 Favours treatment Favours control 149

CHAPTER 7.

FINAL SUMMARY AND CONCLUSION

The overall objective of this thesis was to investigate factors including type of plant sterol preparation, frequency of consumption and time of intake of single dose of plant sterols as well as baseline subjects' characteristics, which may affect plant sterols as cholesterol- lowering agents. To meet this objective three randomized, placebo-controlled, crossover- feeding trials were conducted. The use of crossover design minimizes potential confounding because every subject serves as his/her own control. Thus, the design used in this thesis implements many of the expectations of a well-conducted dietary intervention study. The clinical trials score 3 out of 5 on Jadad scale due to the absence of a double blind design as the candidate who was in contact with the subjects, was also responsible about preparing the treatments and the diet cards. All diets fed in the three clinical trials were healthy in terms of fat and cholesterol contents. Thus, we followed the suggestions of consuming plant sterols in adjunct to a healthy diet. Moreover, compliance to the study products was optimal since subjects consumed the control and the plant sterol- containing products under supervision. In addition, subjects' weight did not differ between different study periods and thus any differences observed in lipid profile are solely due to the study treatments. The following sections summarize the key points of the findings of this thesis, discuss the implications of the findings, the limitations and provide suggestions for future work.

In reviewing the literature on plant sterols, human studies have been carried out using sterols esterified to fatty acids from plant oils, mainly rape seed, sunflower or soybean oil. 150

Alternatively, a novel approach to enhance plant sterol solubility in food matrices is to

esterify plant sterols to fatty acids derived from fish oil. Esterification of plant sterols to

fatty acids from fish oil will also increase the intake of these healthy fatty acids.

Therefore, the objective of the first clinical trial was to examine the effects on plasma

lipids of traditional as well as novel forms of plant sterols: those combined with, or

esterified to, fish-oil fatty acids. However, since traditional forms of plant sterols did not

reduce blood cholesterol levels; the efficacy of the novel forms of plant sterols could not

be confirmed from the first clinical trial. The lack of efficacy of different plant sterols in

the first study could be attributed to the consumption of plant sterols as a single morning

dose. Plant sterols were given in the morning to ensure compliance as the subjects

consumed only the breakfast meal under supervision.

Thus, to further investigate the efficacy of single dose of plant sterols we conducted a

second clinical trial to examine the effects of plant sterols consumed throughout the day

or once a day in the morning on plasma LDL-cholesterol levels and cholesterol kinetics.

The results from the second study confirmed that consumption of plant sterols once a day

in the morning may not result in the most potential reduction in LDL levels. The second

study also showed that the consumption of 1.8 g/d of plant sterols distributed over the day

for 5 days lowered LDL levels by 0.21 mmol/L, whereas the consumption of the same

dose once a day with breakfast did not significantly lower LDL, in spite of a reduction in

cholesterol absorption efficiency relative to the control as measured by single isotope

single tracer method. We used the single isotope single tracer method because it is less invasive and less expensive than the dual isotope method. Unfortunately, the single 151 isotope single tracer method was not sensitive enough in detecting differences in cholesterol absorption efficiency due to different frequency of consumption. The single stable isotope single tracer method used in this study measures only the efficiency of cholesterol absorption rather than the absolute amount that depends on endogenous biliary cholesterol secretion (Ostlund et al., 2002). In addition, the single stable isotope single tracer method used to assess cholesterol absorption efficiency in this study reflects relative dietary cholesterol absorption efficiency to various test diets and not that of net . sterol, including bile acid. Whether, the frequency of intake of plant sterols affects absolute amount of cholesterol absorption and/or bile acid excretion was not tested in this study. Nevertheless, the third review article presented in Appendix 2 summarizes the relationship between cholesterol absorption and synthesis and shows that when one of these parameters increases the other decreases as a feedback up-regulation. Consequently, since cholesterol fractional synthesis rate (FSR) tended to increase when plant sterols were consumed with each of the 3 daily meals, this increase could be attributed to feedback up-regulation of cholesterol FSR in response to reduced sterol absorption. The second study only looked at cholesterol absorption and synthesis as aspects in cholesterol metabolism. Other aspects in cholesterol metabolism need to be investigated, which may also help in understanding the mechanism of action of plant sterols, in particular the action of single dose vs. multiple doses. Some data indicate that the mechanisms involved in the reduction of total and LDL-cholesterol by plant sterols is not only decreased intestinal absorption of cholesterol as even subcutaneously administrated plant sterols reduced circulating cholesterol levels in hamsters (Vanstone et al., 2001). Other potential mechanism of action of plant sterols as cholesterol lowering agents include effects on production of lipoproteins from the liver and the intestines (Ho and Pal, 2005), and 152 conversion of cholesterol into bile acids (Trautwein et al., 2002). More studies are needed to investigate whether the frequency of intake of plant sterols affect the production of lipoproteins from the liver and the intestine as well as the conversion of cholesterol into bile acids. Moreover, long-term studies are needed to confirm the results of the second study and to test the effect of a single dose consumed at different time points of the day.

Plant sterols are not only incorporated in vegetable oil spread, but also in a wide variety of products including products such as milk, yoghurt and cereal bars. In addition, it is believed that the consumption of plant sterols-containing products once a day rather than in multiple doses will increase consumer compliance to the treatment. Thus, a third study was conducted to compare efficacy of new breakfast plant sterol enriched yoghurt consumed with the morning or the evening meals. This work showed that the consumption of low-fat yoghurt containing 1.6 g/d plant sterol failed to significantly decrease LDL-cholesterol when consumed once a day in morning or evening and resulted in a large individual variability in responses. A substantial reduction in LDL-cholesterol was only observed in subjects with low cholesterol absorption efficiency irrespective of the time of intake. The findings of this study call attention for further investigations on the effect of dosing frequency in improving the efficacy of plant sterols in subjects with different baseline cholesterol absorption efficiency, and to look into genetic components behind such a wide LDL responses to dietary treatments. These suggestions for future work are concur with the trend of moving from universal recommendations towards individualized recommendations. There is limited but suggestive evidence that genetic variation may contribute to the heterogeneity in lipid responsiveness to dietary changes

(Masson and McNeill, 2005). Cholesterol metabolism is regulated by a number of 153 different proteins, many of which have been shown to possess genetic polymorphisms as was described in details in Review Article 3 that appears in Appendix 2. However, there are few studies that looked at genetic variation in genes involved in sterol metabolism and heterogeneity in plasma plant sterols and LDL responsiveness to dietary intervention with plant sterol enriched products. Apolipoprotein E phenotype, which have been shown to be correlated with cholesterol absorption (Kesaniemi et al., 1987), have been reported to affect plant sterol cholesterol lowering efficacy in one study (Vanhanen et al., 1993) but not others (Hallikainen et al., 2000b; Plat and Mensink, 2000). In another study, the genotype of apolipoprotein A-IV, scavenger receptor-BI, 3-hydroxy-3-methyl-coenzyme

A reductase, and cholesterol ester transfer did not affect cholesterol lowering effects of plant stanol (Plat and Mensink, 2002b). With regard to variation in plasma plant sterols levels, variants in ABCG8, but not ABCG5 have been linked to lower plasma sterol concentrations (Berge et al., 2002). In response to dietary plant sterols, the T400K variant in ABCG8 accounted for differences in serum sterol concentration and predicted the subject responsiveness to changes in serum sterols but not serum lipoprotein profile (Plat et al., 2005). A recent investigation identified that various mutations in NPC1L1 were common in individuals with low cholesterol absorption and that variant alleles were associated with a 10% lower LDL-cholesterol (Cohen et al., 2006). However, more detailed examination of the relationship between polymorphisms in ABCG5 and 8 and

NPC1L1 and responsiveness to plant sterol intervention is needed. 154

The degree of placebo-adjusted reduction in LDL-cholesterol levels caused by plant sterols differed also widely between studies, ranging from 5% to 15%. The two review articles included in this thesis showed that clinical investigators have used a variety of food as carriers for plant sterols/stanols; differing concentrations of plant sterols/stanols consumed at differing frequency and time of the day; and subjects with different baseline characteristics. The final work of this thesis was a meta-analysis that aimed to further quantify the effect of consumption of plant sterols on LDL levels and to explore potential causes of heterogeneity in effect size between studies. The performed meta-analysis showed that the consumption of plant sterols/stanols containing food products reduced significantly LDL levels by 0.31 mmol/L. The reductions in LDL levels were larger in individuals with high baseline LDL levels than in those with normal to borderline baseline LDL levels. The reduction in LDL was greater when plant sterols were incorporated into fat spreads, mayonnaise and salad dressing, milk and yoghurt comparing with other food products. Plant sterols consumed a single morning dose did not reduce LDL levels.

A major finding from the clinical trials and the meta-analysis performed in this thesis is that the consumption of plant sterols as single dose in the morning does not reduce LDL levels. The conventional proposed mechanism of action of plant sterols involves plant sterols competing with cholesterol for incorporation into the micelles (Heinemann et al.,

1991) indicative of that plant sterols should be consumed at each meal to achieve full potential cholesterol-lowering effect (Plat et al., 2000). The efficacy of plant sterols as a cholesterol-lowering agent may demonstrate a time-of-day variation, possibly coinciding with the diurnal rhythm of cholesterol metabolism. Diurnal rhythm in cholesterol 155 synthesis has been shown in humans (Cella et al., 1995; Jones et al., 1992; Jones and

Schoeller, 1990) where cholesterol fractional synthetic rate values peaked at 6:00 h and were lowest during the daytime period. Moreover, bile acid synthesis in humans has also a diurnal rhythm that is opposite from the diurnal rhythm of cholesterol synthesis

(Galman et al., 2005). To date, no animal or human studies have looked at the diurnal rhythm in cholesterol absorption. However, since cholesterol synthesis and absorption are inversely related, one can speculate that cholesterol absorption is low early in the morning and increases during the daytime period. The presence of diurnal variation of cholesterol metabolism could be supported by results from rat studies which have shown that hepatic cholesterol synthesis is low at noon (Edwards et al., 1972) and the gene expression and the activity of sterol 12a-hydroxylase (CYP8B) is maximal at 13:00-16:00 h (Ishida et al.,

2000). Sterol 12a-hydroxylase is known to be responsible for synthesis of cholic acid and determination of cholic acid to chenodexycholic acid ratio (Vlahcevic et al., 2000).

Cholic acid is more effective in facilitating cholesterol absorption in the intestine

(Chiang, 2004). Given that it is possible that a single dose of plant sterols taken in the morning may not lead to optimal cholesterol reduction. If cholesterol absorption is low early in the morning, then further lowering of cholesterol absorption might not lead to a decrease in blood cholesterol levels.

Since we were looking at testing the efficacy of novel forms, different frequency regimes, and the efficacy of single dose given at different time of the day, plant sterol interventions were used as a monotherapy. Nevertheless, several studies have combined plant sterols with other components known to have favorable effect on lipid profile. Dietary intake of plant sterol-enriched margarines in combination with statin therapy has been shown to 156 have a cumulative effect on reduction of LDL-cholesterol levels. In a study by Simons

(Simons, 2002), 400 mg of cerivastatin reduced LDL-cholesterol levels by 32% compared to a placebo, whereas 2 g/d of plant sterol margarine consumed for 4 weeks reduced

LDL-cholesterol levels by 8% compared to a placebo. However, the intake of both plant sterol margarine and cerivastatin reduced LDL-cholesterol levels by 39% compared to a placebo (Simons, 2002). Similarly, subjects who consumed plant stanols incorporated into a canola oil-based margarine in combination with statin therapy, showed a 10% greater reduction in LDL-cholesterol than did those who consumed a placebo spread combined with statins for 8 weeks (Blair et al., 2000). This reduction in LDL-cholesterol levels was greater than could be achieved by doubling the dose of statin, which would normally produce a further reduction of about 6% in LDL-cholesterol levels (Blair et al.,

2000). The magnitude of reduction in LDL observed in our studies is about 6 %. A combination of plant sterols with statin therapy will serve as an alternative approach to doubling the statin dose, without producing the associated increase in side effects.

Moreover, the addition of plant sterols/stanols to statin therapy is considered a cost- saving strategy in the reduction of LDL-cholesterol levels (Vorlat et al., 2003).

Plant sterol-enriched spreads have also been used with other dietary components known to improve lipid profiles. A portfolio diet containing 1 g of plant sterols/1000 kcal/d, soy protein, viscous fibers, and almonds, reduced LDL-cholesterol levels in hypercholesterolemic individuals by 28% (Jenkins et al., 2003). Shrestha et al. (Shrestha et al., 2006) showed that dietary treatment with 2.5 g/d plant sterols and 6.68 g/d psyllium for 4 weeks decreased LDL-cholesterol levels and the number of the smaller subtractions of LDL and HDL particles, resulting in a less atherogenic lipoprotein profile. In another 157 study, the combination of consuming a plant sterol-enriched spread with exercise for 8 weeks brought about the most favorable modification in lipid profiles compared to intake of plant sterols or performing exercise alone (Varady et al., 2004). Combining a plant sterol-enriched spread and exercise reduced total cholesterol levels by 8.3% and triacylglycerol levels by 13.3%, and increased HDL- cholesterol levels by 7.5% from baseline (Varady et al., 2004). Plant sterols can thus be used in combination with a healthy diet, statin therapy or physical activity to improve lipid profiles. The key message is that plant sterol/stanol-enriched spreads should be part of a healthy diet and not a substitute for it.

The investigations in the aforementioned studies were not without limitations. The three human studies conducted for this thesis were strictly controlled as food was precisely weighted, all meals were isocaloric and energy intake was constant over the study periods. Therefore, the study designs do not represent a free living situation where caloric intake and macronutrient distribution could vary from meal-to-meal as well as day-to-day.

Nevertheless, the high level of control applied in this thesis allowed us to examine the efficacy of plant sterols given at different dosage frequency and time of the day.

The present thesis explored the effect of frequency dosage of plant sterols and time of intake on cholesterol metabolism over 1-4 weeks. Previous studies have shown that plant sterols start to lower LDL levels as early as 1 day (Hallikainen et al., 2002) or 1 week

(Mensink et al., 2002). However, a longer feeding period would have been preferable to see if a sustainable reduction in LDL could be achieved by single dose of plant sterols. 158

The study population is also an important limitation in the context of its clinical implications. Subjects involved in the clinical studies were middle aged males and postmenopausal females. The study subjects were overweight with borderline high levels of LDL-cholesterol who were not taking any drugs known to affect lipid metabolism. The study population in this thesis may have responded differently to some of the parameters measured. Therefore, results obtained in this thesis may be representative of middle age adults with moderate hypercholesterolemia and hence care must be considered in extrapolation of the results to other individuals.

Another concern is the effect of subjects' genetic make up on the responsiveness of LDL levels to plant sterol interventions. In one study we identified that responders to single dose of plant sterols were those subjects with low baseline cholesterol absorption efficiency. Unfortunately, due to the limited sample size we were not able to further explore what single nucleotide polymorphism is responsible of such a phenotype.

Despite these limitations this thesis has contributed to the field of plant sterols and cholesterol metabolism. In conclusion, data presented here demonstrated that: (i) consumption of single morning dose of traditional and novel forms of plant sterols did not reduce LDL-cholesterol levels; (ii) dosing frequency of plant sterols may affect their action as cholesterol-lowering agents as short-term consumption of 1.8 g/d of plant sterols distributed over the day lowered LDL levels, whereas the consumption of the same dose once a day with breakfast did not lower LDL, in spite of a reduction in cholesterol absorption efficiency; (iii) cholesterol fractional synthesis rate increased when plant sterols were consumed with breakfast, lunch and dinner but not as a single dose with 159 breakfast, which could be attributed to feedback up-regulation of cholesterol FSR in response to reduced sterol absorption related to frequent dosage; (iv) no overall reduction in LDL-cholesterol levels were observed when a 1.6 g/d of single dose of plant sterols provided in yoghurt was consumed with breakfast or dinner; however, a reduction in

LDL-cholesterol was observed in subjects with low cholesterol absorption efficiency irrespective of time of intake of single dose of plant sterols provided in yoghurt; and (v) consumption of plant sterol/stanol containing foods reduced significantly LDL levels. The reduction in LDL was more important in studies conducted on individuals with high baseline LDL levels, when plant sterols were incorporated into fat spreads, mayonnaise and salad dressing, milk and yoghurt compared with other food products, and when plant sterols were consumed in 2-3 portions/d or as single dose with lunch or main meal, but not with breakfast. The work from this thesis has identified some factors that affect efficacy of plant sterols as cholesterol-lowering agents, therefore, these findings could be used to improve plant sterol efficacy, as well as to establish future health claims.

In addition, for future investigation, the findings of this thesis as well the limitations of the current studies should be taken into account to examine what was not explored in this thesis. For example, an alternative design to test our objectives would be to implement a free living situation where subjects would consume the study interventions without changing their habitual diet. Moreover, the thesis objectives could be replicated in other adult populations, i.e. young and elderly adults, and in adults with near or above optimal or high and very high levels of LDL-cholesterol. In order to better understand the mechanism of action behind the effect of dosing frequency and time of intake of single dose of plant sterols; aspects of cholesterol metabolism aside from absorption and 160 synthesis should be investigated in the future. The effect of dosing frequency on bile acid synthesis as well as on molecular aspects of cholesterol metabolism may offer more insights into the mechanism of action of plant sterols. Additional investigation in the area of responsiveness to plant sterols and gene-environment interaction should be considered.

Such studies are to be carried out with adequate sample size, and should explore multiple genes encoding proteins involved in cholesterol metabolism. 161

REFERENCES

- AbuMweis, S.S., Nicolle, C, and Jones, P.J. (2006a). Cholesterol-lowering action

of plant sterol-enriched products. Food Sci Tech Bull 2, 101-110.

- AbuMweis, S.S., Vanstone, C.A., Ebine, N., Kassis, A., Ausman, L.M., Jones,

P.J.H., and Lichtenstein, A.H. (2006b). Intake of a single morning dose of

standard and novel plant sterol preparations for 4 weeks does not dramatically

affect plasma lipid concentrations in humans. J Nutr 136,1012-1016.

- Ahrens, E.H., and Boucher, C.A. (1978). Composition of a simulated American

diet J Am Diet Assoc 73, 613-620.

- Alhassan, S., Reese, K.A., Mahurin, J., Plaisance, E.P., Hilson, B.D., Garner, J.C.,

Wee, S.O., and Grandjean, P.W. (2006). Blood lipid responses to plant stanol ester

supplementation and aerobic exercise training. Metab-Clin Exp 55, 541-549.

- Altmann, S.W., Davis, H.R., Zhu, L.J., Yao, X.R., Hoos, L.M., Tetzloff, G., Iyer,

S.P.N., Maguire, M., Golovko, A., Zeng, M., et al. (2004). Niemann-Pick CI like

1 protein is critical for intestinal cholesterol absorption. Science 303, 1201-1204.

- Amundsen, A.L., Ose, L., Nenseter, M.S., and Ntanios, F.Y. (2002). Plant sterol

ester-enriched spread lowers plasma total and LDL cholesterol in children with

familial hypercholesterolemia. Am J Clin Nutr 76, 338-344.

- Andersson, A., Karlstrom, B., Mohsen, R., and Vessby, B. (1999). Cholesterol-

lowering effects of a stanol ester-containing low-fat margarine used in conjunction

with a strict lipid-lowering diet. Eur Heart J Suppl 1, S80-S90.

- Awad, A.B., and Fink, C.S. (2000). Phytosterols as anticancer dietary

components: Evidence and mechanism of action. J Nutr 130,2127-2130. 162

- Ayesh, R., Weststrate, J.A., Drewitt, P.N., and Hepburn, P.A. (1999). Safety

evaluation of phytosterol esters. Part 5. Faecal short-chain fatty acid and

microflora content, faecal bacterial enzyme activity and serum female sex

hormones in healthy normolipidaemic volunteers consuming a controlled diet

either with or without a phytosterol ester-enriched margarine. Food Chem Toxicol

37,1127-1138.

- Beaumier-Gallon, G., Dubois, C, Senft, M., Vergnes, M.F., Pauli, A.M., Portugal,

H., and Lairon, D. (2001). Dietary cholesterol is secreted in intestinally derived

chylomicrons during several subsequent postprandial phases in healthy humans.

Am J Clin Nutr 73, 870-877.

- Berge, K.E., von Bergmann, K., Lutjohann, D., Guerra, R., Grundy, S.M., Hobbs,

H.H., and Cohen, J.C. (2002). Heritability of plasma noncholesterol sterols and

relationship to DNA sequence polymorphism in ABCG5 and ABCG8. J Lipid Res

43,486-494.

- Berger, A., Jones, P.J., and Abumweis, S.S. (2004). Plant sterols: factors affecting

their efficacy and safety as functional food ingredients. Lipids Health Dis 3, 5.

- Blair, S.N., Capuzzi, D.M., Gottlieb, S.O., Nguyen, T., Morgan, J.M., and Cater,

N.B. (2000). Incremental reduction of serum total cholesterol and low-density

lipoprotein cholesterol with the addition of plant stanol ester-containing spread to

statin therapy. Am J Cardiol 86,46-52.

- Bosner, M.S., Lange, L.G., Stenson, W.F., and Ostlund, R.E. (1999). Percent

cholesterol absorption in normal women and men quantified with dual stable

isotopic tracers and negative ion mass spectrometry. J Lipid Res 40, 302-308. 163

- Bosner, M.S., Ostlund, R.E., Osofisan, O., Grosklos, J., Fritschle, C, and Lange,

L.G. (1993). Assessment of percent cholesterol absorption in humans with stable

isotopes. J Lipid Res 34, 1047-1053.

- Bouic, PJ.D. (2001). The role of phytosterols and phytosterolins in immune

modulation: a review of the past 10 years. Curr Opin Clin Nutr Metab Care 4,

471-475.

- Bouic, PJ.D. (2002). Sterols and sterolins: new drugs for the immune system?

Drug Discov Today 7, 775-778.

- Bruckner, G. (2000). Fatty acids and cardiovascular diseases (New York, Marcel

Dekker Inc).

- Calpe-Berdiel, L., Escola-Gil, J.C., Ribas, V., Navarro-Sastre, A., Garces-Garces,

J., and Blanco-Vaca, F. (2005). Changes in intestinal and liver global gene

expression in response to a phytosterol-enriched diet. Atherosclerosis 181, 75-85.

- Cater, N.B., Garcia-Garcia, A.B., Vega, G.L., and Grundy, S.M. (2005).

Responsiveness of plasma lipids and lipoproteins to plant stanol esters. Am J

Cardiol 96,23D-28D.

- Cella, L.K., Vancauter, E., and Schoeller, D.A. (1995). Effect of meal timing on

diurnal rhythm of human cholesterol synthesis. Am J Physiol-Endocrinol Metab

32, E878-E883.

- Chen, H.C. (2001). Molecular mechanisms of sterol absorption. J Nutr 131, 2603-

2605.

- Chiang, J.Y.L. (2004). Regulation of bile acid synthesis: pathways, nuclear

receptors, and mechanisms. J Hepatol 40, 539-551. 164

- Christiansen, L.I., Lahteenmaki, P.L.A., Mannelin, M.R., Seppanen-Laakso, T.E.,

Hiltunen, R.V.K., and Ylirausi, J.K. (2001). Cholesterol-lowering effect of

spreads enriched with microcrystal line plant sterols in hypercholesterolemic

subjects. Eur J Nutr 40, 66-73.

- Cleeman, J.I., Grundy, S.M., Becker, D., Clark, L.T., Cooper, R.S., Denke, M.A.,

Howard, W.J., Hunninghake, D.B., Illingworth, D.R., Luepker, R.V., et al. (2001).

Executive summary of the Third Report of the National Cholesterol Education

Program (NCEP) expert panel on detection, evaluation, and treatment of high

blood cholesterol in adults (Adult Treatment Panel III). JAMA-J Am Med Assoc

285, 2486-2497.

- Cleghorn, C.L., Skeaff, CM., Mann, J., and Chisholm, A. (2003). Plant sterol-

enriched spread enhances the cholesterol-lowering potential of a fat-reduced diet.

Eur J Clin Nutr 57, 170-176.

- Clifton, P.M., Noakes, M., Sullivan, D., Erichsen, N., Ross, D., Annison, G.,

Fassoulakis, A., Cehun, M., and Nestel, P. (2004). Cholesterol-lowering effects of

plant sterol esters differ in milk, yoghurt, bread and cereal. Eur J Clin Nutr 58,

503-509.

- Cohen, J.C., Pertsemlidis, A., Fahmi, S., Esmail, S., Vega, G.L., Grundy, S.M.,

and Hobbs, H.H. (2006). Multiple rare variants in NPC1L1 associated with

reduced sterol absorption and plasma low-density lipoprotein levels. Proc Natl

Acad Sci U S A 103,1810-1815.

- Colgan, H.A., Floyd, S., Noone, E.J., Gibney, M.J., and Roche, H.M. (2004).

Increased intake of fruit and vegetables and a low-fat diet, with and without low- 165

fat plant sterol-enriched spread consumption: effects on plasma lipoprotein and

carotenoid metabolism. J Hum Nutr Diet 17, 561-569.

- Connor, W.E. (2000). Importance of n-3 fatty acids in health and disease. Am J

Clin Nutr 77, 171S-175S.

- Danesh, J., Collins, R., and Peto, R. (2000). Lipoprotein(a) and coronary heart

disease - Meta-analysis of prospective studies. Circulation 102, 1082-1085.

- Davidson, M.H., Maki, K.C., Umporowicz, D.M., Ingram, K.A., Dicklin, M.R.,

Schaefer, E., Lane, R.W., McNamara, J.R., Ribaya-Mercado, J.D., Perrone, G., et

al. (2001). Safety and tolerability of esterified phytosterols administered in

reduced-fat spread and salad dressing to healthy adult men and women. J Am Coll

Nutr 20, 307-319.

- Davis, H.R., Zhu, L.J., Hoos, L.M., Tetzloff, G., Maguire, M., Liu, J.J., Yao,

X.R., Iyer, S.P.N., Lam, M.H., Lund, E.G., et al. (2004). Niemann-Pick CI like 1

(NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key

modulator of whole-body cholesterol homeostasis. J Biol Chem 279, 33586-

33592.

- de Graaf, J., Nolting, P., van Dam, M., Belsey, E.M., Kastelein, J.J.P., Pritchard,

P.H., and Stalenhoef, A.F.H. (2002). Consumption of tall oil-derived phytosterols

in a chocolate matrix significantly decreases plasma total and low-density

lipoprotein-cholesterol levels. Br J Nutr 88,479-488.

- de Vries, J.H.M., Jansen, A., Kromhout, D., Bovenkamp, P.v.d., Staveren,

W.A.V., Mensink, R.P., and Katan, M.B. (1997). The fatty acid and sterol content 166

of food composites of middle-aged men in seven countries. J Food Comp Anal 10,

115-141.

- Deeks, J.J., Higgins, J.P.T., Altman, D.G., and editors. (2005). Analysing and

presenting results. In Cochrane Handbook for Systematic Reviews of

Interventions 425 [updated May 2005]; Section 8

http://wwwcochraneorg/resources/handbook/hbookhtm, J.P.T. Higgins, S. Greens,

and editors., eds.

- Denke, M.A. (1995). Lack of efficacy of low-dose sitostanol therapy as an adjunct

to a cholesterol-lowering diet in men with moderate hypercholesterolemia. Am J

Clin Nutr 61, 392-396.

- Devaraj, S., Autret, B.C., and Jialal, I. (2006). Reduced-calorie orange juice

beverage with plant sterols lowers C-reactive protein concentrations and improves

the lipid profile in human volunteers. Am J Clin Nutr 84, 756-761.

- Devaraj, S., Jialal, I., and Vega-Lopez, S. (2004). Plant sterol-fortified orange

juice effectively lowers cholesterol levels in mildly hypercholesterolemic healthy

individuals. Arterioscler Thromb Vase Biol 24, E25-E28.

- Doornbos, A.M.E., Meynen, E.M., Duchateau, G., van der Knaap, H.C.M., and

Trautwein, E.A. (2006). Intake occasion affects the serum cholesterol lowering of

a plant sterol-enriched single-dose yoghurt drink in mildly hypercholesterolaemic

subjects. Eur J Clin Nutr 60, 325-333.

- Edwards, P.A., Muroya, H., and Gould, R.G. (1972). In vivo demonstration of the

circadian thythm of cholesterol biosynthesis in the liver and intestine of the rat. J

Lipid Res 13, 396-401. 167

- Ewart, H.S., Cole, L.K., Kralovec, J., Layton, H., Curtis, J.M., Wright, J.L.C., and

Murphy, M.G. (2002). Fish oil containing phytosterol esters alters blood lipid

profiles and left ventricle generation of thromboxane A(2) in adult guinea pigs. J

Nutr 132, 1149-1152.

- Field, F.J., Born, E., and Mathur, S.N. (2004). Stanol esters decrease plasma

cholesterol independently of intestinal ABC sterol transporters and Niemann-Pick

CI-like 1 protein gene expression. J Lipid Res 45, 2252-2259.

- Fisher, R.S., Malmud, L.S., Bandini, P., and Rock, E. (1982). Gastric emptying of

a physiologic mixed solid-liquid meal Clin Nucl Med 7, 215-221.

- Fletcher, B., Berra, K., Ades, P., Braun, L.T., Burke, L.E., Durstine, J.L., Fair,

J.M., Fletcher, G.F., Goff, D., Hayman, L.L., et al. (2005). Managing abnormal

blood lipids - A collaborative approach. Circulation 112, 3184-3209.

- Folch, J., Lees, M., and Stanley, G.H.S. (1957). A simple method for the isolation

and purification of total lipides from animal tissues. J Biol Chem 226, 497-509.

- Friedewald, W.T., Fredrick, D.S., and Levy, R.I. (1972). Estimation Of

Concentration Of Low-Density Lipoprotein Cholesterol In Plasma, Without Use

Of Preparative Ultracentrifuge. Clin Chem 18,499-502.

- Galman, C, Angelin, B., and Rudling, M. (2005). Bile acid synthesis in humans

has a rapid diurnal variation that is asynchronous with cholesterol synthesis.

Gastroenterology 129,1445-1453.

- Gylling, H., and Miettinen, T.A. (1994). Serum-cholesterol and cholesterol and

cipoprotein metabolism in hypercholesterolemic NIDDM patients before and

during sitostanol ester-margarine treatment. Diabetologia 37, 773-780. - Gylling, H., and Miettmen, T.A. (1999). Cholesterol reduction by different plant

stanol mixtures and with variable fat intake. Metab-Clin Exp 48, 575-580.

- Gylling, H., and Miettinen, T.A. (2002). Baseline intestinal absorption and

synthesis of cholesterol regulate its response to hypolipidaemic treatments in

coronary patients. Atherosclerosis 160, 477-481.

- Gylling, H., Radhakrishnan, R., and Miettinen, T.A. (1997). Reduction of serum

cholesterol in postmenopausal women with previous myocardial infarction and

cholesterol malabsorption induced by dietary sitostanol ester margarine - Women

and dietary sitostanol. Circulation 96, 4226-4231.

- Hallikainen, M., Sarkkinen, E., Wester, I., and Uusitupa, M. (2002). Short-term

LDL cholesterol-lowering efficacy of plant stanol esters. BMC Cardiovascular

Disordorders 2,14.

- Hallikainen, M.A., Sarkkinen, E.S., Gylling, H., Erkkila, A.T., and Uusitupa,

M.I.J. (2000a). Comparison of the effects of plant sterol ester and plant stanol

ester-enriched margarines in lowering serum cholesterol concentrations in

hypercholesterolaemic subjects on a low-fat diet. Eur J Clin Nutr 54, 715-725.

- Hallikainen, M.A., Sarkkinen, E.S., and Uusitupa, M.I.J. (1999). Effects of low-

fat stanol ester enriched margarines on concentrations of serum carotenoids in

subjects with elevated serum cholesterol concentrations. Eur J Clin Nutr 53, 966-

969.

- Hallikainen, M.A., Sarkkinen, E.S., and Uusitupa, M.I.J. (2000b). Plant stanol

esters affect serum cholesterol concentrations of hypercholesterolemic men and

women in a dose-dependent manner. J Nutr 130,161-116. 169

- Hallikainen, M.A., and Uusitupa, M.I.J. (1999). Effects of 2 low-fat stanol ester-

containing margarines on serum cholesterol concentrations as part of a low-fat

diet in hypercholesterolemic subjects. Am J Clin Nutr 69, 403-410.

- Hamprech, B., Nussler, C, and Lynen, F. (1969). Rhythmic changes of

hydroxymethylglutaryl coenzyme a reductase activity in livers of fed and fasted

rats. FEBS Lett 4, 117-121.

- Heinemann, T., Kullakublick, G.A., Pietruck, B., and Vonbergmann, K. (1991).

Mechanisms of Action of Plant Sterols on Inhibition of Cholesterol Absorption -

Comparison of Sitosterol and Sitostanol. Eur J Clin Pharmacol 40, S59-S63.

- Hendriks, H.F.J., Brink, E.J., Meijer, G.W., Princen, H.M.G., and Ntanios, F.Y.

(2003). Safety of long-term consumption of plant sterol esters-enriched spread.

Eur J Clin Nutr 57, 681-692.

- Hendriks, H.F.J., Weststrate, J.A., van Vliet, T., and Meijer, G.W. (1999). Spreads

enriched with three different levels of vegetable oil sterols and the degree of

cholesterol lowering in normocholesterolaemic and mildly hypercholesterolaemic

subjects. Eur J Clin Nutr 53, 319-327.

- Hirai, K., Shimazu, C, Takezoe, R., and Ozeki, Y. (1986). Cholesterol,

phytosterol and polyunsaturated fatty acid levels in 1982 and 1957 Japanese diets.

J Nutr Sci Vitaminol 32, 363-372.

- Ho, K.J. (1975). Effect of cholesterol feeding on circadian rhythm of hepatic and

intestinal cholesterol biosynthesis in hamsters. Proc Soc Exp Biol Med 150, 271-

277. - Ho, K.J. (1979). Orcadian rhythm of cholesterol biosynthesis: dietary regulation

in the liver and small intestine of hamsters. Int J Chronobiol 6, 39-50.

- Ho, S.S., and Pal, S. (2005). Margarine phytosterols decrease the secretion of

atherogenic lipoproteins from HepG2 liver and Caco2 intestinal cells.

Atherosclerosis 182, 29-36.

- Homma, Y., Ikeda, I., Ishikawa, T., Tateno, M., Sugano, M., and Nakamura, H.

(2003). Decrease in plasma low-density lipoprotein cholesterol, apolipoprotein B,

cholesteryl ester transfer protein, and oxidized low-density lipoprotein by plant

stanol ester-containing spread: A randomized, placebo-controlled trial. Nutrition

19, 369-374.

- Hyun, Y.J., Kim, O.Y., Kang, J.B., Lee, J.H., Jang, Y., Liponkoski, L., and Salo,

P. (2005). Plant stanol esters in low-fat yogurt reduces total and low-density

lipoprotein cholesterol and low-density lipoprotein oxidation in

normocholesterolemic and mildly hypercholesterolemic subjects. Nutr Res 25,

743-753.

- Ishida, H., Yamashita, C, Kuruta, Y., Yoshida, Y., and Noshiro, M. (2000).

Insulin is a dominant suppressor of sterol 12 alpha-hydroxylase P450 (CYP8B)

expression in rat liver: Possible role of insulin in circadian rhythm of CYP8B. J

Biochem 127, 57-64.

- Jadad, A.R., Moore, R.A., Carroll, D., Jenkinson, C, Reynolds, D.J.M.,

Gavaghan, D.J., and McQuay, H.J. (1996). Assessing the quality of reports of

randomized clinical trials: Is blinding necessary? Controlled Clin Trials 17,1-12. 171

- Jakulj, L., Trip, M.D., Sudhop, T., von Bergmann, K., Kastelein, J.J.P., and

Vissers, M.N. (2005). Inhibition of cholesterol absorption by the combination of

dietary plant sterols and ezetimibe: effects on plasma lipid levels. J Lipid Res 46,

2692-2698.

- Jauhiainen, T., Salo, P., Niittynen, L., Poussa, T., and Korpela, R. (2006). Effects

of low-fat hard cheese enriched with plant stanol esters on serum lipids and

apolipoprotein B in mildly hypercholesterolaemic subjects. Eur J Clin Nutr 60,

1253-1257.

- Jenkins, D.J.A., Kendall, C.W.C., Marchie, A., Faulkner, D.A., Wong, J.M.W., de

Souza, R., Emam, A., Parker, T.L., Vidgen, E., Lapsley, K.G., et al. (2003).

Effects of a dietary portfolio of cholesterol-lowering foods vs lovastatin on serum

lipids and C-reactive protein. JAMA-J Am Med Assoc 290, 502-510.

- Jenkins, D.J.A., Kendall, C.W.C., Popovich, D.G., Vidgen, E., Mehling, C.C.,

Vuksan, V., Ransom, T.P.P., Rao, A.V., Rosenberg-Zand, R., Tariq, N., et al.

(2001). Effect of a very-high-fiber vegetable, fruit, and nut diet on serum lipids

and colonic function. Metab-Clin Exp 50, 494-503.

- Jones, P.J., Raeini-Sarjaz, M., Jenkins, D.J.A., Kendall, C.W.C., Vidgen, E.,

Trautwein, E.A., Lapsley, K.G., Marchie, A., Cunnane, S.C., and Connelly, P.W.

(2005). Effects of a diet high in plant sterols, vegetable proteins, and viscous

fibers (dietary portfolio) on circulating sterol levels and red cell fragility in

hypercholesterolemic subjects. Lipids 40, 169-174. 172

- Jones, P.J., Raeini-Sarjaz, M., Ntanios, F.Y., Vanstone, C.A., Feng, J.Y., and

Parsons, W.E. (2000). Modulation of plasma lipid levels and cholesterol kinetics

by phytosterol versus phytostanol esters. J Lipid Res 41, 697-705.

- Jones, P.J.H., Ausman, L.M., Croll, D.H., Feng, J.Y., Schaefer, E.A., and

Lichtenstein, A.H. (1998a). Validation of deuterium incorporation against sterol

balance for measurement of human cholesterol biosynthesis. J Lipid Res 39, 1111-

1117.

- Jones, P.J.H., Howell, T., MacDougall, D.E., Feng, J.Y., and Parsons, W. (1998b).

Short-term administration of tall oil phytosterols improves plasma lipid profiles in

subjects with different cholesterol levels. Metab-Clin Exp 47, 751-756.

- Jones, P.J.H., Leitch, C.A., Li, Z.C., and Connor, W.E. (1993). Human

cholesterol-synthesis measurement using deuterated water - Theoretical and

procedural considerations. Arterioscler Thromb 13, 247'-253.

- Jones, P.J.H., MacDougall, D.E., Ntanios, F., and Vanstone, C.A. (1997). Dietary

phytosterols as cholesterol-lowering agents in humans. Can J Physiol Pharmacol

75, 217-227.

- Jones, P.J.H., Ntanios, F.Y., Raeini-Sarjaz, M., and Vanstone, C.A. (1999).

Cholesterol-lowering efficacy of a sitostanol-containing phytosterol mixture with

a prudent diet in hyperlipidemic men. Am J Clin Nutr 69, 1144-1150.

- Jones, P.J.H., Pappu, A.S., Illingworth, D.R., and Leitch, C.A. (1992).

Correspondence between plasma mevalonic acid levels and deuterium uptake in

measuring human cholesterol synthesis. Eur J Clin Invest 22, 609-613. - Jones, P.J.H., and Schoeller, D.A. (1990). Evidence for diurnal periodicity in

human cholesterol-synthesis. J Lipid Res 31, 667-673.

- Jones, P.J.H., Vanstone, C.A., Raeini-Sarjaz, M., and St-Onge, M.P. (2003).

Phytosterols in low- and nonfat beverages as part of a controlled diet fail to lower

plasma lipid levels. J Lipid Res 44, 1713-1719.

- Judd, J.T., Baer, D.J., Chen, S.C., Clevidence, B.A., Muesing, R.A., Kramer, M.,

and Meijer, G.W. (2002). Plant sterol esters lower plasma lipids and most

carotenoids in mildly hypercholesterolemic adults. Lipids 37, 33-42.

- Jurevics, H., Hostettler, J., Barrett, C, Morell, P., and Toews, A.D. (2000).

Diurnal and dietary-induced changes in cholesterol synthesis correlate with levels

of mRNA for HMG-CoA reductase. J Lipid Res 41, 1048-1053.

- Katan, M.B., Grundy, S.M., Jones, P., Law, M., Miettinen, T., and Paoletti, R.

(2003). Efficacy and safety of plant stanols and sterols in the management of

blood cholesterol levels. Mayo Clin Proc 78, 965-978.

- Kesaniemi, Y.A., Ehnholm, C, and Miettinen, T.A. (1987). Intesitnal cholesterol

absorption efficiency in man is related to apoprotein-E phynotype. J Clin Invest

£0,578-58!.

- Ketomaki, A.M., Gylling, H., Antikainen, M., Siimes, M.A., and Miettinen, T.A.

(2003). Red cell and plasma plant sterols are related during consumption of plant

stanol and sterol ester spreads in children with hypercholesterolemia. J Pediatr

742,524-531.

- Korpela, R., Tuomilehto, J., Hogstrom, P., Seppo, L., Piironen, V., Salo-

Vaananen, P., Toivo, J., Lamberg-Allardt, C, Karkkainen, M., Outila, T., et al. 174

(2006). Safety aspects and cholesterol-lowering efficacy of low fat dairy products

containing plant sterols. Eur J Clin Nutr 60, 633-642.

- Kratz, M., Kannenberg, F., Gramenz, E., Berning, B., Trautwein, E., Assmann,

G., and Rust, S. (2007). Similar serum plant sterol responses of human subjects

heterozygous for a mutation causing sitosterolemia and controls to diets enriched

in plant sterols or stands. Eur J Clin Nutr Epub ahead of print

- Krauss, R.M., Eckel, R.H., Howard, B., Appel, L.J., Daniels, S.R., Deckelbaum,

R.J., Erdman, J.W., Kris-Etherton, P., Goldberg, I.J., Kotchen, T.A., et al. (2001).

Revision 2000: A statement for healthcare professionals from the nutrition

committee of the American Heart Association. J Nutr 131, 132-146.

- Kris-Etherton, P.M., Harris, W.S., and Appel, L.J. (2002). Fish consumption, fish

oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106, 2747-2757.

- Lau, V.W.Y., Journoud, M., and Jones, P.J.H. (2005). Plant sterols are efficacious

in lowering plasma LDL and non-HDL cholesterol in hypercholesterolemic type 2

diabetic and nondiabetic persons. Am J Clin Nutr 81, 1351-1358.

- Law, M. (2000). Plant sterol and stanol margarines and health. Br Med J 320, 861-

864.

- Law, M.R., Wald, N.J., and Thompson, S.G. (1994). By how much and how

quickly does reduction in serum-cholesterol concentration lower risk of ischemic-

heart-disease.. Br Med J 308, 367-373.

- Lee, Y.M., Haastert, B., Scherbaum, W., and Hauner, H. (2003). A phytosterol-

enriched spread improves the lipid profile of subjects with type 2 diabetes 175

mellitus. A randomized controlled trial under free-living conditions. Eur J Nutr

42, 111-117.

- Lichtenstein, A.H. (2002). Plant sterols and blood lipid levels. Curr Opin Clin

Nutr Metab Care 5, 147-152.

- Lichtenstein, A.H., Erkkila, A.T., Lamarche, B., Schwab, U.S., Jalbert, S.M., and

Ausman, L.M. (2003). Influence of hydrogenated fat and butter on CVD risk

factors: remnant-like particles, glucose and insulin, blood pressure and C-reactive

protein. Atherosclerosis 171, 97-107.

- Lichtenstein, A.H., Jalbert, S.M., Adlercreutz, H., Goldin, B.R., Rasmussen, H.,

Schaefer, E.J., and Ausman, L.M. (2002). Lipoprotein response to diets high in

soy or animal protein with and without isoflavones in moderately

hypercholesterolemic subjects. Arterioscler Thromb Vase Biol 22, 1852-1858.

- Lichtenstein, A.H., Matthan, N.R., Jalbert, S.M., Resteghini, N.A., Schaefer, E.J.,

and Ausman, L.M. (2006). Novel soybean oils with different fatty acid profiles

alter cardiovascular disease risk factors in moderately hyperlipidemic subjects.

Am J Clin Nutr 84, 497-504.

- Lottenberg, A.M., Nunes, V.S., Nakandakare, E.R., Neves, M., Bernik, M.,

Lagrost, L., dos Santos, J.E., and Quintao, E. (2003). The human cholesteryl ester

transfer protein I405V polymorphism is associated with plasma cholesterol

concentration and its reduction by dietary phytosterol esters. J Nutr 133, 1800-

1805.

- Lundberg, K., Josefsson, A., and Nordin, C. (2007). Diurnal and seasonal

variation of cholecystokinin peptides in humans. Neuropeptides 41, 59-63. 176

- Lutjohann, D., Meese, CO., Grouse, J.R.d., and von Bergmann, K. (1993).

Evaluation of deuterated cholesterol and deuterated sitostanol for measurement of

cholesterol absorption in humans. J Lipid Res 34, 1039-1046.

- Mackay, J., and Mensah, G.A. (2004). The Atlas of Heart Disease and Stroke

(World Health Organization, Geneva).

- Maki, K.C., Davidson, M.H., Umporowicz, D.M., Schaefer, E.J., Dicklin, M.R.,

Ingram, K.A., Chen, S., McNamara, J.R., Gebhart, B.W., Ribaya-Mercado, J.D.,

et al. (2001). Lipid responses to plant-sterol-enriched reduced-fat spreads

incorporated into a National Cholesterol Education Program Step I diet. Am J Clin

Nutr 74, 33-43.

- Masson, L.F., and McNeill, G. (2005). The effect of genetic variation on the lipid

response to dietary change: recent findings. Curr Opin Lipidology 16, 61-67.

- Matvienko, O.A., Lewis, D.S., Swanson, M., Arndt, B., Rainwater, D.L., Stewart,

J., and Alekel, D.L. (2002). A single daily dose of soybean phytosterols in ground

beef decreases serum total cholesterol and LDL cholesterol in young, mildly

hypercholesterolemic men. Am J Clin Nutr 76, 57-64.

- Mensink, R.P., Ebbing, S., Lindhout, M., Plat, J., and van Heugten, M.M.A.

(2002). Effects of plant stanol esters supplied in low-fat yoghurt on serum lipids

and lipoproteins, non-cholesterol sterols and fat soluble antioxidant

concentrations. Atherosclerosis 160, 205-213.

- Miettinen, T.A., and Gylling, H. (1999). Regulation of cholesterol metabolism by

dietary plant sterols. Curr Opin Lipidology 10, 9-14. 177

- Miettinen, T.A., Tilvis, R.S., and Kesaniemi, Y.A. (1989). Serum cholestanol and

plant sterol levies in relation to cholesterol metabolism in middle aged men

Metab-Clin Exp 38, 136-140.

- Miettinen, T.A., Tilvis, R.S., and Kesaniemi, Y.A. (1990). Serum plant sterols and

cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of

a randomly selected male-population. Am J Epidemiol 131, 20-31.

- Miettinen, T.A., and Vanhanen, H. (1994). Dietary sitostanol related to

absorption, synthesis and serum Level of cholesterol in different apolipoprotein-E

phenotypes. Atherosclerosis 105, 217-226.

- Miettinen, T.A., Vuoristo, M., Nissinen, M., Jarvinen, H.J., and Gylling, H.

(2000). Serum, biliary, and fecal cholesterol and plant sterols in colectomized

patients before and during consumption of stanol ester margarine. Am J Clin Nutr

77,1095-1102.

- Mifflin, M.D., Stjeor, ST., Hill, L.A., Scott, B.J., Daugherty, S.A., and Koh, Y.O.

(1990). A new predictive equation for resting energy expenditure in health

individuals Am J Clin Nutr 51, 241-247.

- Moreau, R.A., Whitaker, B.D., and Hicks, K.B. (2002). Phytosterols,

phytostanols, and their conjugates in foods: structural diversity, quantitative

analysis, and health-promoting uses. Prog Lipid Res 41, 457-500.

- Morton, G.M., Lee, S.M., Buss, D.H., and Lawrance, P. (1995). Intakes and major

dietary sources of cholesterol and phytosterols in the British diet. J Hum Nutr Diet

8, 429-440. 178

- Moruisi, K.G., Oosthuizen, W., and Opperman, A.M. (2006). Phytosterols/stanols

lower cholesterol concentrations in familial hypercholesterolemic subjects: A

systematic review with meta-analysis. J Am Coll Nutr 25, 41-48.

- Mussner, M.J., Parhofer, K.G., von Bergmann, K., Schwandt, P., Broedl, U., and

Otto, C. (2002). Effects of phytosterol ester-enriched margarine on plasma

lipoproteins in mild to moderate hypercholesterolemia are related to basal

cholesterol and fat intake. Metab-Clin Exp 57, 189-194.

- National Cholesterol Education Program Expert Panel (2002). Third Report of the

National Cholesterol Education Program (NCEP) Expert Panel on Detection,

Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment

Panel III) Final Report. Circulation 106, 3143-3421.

- Naumann, E., Plat, J., and Mensink, R.P. (2003). Changes in serum concentrations

of noncholesterol sterols and lipoproteins in healthy subjects do not depend on the

ratio of plant sterols to stanols in the diet. J Nutr 133, 2741-2747.

- Neil, H.A.W., and Huxley, R.R. (2002). Efficacy and therapeutic potential of plant

sterols. Atheroscler Suppl 3, 11-15.

- Neil, H.A.W., Meijer, G.W., and Roe, L.S. (2001). Randomised controlled trial of

use by hypercholesterolaemic patients of a vegetable oil sterol-enriched fat spread.

Atherosclerosis 156, 329-337.

- Nestel, P., Cehun, M., Pomeroy, S., Abbey, M., and Weldon, G. (2001).

Cholesterol-lowering effects of plant sterol esters and non-esterified stanols in

margarine, butter and low-fat foods. Eur J Clin Nutr 55, 1084-1090. 179

- Nguyen, T.T., Dale, L.C., von Bergmann, K., and Croghan, I.T. (1999).

Cholesterol-lowering effect of stanol ester in a US population of mildly

hypercholesterolemic men and women: A randomized controlled trial. Mayo Clin

Vroc 74, 1198-1206.

- Nigon, F., Serfaty-Lacrosniere, C, Beucler, I., Chauvois, D., Neveu, C, Giral, P.,

Chapman, M.J., and Bruckert, E. (2001). Plant sterol-enriched margarine lowers

plasma LDL in hyperlipidemic subjects with low cholesterol intake: Effect of

fibrate treatment. Clin Chem Lab Med 39, 634-640.

- Noakes, M., Clifton, P., Ntanios, F., Shrapnel, W., Record, I., and Mclnerney, J.

(2002). An increase in dietary carotenoids when consuming plant sterols or stanols

is effective in maintaining plasma carotenoid concentrations. Am J Clin Nutr 75,

79-86.

- Noakes, M., Clifton, P.M., Doornbos, A.M.E., and Trautwein, E.A. (2005). Plant

sterol ester-enriched milk and yoghurt effectively reduce serum cholesterol in

modestly hypercholesterolemic subjects. Eur J Nutr 44, 214-222.

- Normen, L., Dutta, P., Lia, A., and Andersson, H. (2000). Soy sterol esters and

beta-sitostanol ester as inhibitors of cholesterol absorption in human small bowel.

Am J Clin Nutr 71, 908-913.

- Ntanios, F. (2001). Plant sterol-ester-enriched spreads as an example of a new

functional food. Eur J Lipid Sci Technol 103, 102-106.

- Ntanios, F.Y., Homma, Y., and Ushiro, S. (2002). A spread enriched with plant

sterol-esters lowers blood cholesterol and lipoproteins without affecting vitamins 180

A and E in normal and hypercholesterolemic Japanese men and women. J Nutr

132, 3650-3655.

- Ntanios, F.Y., and Jones, P.J.H. (1998). Effects of variable dietary sitostanol

concentrations on plasma lipid profile and phytosterol metabolism in hamsters.

Biochim Biophys Acta-Lipids Lipid Metab 1390, 237-244.

- O'Neill, F.H., Patel, D.D., Knight, B.L., Neuwirth, C.K.Y., Bourbon, M., Soutar,

A.K., Taylor, G.W., Thompson, G.R., andNaoumova, R.P. (2001). Determinants

of variable response to statin treatment in patients with refractory familial

hypercholesterolemia. Arterioscler Thromb Vase Biol 21, 832-837.

- Ostlund, R.E. (2002). Phytosterols in human nutrition. Annu Rev Nutr 22, 533-

549.

- Ostlund, R.E. (2004). Phytosterols and cholesterol metabolism. Curr Opin

Lipidology 15, 37-41.

- Ostlund, R.E., Racette, S.B., Okeke, A., and Stenson, W.F. (2002). Phytosterols

that are naturally present in commercial corn oil significantly reduce cholesterol

absorption in humans. Am J Clin Nutr 75, 1000-1004.

- Ostlund, R.E., Spilburg, C.A., and Stenson, W.F. (1999). Sitostanol administered

in lecithin micelles potently reduces cholesterol absorption in humans. Am J Clin

Nutr 70,826-831.

- Pai, M., McCulloch, M., Gorman, J.D., Pai, N., Enanoria, W., Kennedy, G.,

Tharyan, P., and Colford, J.M. (2004). Systematic reviews and meta-analyses: An

illustrated, step-by-step guide. Natl Med J India 17, 86-95. 181

- Pelletier, X., Belbraouet, S., Mirabel, D., Mordret, F., Perrin, J.L., Pages, X., and

Debry, G. (1995). A diet moderately enriched in phytosterols lowers plasma-

cholesterol concentrations in normocholesterolemic humans. Ann Nutr Metab 39,

291-295.

- Piironen, V., Lindsay, D.G., Miettinen, T.A., Toivo, J., and Lampi, A.M. (2000).

Plant sterols: biosynthesis, biological function and their importance to human

nutrition. J Sci Food Agric 80, 939-966.

- Pineda, J.A., Ranedo, M.J.C., Anda, J.A., and Terreros, S.F. (2005).

Hypocholesteremic effectiveness of a yogurt containing plant stanol esters. Rev

Clin Esp 205, 63-66.

- Plat, J., Bragt, M.C.E., and Mensink, R.P. (2005). Common sequence variations in

ABCG8 are related to plant sterol metabolism in healthy volunteers. J Lipid Res

46, 68-75.

- Plat, J., and Mensink, R.P. (2000). Vegetable oil based versus wood based stanol

ester mixtures: effects on serum lipids and hemostatic factors in non-

hypercholesterolemic subjects. Atherosclerosis 148, 101-112.

- Plat, J., and Mensink, R.P. (2002a). Increased intestinal ABCA1 expression

contributes to the decrease in cholesterol absorption after plant stanol

consumption. Faseb J 16, 1248-1253.

- Plat, J., and Mensink, R.P. (2002b). Relationship of genetic variation in genes

encoding apolipoprotein A-IV, scavenger receptor BI, HMG-CoA reductase,

CETP and apolipoprotein E with cholesterol metabolism and the response to plant

stanol ester consumption. Eur J Clin Invest 32, 242-250. 182

- Plat, J., van Onselen, E.N.M., van Heugten, M.M.A., and Mensink, R.P. (2000).

Effects on serum lipids, lipoproteins and fat soluble antioxidant concentrations of

consumption frequency of margarines and shortenings enriched with plant stanol

esters. Eur J Clin Nutr 54, 671-677.

- Polagruto, J.A., Wang-Polagruto, J.F., Braun, M.M., Lee, L., Kwik-Uribe, C, and

Keen, C.L. (2006). Cocoa flavanol-enriched snack bars containing phytosterols

effectively lower total and low-density lipoprotein cholesterol levels. J Am Diet

Assoc 106, 1804-1813.

- Quilez, J., Rafecas, M., Brufau, G., Garcia-Lorda, P., Megias, I., Bullo, M., Ruiz,

J.A., and Salas-Salvado, J. (2003). Bakery products enriched with phytosterol

esters, alpha-tocopherol and beta-carotene decrease plasma LDL-cholesterol and

maintain plasma beta-carotene concentrations in normocholesterolemic men and

women. J Nutr 133, 3103-3109.

- Raeini-Sarjaz, M., Ntanios, F.Y., Vanstone, C.A., and Jones, P.J.H. (2002). No

changes in serum fat-soluble vitamin and carotenoid concentrations with the

intake of plant sterol/stanol esters in the context of a controlled diet. Metab-Clin

Exp 51, 652-656.

- Repa, J. J., Dietschy, J.M., and Turley, S.D. (2002). Inhibition of cholesterol

absorption by SCH 58053 in the mouse is not mediated via changes in the

expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte. J Lipid

Res 43, 1864-1874.

- Rifai, N., and Ridker, P.M. (2001). High-sensitivity C-reactive protein: A novel

and promising marker of coronary heart disease. Clin Chem 47, 403-411. 183

- Robins, S.J., Fasulo, J.M., Pritzker, C.R., and Patton, G.M. (1996). Hepatic

transport and secretion of unesterified cholesterol in the rat is traced by the plant

sterol, sitostanol. J Lipid Res 37, 15-21.

- Ros, E. (2000). Intestinal absorption of triglyceride and cholesterol. Dietary and

pharmacological inhibition to reduce cardiovascular risk. Atherosclerosis 151,

357-379.

- Rozner, S., and Garti, N. (2006). The activity and absorption relationship of

cholesterol and phytosterols. Colloid Surf A-Physicochem Eng Asp 282, 435-456.

- Russell, J.C., Ewart, H.S., Kelly, S.E., Kralovec, J., Wright, J.L.C., and Dolphin,

P.J. (2002). Improvement of vascular dysfunction and blood lipids of insulin-

resistant rats by a marine oil-based phytosterol compound. Lipids 37, 147-152.

- Saito, S., Takeshita, M., Tomonobu, K., Kudo, N., Shiiba, D., Hase, T.,

Tokimitsu, I., and Yasukawa, T. (2006). Dose-dependent cholesterol-lowering

effect of a mayonnaise-type product with a main component of diacylglycerol-

containing plant sterol esters. Nutrition 22, 174-178.

- Schmitz, G., Langmann, T., and Heimerl, S. (2001). Role of ABCG1 and other

ABCG family members in lipid metabolism. J Lipid Res 42, 1513-1520.

- Seki, S., Hidaka, I., Kojima, K., Yoshino, H., Aoyama, T., Okazaki, M., and

Kondo, K. (2003). Effects of phytosterol ester-enriched vegetable oil on plasma

lipoproteins in healthy men. Asia Pac J Clin Nutr 12, 282-291.

- Seman, L.J., DeLuca, C, Jenner, J.L., Cupples, L.A., McNamara, J.R., Wilson,

P.W.F., Castelli, W.P., Ordovas, J.M., and Schaefer, E.J. (1999). Lipoprotein(a)- 184

cholesterol and coronary heart disease in the Framingham Heart Study. Clin Chem

45, 1039-1046.

- Shin, M.J., Lee, J.H., Jang, Y., Lee-Kim, Y.C., Park, E., Kim, K.M., Chung, B.C.,

and Chung, N. (2005). Micellar phytosterols effectively reduce cholesterol

absorption at low doses. Ann Nutr Metab 49, 346-351.

- Shrestha, S., Volek, J.S., Udani, J., Wood, R.J., Greene, CM., Aggarwal, D.,

Contois, J.H., Kavoussi, B., and Fernandez, M.L. (2006). A combination therapy

including psyllium and plant sterols lowers LDL cholesterol by modifying

lipoprotein metabolism in hypercholesterolemic individuals. J Nutr 136, 2492-

2497.

- Sierksma, A., Weststrate, J.A., and Meijer, G.W. (1999). Spreads enriched with

plant sterols, either esterified 4,4-dimethylsterols or free 4-desmethylsterols, and

plasma total- and LDL-cholesterol concentrations. Br J Nutr 82, 273-282.

- Simons, L.A. (2002). Additive effect of plant sterol-ester margarine and

cerivastatin in lowering low-density lipoprotein cholesterol in primary

hypercholesterolemia. Am J Cardiol 90,131-140.

- Sleight, P. (2003). Current options in the management of coronary artery disease.

Am J Cardiol 92, 4N-8N.

- Sniderman, A.D., Furberg, CD., Keech, A., van Lennep, J.E.R., Frohlich, J.,

Jungner, I., and Walldius, G. (2003). Apolipoproteins versus lipids as indices of

coronary risk and as targets for statin treatment. Lancet 361, 111-ISO.

- Spilburg, C.A., Goldberg, A.C., McGill, J.B., Stenson, W.F., Racette, S.B.,

Bateman, J., McPherson, T., and Ostlund, R.E. (2003). Fat-free foods 185

supplemented with soy stanol-lecithin powder reduce cholesterol absorption and

LDL cholesterol. J Am Diet Assoc 103, 577-581.

- St-Onge, M.P., and Jones, P.J.H. (2003). Phytosterols and human lipid

metabolism: Efficacy, safety, and novel foods. Lipids 38, 367-375.

- Tammi, A., Ronnemaa, T., Miettinen, T.A., Gylling, H., Rask-Nissila, L., Viikari,

J., Tuominen, J., Marniemi, J., and Simell, O. (2002). Effects of gender,

apolipoprotein E phenotype and cholesterol-lowering by plant stanol esters in

children: The STRIP study. Acta Paediatr 91, 1155-1162.

- Temme, E.H.M., Van Hoydonck, P.G.A., Schouten, E.G., and Kesteloot, H.

(2002). Effects of a plant sterol-enriched spread on serum lipids and lipoproteins

in mildly hypercholesterolaemic subjects. Acta Cardiol 57, 111-115.

- Thomsen, A.B., Hansen, H.B., Christiansen, C, Green, H., and Berger, A. (2004).

Effect of free plant sterols in low-fat milk on serum lipid profile in

hypercholesterolemic subjects. Eur J Clin Nutr 58, 860-870.

- Thuluva, S.C., Igel, M., Giesa, U., Lutjohann, D., Sudhop, T., and von Bergmann,

K. (2005). Ratio of lathosterol to campesterol in serum predicts the cholesterol-

lowering effect of sitostanol-supplemented margarine. Int J Clin Pharmacol Ther

43,305-310.

- Tilvis, R.S., and Miettinen, T.A. (1986). Serum plant sterols and their relation to

cholesterol absorption. Am J Clin Nutr 43, 92-91.

- Trautwein, E.A., Schulz, C, Rieckhoff, D., Kunath-Rau, A., Erbersdobler, H.F.,

de Groot, W.A., and Meijer, G.W. (2002). Effect of esterified 4-desmethylsterols 186

and -stanols or 4,4 '-dimethylsterols on cholesterol and bile acid metabolism in

hamsters. Br J Nutr 87, 227-237.

- Vanhanen, H. (1994). Cholesterol malabsorption caused by sitostanol Ester

feeding and neomycin in pravastatin-treated hypercholesterolemic patients. Eur J

Clin Pharmacol 47, 169-176.

- Vanhanen, H.T., Blomqvist, S., Ehnholm, C, Hyvonen, M., Jauhiainen, M.,

Torstila, I., and Miettinen, T.A. (1993). Serum-Cholesterol, Cholesterol

Precursors, and Plant Sterols in Hypercholesterolemic Subjects with Different

Apoe Phenotypes During Dietary Sitostanol Ester Treatment. J Lipid Res 34,

1535-1544.

- Vanstone, C.A., Raeini-Sarjaz, M., and Jones, P.J.H. (2001). Injected

phytosterols/stanols suppress plasma cholesterol levels in hamsters. J Nutr

Biochem 12, 565-574.

- Vanstone, C.A., Raeini-Sarjaz, M., Parsons, W.E., and Jones, P.J. (2002).

Unesterified plant sterols and stanols lower LDL-cholesterol concentrations

equivalently in hypercholesterolemic persons. Am J Clin Nutr 76, 1272-1278.

- Varady, K.A., Ebine, N., Vanstone, C.A., Parsons, W.E., and Jones, P.J.H. (2004).

Plant sterols and endurance training combine to favorably alter plasma lipid

profiles in previously sedentary hypercholes terolemic adults after 8 wk. Am J

Clin Nutr 80, 1159-1166.

- Varady, K.A., and Jones, P.J.H. (2006). Decrease in intestinal cholesterol

absorption by plant sterols and exercise improves lipid profiles in

hypercholesterolemic adults. Faseb J 20, A1025-A1025. 187

- Vissers, M.N., Zock, P.L., Meijer, G.W., and Katan, M.B. (2000). Effect of plant

sterols from rice bran oil and triterpene alcohols from sheanut oil on serum

lipoprotein concentrations in humans. Am J Clin Nutr 72, 1510-1515.

- Vlahcevic, Z.R., Eggertsen, G., Bjorkhem, I., Hylemon, P.B., Redford, K., and

Pandak, W.M. (2000). Regulation of sterol 12 alpha-hydroxylase and cholic acid

biosynthesis in the rat. Gastroenterology 118, 599-607.

- Volpe, R., Niittynen, L., Korpela, R., Sirtori, C, Bucci, A., Fraone, N., and

Pazzucconi, F. (2001). Effects of yoghurt enriched with plant sterols on serum

lipids in patients with moderate hypercholesterolaemia. Br J Nutr 86, 233-239.

- Vorlat, A., Conraads, V.M., and Vrints, C.J. (2003). Regular use of margarine-

containing stanol/sterol esters reduces total and low-density lipoprotein (LDL)

cholesterol and allows reduction of statin therapy after cardiac transplantation:

Preliminary observations. J Heart Lung Transplant 22, 1059-1062.

- Vuorio, A.F., Gylling, H., Turtola, H., Kontula, K., Ketonen, P., and Miettinen,

T.A. (2000). Stanol ester margarine alone and with simvastatin lowers serum

cholesterol in families with familial hypercholesterolemia caused by the FH-North

Karelia mutation. Arterioscler Thromb Vase Biol 20, 500-506.

- Wang, Y.W., Vanstone, C.A., Parsons, W.D., and Jones, P.J.H. (2004). Validation

of a single-isotope-labeled cholesterol tracer approach for measuring human

cholesterol absorption. Lipids 39, 87-91.

- Wester, I. (2000). Cholesterol-lowering effect of plant sterols. Eur J Lipid Sci

Technol 102, 37-44. 188

- Weststrate, J.A., and Meijer, G.W. (1998). Plant sterol-enriched margarines and

reduction of plasma total- and LDL-cholesterol concentrations in

normocholesterolaemic and mildly hypercholesterolaemic subjects. Eur J Clin

Nutr 52, 334-343.

- Whelan, A.M., Jurgens, T.M., and Bowles, S.K. (2006). Natural health products in

the prevention and treatment of osteoporosis: Systematic review of randomized

controlled trials. Ann Pharmacother 40, 836-849.

- Yoshida, M., Vanstone, C.A., Parsons, W.A., Zawistowski, J., and Jones, PJ.H.

(2006). Effect of plant sterols and glucomannan on lipids in individuals with and

without type II diabetes. Eur J Clin Nutr 60, 529-537. 189

APPENDICES 190

Appendix 1. Published version of Review Article 2: Plant sterols: factors affecting their efficacy and safety as functional food ingredients 191

O Lipids in Health and Disease BioMed Central

Review Open Acces Plant sterols: factors affecting their efficacy and safety as functional food ingredients Alvin Berger1, Peter JH Jones*2 and Suhad S Abumweis2

Address: 'Head, Biochemical Profiling, Paradigm Genetics, P.O. Box 14528, Research Triangle Park, North Carolina, 27709-4528, USA and 2School of Dietetics and Human Nutrition, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, H9X3V9, Canada Email: Alvin Berger - [email protected]; Peter JH Jones* - [email protected]; Suhad S Abumweis - [email protected] * Corresponding author

Published: 07 April 2004 Received: 13 February 2004 Accepted: 07 April 2004 Lipids in Health and Disease 2004, 3:5 This article is available from: http://www.lipidworld.eom/contertt/3/l/5 © 2004 Berger et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted In all media for any purpose, provided this notice is preserved along with the article's original URL.

Abstract Plant sterols are naturally occurring molecules that humanity has evolved with. Herein, we have critically evaluated recent literature pertaining to the myriad of factors affecting efficacy and safety of plant sterols in free and esterified forms. We conclude that properly solubilized 4-desmetyl plant sterols, in ester or free form, in reasonable doses (0.8-1.0 g of equivalents per day) and in various vehicles including natural sources, and as part of a healthy diet and lifestyle, are important dietary components for lowering low density lipoprotein (LDL) cholesterol and maintaining good heart health. In addition to their cholesterol lowering properties, plant sterols possess anti-cancer, anti­ inflammatory, anti-atherogenicity, and anti-oxidation activities, and should thus be of clinical importance, even for those individuals without elevated LDL cholesterol. The carotenoid lowering effect of plant sterols should be corrected by increasing intake of food that is rich in carotenoids. In pregnant and lactating women and children, further study is needed to verify the dose required to decrease blood cholesterol without affecting fat-soluble vitamins and carotenoid status.

Background 0.1-0.14% of cholesterol levels [13]. Due to their struc­ Plant sterols are plant compounds with similar chemical tural similarity to cholesterol, plant sterols were first and structure and biological functions as cholesterol [1]. Plant foremost studied for their cholesterol absorption inhibi­ sterols contain an extra methyl, ethyl group or double tion properties. In addition to their cholesterol lowering bond. The most abundant plant sterols are sitosterol, effect, plant sterols may possess anti-cancer [14], anti- campesterol and stigmasterol [2]. The daily dietary intake atherosclerosis [15,16], anti-inflammation [17] and anti- of plant sterol is 160-400 mg among different popula­ oxidation activities [18]. The objective of the present tions [3-9J. However, in the earlier stages of human evo­ review is to assess the evidence supporting the various lution, some 5-7 million years ago, plant sterol intake in physiological effects of plant sterols with emphasis on Myocene diets would have been considerably higher, up recent advances in knowledge. to 1 g/d [10]. Dietary sources include vegetable oils (espe­ cially unrefined oils), nuts, seeds and grains [1]. Absorp­ Physiological effects of plant sterols tion efficiency for plant sterols in humans is considerably Cholesterol lowering actions less than that of cholesterol. Percent absorption of the The cholesterol lowering effect of plant sterols is well doc­ former is 2-5% [11] versus 60% for the tatter [12]. Conse­ umented in the literature. It is now accepted, after much quently, blood levels of plant sterols in humans are only earlier scientific debate and study, that 4-desmethyl plant

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Table I: Effects of free sterols and stands on LDL cholesterol and cholesterol absorption parameters.

Reference Products Vehicle Subject Condition: Daily Doseg gServings/d Outcome; Where possible, Starting mean Free sterol/ Piacebo-adjusted % decrease LI>L TCroM stanol is given r % decrease with sterol Equivalents (T0 vs Jj) % decrease with platjebo (T0 vs Tf). Cabs also indicated

m A2 Spread 9 Normocholesterol 1.0 1 Cabs 42.0% C2 ernlc 1.3 1 Cabs 33.0%

[I76] B2 Sunflower oil 3 M, 3 W Hyperchoiesterole 1.5 i LDL 14.4% (Capsule) mic

P7] Al Mayonnaise 24M8.W 6.5 0.96 Habitual usage •lTC5,7% Bl 1.0 i TC X4%

pig Al Mayonnaise 22M.9W >6.0 1.0 Habitual usage 4-LDL 6.2% Bl 1.0 1 LDL 5.1% Dl 1.2 I LDL 7.7%

pi] Bl+step 1 diet Capsules with 33 M 6.2 3.0 3 1 LDL 1.8% sterols 3,0with:8;g consumed with cholestyramine 8 g food of cholestyramine

[33] A2 Spread 12 M Normocholesterol 0.7 i, LDL 6,1% emlc

[I77] A2 olive oil 8M.8W 37M.4.IW 0.4g/IOOOKcal 3 I LDL 2.8%

[103] Al Spread 22 SOmfrlmum 1.6 I LDL 15 % Normocholesterolemks i LDL 4.7% hypcrdioiesterolmlcs

[97] Bl+prudercdlet Spread 32M 6.7-48 1.7 3 i LDL 15%

P2] B2 +soyl»clthin Spread 4M.2W 5.2 I.0JB2 powder I Cabs 11.0% 0;3-0.7-B2fledthin 1 Cabs 34.4% at 0.3 .g miceilar mix J-Cabs 36.7% at0.7 g

po] A2 Spread 39M.37W -1.9-5.1 0.8 Habitual usage 4-LDL6%.iTC9%

£»] Bl Liquid emulsion. 12 M 6.9 3 3 i Cabs 40%

[100] Al Butter fat ± 9M.6W 6.5 1.8 3 I LDL 13.3%. 1 Cobs 56.IX Bl sterols | IDL 13.454. A Cabs, 33.3% 1:1 mixture iLDLl6.0X.J.C^bj.49.2% AIBI

trt A2 VegetabjeoiL 18 M 7X> 13 2 I Cabs 39% partly filled milk

PI] A? Vegetable: oiL J3M <5.2 2.2 2 A Cabs 51,1% partly ill W milk

[*?] A? VegetableoU 18 M. 51 F 7.0 1,2 or 1.6 2 i LDL 71% at 1.2 g partly filled i LDL 9:6% at 1.6 g milk

Abbreviation; C-sbs, chole^terdabsorption; LDUG, low density lipppfctelh ch6)esrerpli TG; total cholesterol; Taancl Tjrefertdstart aridend points of the study," M, men; VV, women; A, free plant sterol; 6, free plant stanol; Cj plant sterol ester; D, plant stanol ester; I, tall sterol; 2, soy sterol; 3, shea sterol; 4, corn steroijS, rice sterol.

sterols or stands, either in their free or esteriffcd form, ally speaking, properly solubilized free sterols and decrease blood levels of total cholesterol and LDL-eholes* esterified sterols possess similar cholesterol lowering abil- terbl through reduction of cholesterol absorption. Gener- ity [19,20). In some studies such comparisons have been

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Table 2: Effects of estefitted sterols and stanols on LDL cholesterol and cholesterol absorption.

Reference Product* Vehicle Subject Condition: Daily Dose g Free tfServings id Outcome: Where possible, Starting mean sterol/stanol Placebo-adjusted % decrease TC mM<»*ange) Equivalents LDL is given —^decrease With sterol (To vsT$% decrea$ewith placebo (T»vsTf)

[32] Dl Mayonnaise II M, 4 W >6.0 0,82.0 Habitual usage 4 LDL 7.7% 4 LDL 15.0%

[I02] Dl Spread rapeseed 153 M*W 59-5.9 1.8 or 16 3 J. LDL 10.2%

[25] C2 (66% Spread 100 <80 3.3DI-2 2 4 LDL 13% esterified),DI-Z 4 LDL 13% C3.CS wLDL I.SC5 **LDL

[176] CDI Spread 23W 5.5-8.0 2.4,3.2.3.2 4 LDL 8-10% t HDL 5-6%

[104] D2 Spread, low fat 20M, 35W 6.1-6.6 4. LDL 13.7% DI-2 diet n 4 LDL 8.6 X

[104] C2 Spread 42M.S8W 52(17-7.4) 2 4 LDL 6.2 % 4 LDL 9.2 % § 4 LDL 9.8%

[76] CI Spread 34M*W 4.8-7.0 2.0 2 4- LDL 10.4% Dl 4 LDL 117%

[37] DI-2 Spread I4M.8W 69(5.0-8.5) 0.8 0-3 4 LDL 1.7% 1.6 4 LDL 5.6% 14 4 LDL 9.7% 3.0 4 LDL 10.4%

[98] DI-2 Spread 105 6.0-6.6.1 2.0 2 4 LDL 8.9% 3;0 3 4 LDL 6.7%

[77] CI Spread ISM 64-65(6.0-10.0) 1.8 2-3 J. LDL 13.4%, 4 Cabs 362% Dl 1.8 i LDL 6:4%, I.C-abs 25.9%

[179] C2.DI-2 Spread 5M.2W llleostomy 1.5 4 Cabs 38-39%

[99] D2 Spread 4IM.7I W 5.0-5.1 3.8 3 4 LDL 126% DI-2 4.0 4 LDL 1.6%

[96] DM Spread IIM.28W 491447 W 15-dlfferent# 4 LDL 9.9 servings: 3 4 LDL 10.2%

[38] C2 SpreadiStep 1 324'MftW 62 I.I 2 4 LDL 4,9%, 4 TC 2.6% diet 4 LDL 5:4%,4 TC40%

C?8] C(rram|r2) Spread 34t*28W 7.2(iontrol)6.S 2.5, 4IDL 10-15% (treatment)

[73] C2 Beef 34M 5.9 .2.4: 1 4 LDL 15%;

[70] p Yoghurt |6M,44W 51 3.0 3 4 LDL 14 %

[65] C Spread 25M.J8W 61 1:8 2 4 LDL £4%, l TC 3.4%

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Table 2: Effects of esterified sterols and stands on LDL cholesterol and cholesterol absorption. (Continued)

[7?] G (vegetable oil) Spread.Japanese 26M.27W S5 1.8 2 4-LDL 9.2%, 4 TC S.8% basal diet

[80] C (vegetable oil) Spread I9M.3IW 4.0 It I LDL 113%, i TC 8.«

[180] D Spread 38M,22W 6.1 (2 g group! 2.0 2 UDL9;«.lTC6.3% 4-0 (3 g group) 3.6 3 .1 LDL 7,3%,: 4 TC 5.5%

[71] C3 Vegetable oil. 33 M <5.2 2.2 2 4. C-abs 57.4% partly filial milk

See Table I for abbreviations. flawed because the free sterols were not properly solubi- basal spread, probably consumed in 2-3 doses, did not lized [21]. Ostlurid et al. [22] showed that emulsions of result in reduction in total cholesterol compared to con­ sitostanol, mixed with lecithin containing 0.7 g of sterol, trol group. LDL cholesterol typically follows changes in reduced cholesterol absorption considerably, whereas less total cholesterol, sometimes being more responsive to effect was seen with sitosterol in crystalline form. plant sterol modulation. Since the control spread con­ tained 0.36 g of rape seed oil derived sterols, a level of This review will focus on the effects of 4-desmethyl ster­ consumption by vegetarians, the study is essentially com­ ols, stands, and esterified forms. Methylated sterols (4a- paring vegetarian levels of consumption of plant sterols to monomefhyl and 4,4-dimefhyl) in sources such as shea a 3-fold higher level. The conclusion from this study alone and M. dpina fungi for example, and'.'those sterols esteri­ would be that a higher dose man 0.95 g of free sterols fied to non-fatty acids such as ferulate (such as the sterols should be considered to achieve a more consistent and in rice bran oil), may not be equivalentih cholesterol low­ effective lowering of LDL cholesterol levels. In another ering ability compared with the forms present in tall and study with a design similar to that of Vanhanen and Miet­ soybean oils [19,20,23-26]. tinen [27], the absolute reduction in LDL cholesterol was only statistically significant for the sitostanol esters, which Important issues that remain to be verified regarding the showed slightly better efficacy than the free sterols and cholesterol lowering effect of plant sterols includes (i) effi­ stanols [28]. The dose of 1.0 g sitosterol reduced choles­ cacy of low dose of plant sterols, (ii) the effect diet back­ terol absorption more effectively than die controlspread. ground on plant sterol efficacy, (iii) the efficacy of plant This is not surprising because absorption is known to be sterols when incorporated in food other than fat spread an extremely sensitive marker that does not necessarily (iv) the optimal number of plant sterols servings and (v) correlate to changes in LDL cholesterol levels [7,29-31]. the relative efficacy of plant sterols among different Even basal levels of consumption of plant sterol are corre­ populations. lated with cholesterol absorption.

Efficacy of low dose of plant sterols Vanhanen et al [32] showed that in mildly hypercholeste- Tables 1 and 2 summarize recent human intervention rolemic men and women of age 33-60, 1.2 g of free tall clinical trials assessing the effects of 4-desmethyl free and stanol equivalents in mayonnaise decreased LDL levels by esterified plant sterols. Doses of plant sterols reported in 7.7%. Relative to starting levels for this group, this reduc­ literature are often difficult to comprehend, particularly tion was statistically significant, but the absolute lowering those reported in earlier literature. Herein, all doses refer of LDL was not statistically significant after accounting for to free plant sterol equivalent doses. If the contribution of reductions in LDL cholesterol observed with the control naturally occurring plant sterols in the food vehicle was group. The lack of statistical significance in LDL lowering reported, this is then added to the free plant sterol dose. is not surprising because the total sample.size was only 15 Ideally, the free plant sterol dose should be calculated persons, and there was appreciable plant sterol, about 0.4 experimentally using the average mol% of fatty acids rela­ g, in the control mayonnaise, which probably reduced tive to free sterols. LDL cholesterol as well. The quantity of plant sterols in the control spread complicates interpretation of results Selected studies 1990-1994 and makes comparisons to other scientific studies more Vanhanen and Miettinen [27] in 1992 found a dose of difficult. 0.95 g of sitosterol per day, including the contribution of free sterols present in the canola oil used to prepare the

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Selected studies 1995-1999 Maki et al. [38] administered mildly hypercholestero­ Pelletier et al. (33] demonstrated that 0.7 g of soy sterols lemic men and women 1.1 g/d of free tall sterol equiva­ in spreads fed to 12 normocholesterolemic individuals lents in spreads, fed as sterol esters in two doses, which reduced LDL cholesterol by 15.2% relative to the control. decreased LDL levels by 4.9% while 2.2 g/d of sterol In another study [20], a dose of 0.8 g of soy sterols fed to equivalents decreased LDL by 5.4%. 76 normocholesterolemic individuals reduced LDL cho­ lesterol by 6.1% relative to the control, and more impor­ Christiansen et al. [39] reported an 11.3% reduction in tantly, did not reduce carotenoids or carotenoids LDL cholesterol after 6 months with 1.5 g/d of rnicrocrys- normalized to cholesterol, as reported by Hendriks et al. talline free sterols in spreads. No additional improvement [191 with a similar dose of soy free sterol equivalents, was seen with 3,0 g/d of plant sterol. administered as an ester, The LDL reductions reported by Sierksma et al. [20] were less than that seen in the Pelletier Volpe et al. [40] reported a 6.3% placebo-adjusted et al. [33] study, which used a similar dosage. The decrease in LDL cholesterol after 4 wks with 1.0 g/d and a reduction in LDL in the Seerksma study was not seen in all greater reduction of about 12.2% with 2 g/d after 4 wks. subjects because of the well-known within person LDL DeGraafetal. [105] found an intake of 1.8; g/d of free ster­ variation of 10% [34| or solubilization issues. Neverthe­ ols in chocolates to decrease LDL cholesterol 8.9% relative less, this 6% reduction in LDL correlates with a 15% to baseline. reduction in CHD risk at age 40, and a 6% reduction at age 70 [35] or a 10% reduction [36]. Thomsen et al. [42] examined effects of non-esterified, non-hydrogenated, soybean derived plant sterols, solubi- Hendriks etal (.19) showed that in men and women with lized in a partly vegetable oil filled low fat milk oh semm a wide range of ages and starting total cholesterol from LDL cholesterol in 81 mildly hyperchalesterolermc Dan­ low/normal to high, 0.83 g of free soy sterol equivalents ish patients, in a double-blind, randomised, placebo-con­ in spreads decreased LDL cholesterol 6.2%. Interestingly, trolled 3-arm cross-over study. Subjects consumed 0.83 g was less effective than the two higher doses, 1.6 and habitual diets, with some restrictions on consumption of 3.2 g of sterol equivalents, at reducing LDL cholesterol, fat and cholesterol rich foods, Subjects received 0,1.2, or but the differences amongst the three doses were not sta­ 1.6 g/d of sterols in two servings of 250 mL milk for 12 tistically significant This study thus gives strong indica­ wks (4 wks/dose). The placebo-adjusted mean reduction tion that a 0.8 g free sterol equivalent dose of plant sterols in LDL was 7.1 ± 12.3 and 9.6 ± 12.4% (mean ± SD) for can efficaciously diminish LDL cholesterol. Nevertheless, groups receiving 1.2 and 1.6 g of plant sterols, respec­ the authors concluded that the 1.6 gdosage is most desir­ tively, with no differences between sexes. There was no able because of the lack of effect on lipid normalized car­ statistically significant difference in LDL lowering otene, and the quantitatively greater reduction in LDL amongst the 1.2 and 1.6 g/d groups, although Apo B was cholesterol. decreased more with 1.6 than 1.2 g/d of sterols. Apo B is an index of LDL particle number, thus the higher dose Selected studies 2000-2004 may have decreased numbers of LDL particles more dian Hallikainen et al. (37) showed that in normal to mildly the lower dose. Differences in numbers of small, dense, hypercholesterolemic men and women, 0.8 g of free tall/ atherogenic LDL particles and LDL oxidization (43) are vegetable stanol equivalents in spreads decreased LDL other important future parameters to assess. It is notewor­ non-significantly by only 1.6%. This 0.8 g dose did reduce thy that there were 20-23% non-responders in the two the number of apo B particles by 8.7%, indicating a sterol groups, which was partially consistent with the large reduced number of LDL particles. Similar to Vanhanen et differences in cholesterol absorption inhibition observed al. [32], the higher dose of 1.6 g of stanol equivalents with similar milk products containing plant sterols [44]. reduced LDL cholesterol to a greater extent of 6.1%, and Thus, renewed attention should be given to the issue of two higher doses (2.4 and 3.2 g/d) reduced LDL choles­ non-responders. Another noteworthy observation was the terol 10.6-11.5%. The three higher doses (1.6-3.2 g/d) randomization order in which the three milk products lowered LDL cholesterol in a statistically significant man­ affected the magnitude of the LDL lowering results, but ner. A caveat in this study was diat the 0.8 g/d dose was not the overall statistical findings. The placebo-adjusted given after cholesterol was already lowered by 3 subse­ mean percentage decrease in LDL was more pronounced quent plant sterol treatments, possibly producing bias with certain randomixation sequences compared to oth­ against seeing a reduction in LDL cholesterol with 0.8 g/d. ers. This consideration is typically ignored in reporting Albeit the above experimental weakness, the conclusion results of plant sterol clinical trials examining cholesterol would be that the dose of 1.6 g/d of stanol equivalents is lowering efficacy. a more optimal dose for LDL cholesterol reduction.

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In a very recent study still in press [45], 72 men and ing to more statistically significant results [32,37,40]. women aged 20-73 received two 8 ounce servings of Increasing the dosage beyond 1000 mg per day of free Minute Maid brand non-fat orange juice with breakfast sterol equivalents did not further increase LDL cholesterol and dinner meals, providing 2 g/d of Gargill CoroWise lowering efficacy [19]. plantsterols for 8 wks. LDL was reduced 12.4% compared to baseline and placebo; HDL and triacylglycerol levels In humans, there is a good likelihood that a dose of 0.8- were not changed. The authors speculate that the fat in the 1,0 g of free sterol equivalents per day, properly solubi- meals may help to emulsify the plant sterols in the orange lized, administered in 2-3 servings with a meal, will juice. reduce LDL cholesterol by 5% or more and that this reduc­ tion in LDL cholesterol will correlate with an approximate Effects of naturally occurring plant sterols 6-10% reduction in CHD risk atage 70 [35,36], However, The effects of naturally-occurring plantsterols on choles­ at this dosage level, it is likely that not all individuals will terol metabolism have also been studied in both older achieve a 5% reduction in LDL cholesterol [20]. and more recent literature. It was reported that the differences between effects of different plant oils on blood Clinical relevance ofLDL-cholesterol-loweringby plant sterols lipid profiles may be related to their content of plant ster­ As previously noted, it is generally agreed that high blood ols [46-49]. Indeed, there has been renewed interest in the cholesterol level (especially LDL cholesterol) is a risk fac­ cholesterol lowering properties of speciality grains and tor for coronary heart disease (CHD). Oxidation of excess unprocesses oils rich in plant sterols including amaranth LDL cholesterol leads to arterial wall plaque build tip, oil [50,51], rice bran oil [52] (Berger et al., submitted), which then restricts blood flow and increases blood pres­ avocado oil [53], extra virgin olive oil [54], macadamian sure. Unless, hypercholesterolemia and hypertension are nut [55], and argan oil [56]. treated, these factors are associated with increased risk of coronary heart disease (myocardial infarction) and stroke Ostlund et al, [49] showed that doses as low as 150-300 [35], mgof naturally present com oil-derived phytoslerols can reduce dietary cholesterol absorption. Also, it was shown Therefore, the clinical relevance of LDL-cholesterol lower­ that the consumption of original wheat germ, which con^ ing lies in the potential for plant sterols to reduce the tains about 328 mg plant sterols, reduced the cholesterol actual risk of CHD. As already described, there is an absorption by 42.8 % compared to plant sterol4ree wheat impressive body of scientific data demonstrating choles­ germ [57]. These results indicate that naturally available terol-lowering by plant sterols. However, it is pertinent to plant sterols are biologically effective as plant sterol sup­ tease out from published studies, those providing the plementation in reducing cholesterol absorption, and that highest level of evidence for a clinically-important effect. natural plantsterols have important effects on cholesterol Two reviews have addressed this issue [60,61]. Law [60] metabolism [57|. estimated that consumption of 2 g of equivalents of plant sterol or stanol per day would reduce heart disease risk Summary of biologically active dose of plant sterols for optimal 25%. But only a randomized clinical trial using CHD as cholesterol lowering an endpoint, could provide certainty of die effectiveness Several studies [19,20,28,32,33,40,58] using intakes of of plant sterols in reducing heart disease incidence. But for 800-1000 mg of plant sterols per day have shown biolog­ a clinical trial to detect a 12-20% reduction in coronary ically/clinically significant (5% or more) reductions in heart disease incidence would require 10,000-15,000 LDL cholesterol levels, relative to control, or at least patients with CHD (and more for healthy people). Even if showed a statistically significant treatment effect relative such a trial were feasible, it would probably still be under­ to the starting LDL cholesterol level at the beginning of powered to detect any rare adverse events (undesirable the treatment period, independent of control. Other stud­ side effects) [61]. Thus, we must judge the effectiveness of ies [27,37] with a similar dosage range did not meet the plant sterol doses on their theorized ability to reduce above criteria for biological reduction of LDL lewis, or CHD incidence, using LDL cholesterol as a marker. achieve statistical significance. Some studies showed that 800-1000 mg/d of free plant sterol equivalents can Low fat versus high fat background diet decrease the absorption of cholesterol, which is indica­ Dietary cholesterol consumption is 250-500 mg/d, and tive, but not necessarily predictive, of actual LDL choles­ normally half is absorbed, while bilary cholesterol pro­ terol lowering [22,28,32,59[. duction is 600-1000 mg/d. Since plantsterols impair the absorption of bom bilary and dietary cholesterol, it is not 11 has been shown that increasing die dosage beyond 1000 surprising that they are effective even when consumed in mg per day of free sterol equivalents increased LDL cho­ low fat diets [62,63], although evidence from some stud­ lesterol lowering efficacy or consistency of response lead- ies suggests them to be more effective when consumed

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with diets containing cholesterol [21,64,65]. In a study by the plant sterols solubilize, or remain as small crystals Denke [21], plant stands were given in capsules and not over time. In products such as milk, the milk fat globule blended with fatty matrix, which limits their cholesterol- membrane components may enhance the absorption of lowering action. In addition, compliance was monitored cholesterol[44,68,69],butalsoaidin solubilization. Pou- by capsule counting and not by direct supervision, which teau et al. [44] described a rapid filtration and detection decreased compliance monitoring. Mussner et al. [65] method to quantify plant sterol crystals. The authors also found esterified phytosterols in spreads to reduce LDL used light scattering techniques to quantify the size of the cholesterol about 5.4%, but the reduction was 11.6% in crystals as a function of storage time of the milk products. those tertiles having the highest intake of dietary choles­ The method of dispersion, processing, and use of emulsi- terol. Recent studies have shown plant sterols to be effec­ fiers, surfactants, and crystal habit modifiers will affect the tive even if consumed with Step I diets [38,40,66], success of plant sterols in non-spread vehicles Similarly, Judd et al. [67] showed 'that high doses of vege­ [42,44,71,84,85], table oil sterol esters lowered LDL cholesterol to about the same level, whether the basal diet was a typical American Commercially, plant sterols are currently contained in diet or a Step I type of diet, suggesting dramatic changes in bars (Logicol-Australia, Benecol-UK), vegetable oils usual fat intake are not necessary, if plant sterols are con­ (Ekonarjapan; NutraLease Canola Active-Israel), orange sumed concurrently. juice (Minute Maid Heart Wise containing Cargill CoroW- ise plant sterols) [45], mayonnaises (Logicol-Australia), Vehicle for delivering plant sterol milk (Benecol-UK, Logicol-Australia, SereCol-Argente- Most clinical trials have been conducted using plant ster­ nia), yogurt (Logicol-Australia; Benecol-UK), yogurt ols or stands added to spreads. As long as plant sterols are drinks (Benecol), soy milk (Pacific Foods), meat and consumed with a meal to stimulate biliary flow, they can soups (Raisio-Finland),and green teas (Choi zero, Korea). effectively lower LDL cholesterol on die background of Plant sterols are also being sold or developed mixed with various types of basal diets and food vehicles. Plant sterols other functional ingredients such as: fiber (Unilever Fruit are efficacious when consumed in: oil: water emulsions D'or-France); healthy oils (Benecol Olive Spread-UK); [68,69|; water as lecithin micelles [22]; yogurt [40,70]; non-absorbable diacylglycerol (Kao-ADM Econa Healthy low fat filled milks [42,44,71]; chocolate [105]; cereal; Cooking Oil; Enzymotec MultOil Platform, ArteriCare snack bars, breads, and beverages [66,72]; and beef/ham­ products, Israel); almonds, soy protein and viscous fibers burger [73,74]. Efficacy of a soy stanol-lecithin powder in [86]; and minerals [87-89|. There is also interest to com­ reducing cholesterol absorption and LDL-C has been bine plant sterols with antioxidants, such as flavonoids, evaluated in a randomized, double-blind parallel study quercetins, and catechin; and a spice mixture developed [75]. The subjects who followed a Step I diet consumed by Selako, and marketed as Flavomare in Scandanavia. It soy stanol-lecithin powder in a beverage. The provided is only a matter of time before ingredients such as conju­ daily dose of plant stands was 1.9 g. The reductions in gated linoleic acid (CLA) are mixed with plant sterols in blood cholesterol and LDL cholesterol were 10.1 and various vehicles (e.g., Clarinol's CLA has received GRAS 14.4%, respectively. In another group of subjects, choles­ status for addition to milks, yogurts, bars, etc.). terol absorption was measured using 625 mg stanols pro­ vided in beverage or egg whites. Stanol-lecithin reduced Various manufacturers also sell plant sterols in supple­ cholesterol absorption by 32.1% and 38.2 % when con­ ment form, and there is interest to develop plant sterols as sumed in beverage and egg white, respectively. drugs (e.g., Forbes' FM-VP4 drug candidate). Plant sterols may also be combined with other drugs that lower choles­ The reduction in LDL cholesterol reported, using the pre­ terol through different mechanisms of action, including vious different vehicles, ranged between 7-14 %, which is statins and ezetimibe [90,91]. Recent evidence suggests close to the reduction in LDL cholesterol reported in stud­ that patients who had previous actue coronary syndrome ies that used fat spread as a vehicle for delivering plant benefited from aggressive LDL lowering with statins to sterol [25,37,76-82]. A recent controlled clinical trial has levels subtstantially below current target levels [92,931. shown that the intake of plant sterols provided in low-fat This finding provides enthusiasm for developing novel and non-fat beverages did not affect lipid profiles in mod­ plant sterol-drug and drug-drug combined strategies to erately hypercholesterolemic individuals [83], The find­ aggressively lower LDL cholesterol in some populations. ing of this study was contrary to the findings of other Despite evidence that plant sterols can effectively reduce studies reporting that plant sterols were effective in reduc­ LDL cholesterol and inhibit cholesterol absorption in ing blood cholesterol even when incorporated into low- vehicles other than spread type vehicles, regulatory agen­ fat or non-fat foods [40,42,44,70,72]. This discrepancy cies have been slow to accept plant sterols in foods other may be related to the fact that the plant sterols must be than spreads in some countries such as the USA [94] and added to the low or non fat food matrix in such a way that Australia |95). Rigorous efforts underway by food

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companies and other highly respected organizations to [19,22,25,37,59,76,96,102,103], hypercholesterolemic allow claims for plantsterols in foods other than spreads, subjects [37,38,40,62,72,73,76,77,80,97,104-106], sub­ jects with familial hypercholesterolemia [78,100], and in Optimal number of servings type II diabetic hypercholesterolemic patients (107,108). It has been suggested that plant sterols should be con­ Further, in a type II diabetic population consuming statin, sumed at each cholesterol containing meal to achieve an plantsterols had a combined effect on lowering LDL cho­ optimal effect Adaily intake of 2.5 g plant stanol esters, lesterol an additional 27%, the combined effect being either consumed once per day at lunch, or divided over 44% [108], The reduction in LDL cholesterol seems to be three portions resulted in a similar decrease in serum total greater in hypercholesterolemic individuals with type II and LDL cholesterol levels [96]. Similar efficacy with a sin­ diabetes. Plant sterols decreased LDL cholesterol in hyper­ gle larger dose sterol esters has also been demonstrated in cholesterolemic individuals with and without type II dia­ two additional studies [73]. A single serving of yogurt, betes by 14.9 % and 29.8 %, respectively (Lau et al. providing 1 g of pure free sterols, resulted in a placebo- unpublished data). adjusted reduction in LDL cholesterol of 6.3% [40J. Con­ sumption of a single dose of 2.4 g/d plant sterols resulted Plant sterols are not recommended for pregnant or lactat- in a 9.3 and 14.6 % reductions in blood total and LDL ing women. However, there has not been a systematic cholesterol levels, respectively, in hypercholesterolemic study testing this issue. Vegetarian women habitually con­ individuals [73], Single doses of plant sterols may have sume up to 500 mg of plant sterols per day. There is no sustained effects on cholesterol absorption via interac­ evidence that such women cannot have normal pregnan­ tions with intestinal proteins (see Section 3.1.5.1 for cies. Certain ethnic groups are known to have high levels details). of plant sterol intake and their pregnancy outcome could be evaluated in future studies. For example, in 372 semi- Nevertheless, as there are a plethora of studies showing acculturated Tarahumara Indians in the Sierra Madre the efficacy of plant sterols distributed in 2-3 meals Occidental Mountains of Mexico, the diet was found to be [ 19,25,37,38,70,76-82,97-100], and only two studies to high in fiber and to contain less than 1.0.0 mg/day of cho­ date demonstrating efficacy with a single larger serving lesterol and over 400 mg/day of plant sterols [4]. Further, [73,96], it seems prudent to remain consistent with the in the earlier stages of human evolution, some 5-7 mil­ more established, conservative recommendation of con­ lion years ago, plant sterol intake in Myocene diets would suming plant sterols in 2-3 doses with food, as adopted have been considerably higher, up to 1 g/d [10]. Such by the United States FDA. diets were not only rich in plant sterols, but also dietary fiber, vegetable protein, and associated phytochemicals; Population understudy but low in saturated and trans-fa tty acids [ 1 o]. To meet the Plant sterol for the adult population body's needs for cholesterol, genetic differences and poly­ Typically, cholesterol lowering properties of plant sterols morphisms Were conserved by evolution, tending to raise are similar in both men and women, although recent seaim cholesterol levels. studies highlight that plant sterols can diminish fat solu­ ble vitamins only in women] 37], Mixed gender studies Plant sterols likely interact with ATP-binding cassette must possess the statistical power to separate men and (ABC) transport proteins to direct cholesterol back into women as a statistical covariant, otherwise, the researcher the intestinal lumen, regulating absorption of cholesterol must assume an identical response across both sexes. and plant sterols 1109-113], Plat and Mensink [114| first hypothesized that plant sterols increased the expression of The recent study of Matvienko et al. [73] demonstrates ABCAl. Thereafter, based on an animal study, it was sug­ that soy sterol esters can effectively decrease LDL choles­ gested diatplant sterols are converted into a liverXrecep- terol in young adults of age 23, suggesting age is not a very lor (LXR) agonist, which activates the expression of ABC critical variable influencing LDL cholesterol lowering proteins [115], Mutations in ABC proteins are responsible properties of plant sterols, as also confirmed in studies for the rare disease sitosterolemia [116]; and polymor­ with children [101]. In contrast, the meta-analysis of Law phisms of ABC proteins may affect cholesterol absorption [60] predicted that plant sterol and stanol esters would based on a preliminary study [117]. Polymorphism of reduce LDL cholesterol more effectively at each dose in ABCG8 gene was found to contribute to blood plant sterol older compared with younger people. However, it should levels in healthy subjects [118] suggesting ABCG8 protein be taken into consideration that older people had higher regulates non-cholesterol sterol absorption. starting circulating lipid lewis, so the percent change did not differ across age ranges. A number of studies have Apolipoprotein E phenotype was originally shown to be shown that plant sterols effectively reduce blood choles­ correlated with cholesterol absorption [119]. It was terol in normocholesterolemic

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shown in one study [120] but not others [37,99] to affect were reported except for lycopene, which decreased by plant sterol cholesterol lowering efficacy in recent trials. 8.1%. This decrease was considered of minor biological and clinical importance] 136]. The authors recommended In addition to the above proteins, cholesterol absorption an increase in the intake of fruit and vegetables to avoid is likely controlled by additional proteins [121], as well a the reduction in lycopene values when plant sterols were putative sterol transporter system [122]; In this context, introduced to Step I diet of children with familial the genotype of apolipoprotein A-1V, scavenger receptor- hypercholesterolemia. 81, 3-hydroxy-3-mediyl-coenzyme A reductase, apolipo^ protein E, and cholesterol ester transfer did not affect cho­ In a crossover study, healthy 2-5 year old children con­ lesterol lowering effects of plant stanol [122|. sumed either 3 g/d plant stanol ester or 5 g/d insoluble wheat bran fiber for 2 weeks, then 10 g/d for the second Plant sterols for children two weeks [ 101 ]. Relative to baseline, LDL cholesterol lev­ Plant sterols are not recommended for normocholestero- els were reduced 15.5% with stanol esters and 4% with the lemic children under fivebecause children who are grow­ fiber diet. Stanol esters did not affect triacylglycerols or ing have a large need for cholesterol for normal HDL cholesterol. The study showed that stanol esters development. There is also the fear that plant sterols, par­ reduced LDL cholesterol in normocholesterolemic chil­ ticularly esters, could affect the absorption of fat soluble dren similarly to that found in normocholesterolemic vitamins, However, no direct evidence points at plant ster­ adults and hypercholesterolemic adults and children. In ols being in some way dangerous for children. Studies healthy 6-year-old children who were on a low-saturated, with small amounts of plant sterols fed to infants have low-cholesterol diet, daily intake of 1.5 g/d of plant stanol shown that neonates have the adaptive ability to increase ester was effective in reducing total cholesterol and LDL their cholesterol synthesis [123-125]. In fact, infants are cholesterol values by 5.4% and 7.5%, respectively [137]. typically fed formula diets containing cholesterol concen­ The intake of plant stanol did not cause any adverse clini­ trations 3-35 times lower than breast milk, with consider­ cal effects, nor did it affect the levels of fat soluble vita­ ably higher levels of plant sterols [126]. There is the mins; however, it did cause a 19% reduction in ratio of p- possibility that cholesterol, received in utero or adminis­ earotene to LDL cholesterol ratio. tered to neonates, could affect gene expression and physi­ ology later in life. This theory was initiaHy based on the Children consuming vegetable oil sterols in margarine for increased atherosclerosis incidence in adults fed formula 13 months had serum concentrations of campesterol and rather than breast milk as infants [127], as well as higher sitosterol that were 75% and 44% higher than uiose in the cholesterol in men fed breast milk for less than 3 months control children, while serum cholesterol precursor sterol as compared to more than 9 months [ 128]. This so called concentrations, indicative of cholesterol synthesis, did "cholesterol imprinting" hypothesis is now being not differ between the two groups [138]. Thus, doubling explored in controlled animal models with microarrays dietary plant sterol intake almost doubles serum plant [ 129]. Children with allergies to dairy routinely consume sterol concentrations in 13-mo-old children, but has no vegetable oils rich in plant sterols and less cholesterol, and effect on endogenous cholesterol synthesis. Relative intes­ thus have less cholesterol absorbed, but compensatory tinal absorption of natural plant sterols from the diet in increases in cholesterol synthesis [130]. early childhood is similar to that in adults. In the older study of Mellies et al. [139], 300-900 mg/d of plant ster­ Most studies examining the effects of plant sterols in chil­ ols led to a large accumulation of plant sterols in the dren have been conducted with hypercholesterolemic plasma (0.44 mM) of normo and hypercholesterolemic children [131-134]. Generally, plant sterols seem to be as children. effective in hypercholesterolemic children as in hypercho­ lesterolemic adults. Some older studies in children must As in adults, in children, the apo E phenotype, could be a be interpreted with caution, as the preparations may have factor affecting the efficacy of plant sterols. Plant sterols, been crystalline [135|. Becker [132|, for example, found as an index of cholesterol absorption, were higher in that severely hypercholesterolemic children could be adults or children with the E4/3 phenotype as compared effectively treated with sitosterol, and that 3 g/d sitosterol with those with other phenotypes [140]. Lathosterol, au combined with a half dose of bezafibrate was an effective index of cholesterol synthesis, was also higher in children way to reduce the bezafibrate dose. Intake of 1.7 g/d of with E4/3 phenotype than in those with E3/3 or E3/2, plant sterols in ester form was effective in reducing total indicating these children both absorb and synthesize cholesterol levels, LDL cholesterol and apo B levels in more cholesterol [140]. The effect of phenotype of apo E children with familial hypercholesterolemia who fol­ on response to sterol intake was investigated in 6-year-old lowed Step I diet without any adverse effects [136], No children [141]. Daily intake of 1.6 g of plant stanol was changes in concentration of lipid-adjusted carotenoids effective in reducing blood cholesterol and LDL

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cholesterol by 65 and 8%, respectively, in these children have anti-atherogenicity activity. In rabbits, sitosatanol regardless ofapo E phenotype. Thus, children with differ^ feeding decreased plaque accumulation in coronary arter­ ent apo E phenotype can achieve a reduction in their cho­ ies within the ascending aorta [ 149 ]. Feeding plant sterols lesterol levels by intake of plant sterol. to apo E-deficient mice decreased platelet counts as well as the susceptibility of red blood cells to hemolysis, From the previous studies, it is clear that plant sterols are decreased plasma fibrinogen [16], and decreased forma-- effective in reducing blood cholesterol in healthy as well tion of atherosclerotic lesions [15,16,150]. In healthy sub­ as in hypercholesterolemic children. The only side effect jects who consumed 4 g/d of wood based stanol ester, the repotted is a reduction in levels in ratio of p-carotene to activity of antithrombin-IJI tended to increase compared LDL cholesterol ratio and lyeopene values, which could be to Control group [99|. Thus, plant sterols may reduce balanced by increasing the intake of fruit and vegetable, atherosclerosis development not only by reducing blood especially those rich in carptenoids, as was the case in cholesterol levels but also by possessing anti-atherogenic­ adult population [1Q6], ity activity.

Plant sterat intake and sitosterolemia Anti-cancer activity Sitosterolemia is a rare autosomal recessively inherited The action of plant sterols as anticancer dietary compo­ disorder which results from absorption of high amounts nents has been recently extensively reviewed [151]. Plant of plant sterol and cholesterol for unclear reasons linked sterols can suppress tumor cell growth (LNCaP and HT- to a locus at chromosome 2p21 [142-144] leading to 29) [152,153]. Compared to cholesterol, p-sitosterol development of coronary heart disease at young age, and caused a 24% decrease in cell growth and a 4-fold increase development of tendonxanmomatosis.Variotiscandidate in apoptosis. In the latter work, the authors were inter­ genes involved in cholesterol absorption have been ested in the effects of p-sitosterol on the sphingomyelin excluded at present [145]. Sitosterolemic persons should cycle, and measured two keys enzymes: protein phos­ avoid food products containing plant sterols. Hydrogen- phatase 2A (PP 2Aj and phospholipase D (PLD). A 50% ated plant sterols may be safer than non'-hydrogenated increase was observed in PP 2A activity in media contain­ plant sterols for this population because the former is less ing 16 n.M of p-sitosterol; however, there were noxhanges absorbed, however, this argument is speculative. A recent in protein levels of PP 2A PLD activity increased in pres­ study found that heterozygous subjects for sitosterolemia ence of phorbol myristate and p-sitosterol. This study sug­ who received sterol esters in a spread providing 3.3 g of gests that the sphingomyelin cycle, \vhich increases cell free sterol equivalents for 4 weeks, had a 10.6*6 reduction apoptosis, is mediated by PLD, PP 2A and possibly, incor­ in LDL cholesterol [146]. Levels of campesterol and sito­ poration of p-sitosterol into the membrane. Another pos­ sterol were increased, but the magnitude of the increase sible mechanism by which p-sitosterol can protect against was not much greater than that observed in normal sub­ cancer is through down-regulation of cholesterol synthe­ jects consuming similar spreads. In another recent study sis, as was found in MDA-MB-231 human breast cancer in 12 subjects who were obligate heterozygotes for sitoste­ cells [14|. In an important in vivo study, SCID mice were rolemia, consumption of plant sterol ester for 6 weeks xenografted with die human breast cancer cell line MDA- resulted in an additional significant reduction of 5.9% in MB-231 [154]. Plant sterol-fed mice had a 33% smaller LDL cholesterol over that provided by a Step I diet alone, tumor size and 20% less metastases in lymph nodes and but no additional significant reduction was found after lungs than cholesterol-fed mice. This finding implied the consumption of plant sterol ester for 12 weeks [147]. possibility that plant sterols may retard the growth and Although plasma levels of plant sterols concentration spread of breast cancer cells. In addition to retarding the were elevated, the increase was similar to that reported in growth of breast cancer cells by plant sterols, there is some normal and mildly hypercholesterolemic subjects who evidence that plant sterols can affect the development of consumed plant sterol esters [ 147|. The increase in plasma prostate, cancer [155]. In a meta-analysis, 519 men were levels of plant sterols reached a plateau, which indicates studied in 4 randomized, placebo-controlled, double- that obligate heterozygotes eliminated the plant sterols blind trials, p-sitosterol improved urinary symptom scores from their body in order to prevent their accumulation. and flow measures, suggesting mat non-glucosidic forms For prudency, it is nevertheless recommended mat per­ of P-sitosterol improve urinary symptoms and flow meas­ sons with sitosterolemia avoid plant sterols. ures. Long term effectiveness, safety, and ability to prevent benign prostatic hyperplasia complications are not Anti-atherogeniclty activity known [155]. In another recent study, there was no evi­ In vitro studies have shown mat plant sterols are effective dence that plant sterol usage at dose of 300 mg/d, in preventing hyperproliferation of vascular smooth mus­ decreased risk of colon and rectal cancers [156], A similar cle cell that play a role in atherosclerosis development conclusion was reached following a rat study in which rats [148]. Animal studies have shown that plant sterols also

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were given the carcinogen methyl-nitroso-urea and then properties. Such antioxidant protection could also benefit monitored for tumor development [157], atherosclerosis [166| and cancer [167] disease state.

Plant sterols have also been found to have a protective Anti-ulcer activity effect against lung cancer [158]. In this study, intake of In a recent study, phytosterol esters, but not sterols, in about 144 mg/d of plant sterols was associated with horse gram (an herb in the genus Dolichos cultivated in reduction in risk for lung cancer even after controlling of India for food and fodder) were protective in a pyloric confounding factors, i.e. tobacco smoking, vegetables, ligation model of ulcer, whereas sterols were protective in fruits, and antioxidant substances. Total dietary plant acute ulcer models using ethanol-induced and cysteam- sterol intake was found to be inversely associated with ine-induced ulceration [168], Phospholipids were protec­ breast ]159|), stomach [160], and esophageal [161] can­ tive in both types of model. Thus, the presence of sterols, cers. It was found that women witii highest quartiles of sterol esters, arid phospholipids in food lipids in staple total dietary intakes of plant sterols (>122 mg/d) had diets may account for the low prevalence of duodenal reduced risk of endometrial cancer (162], and intake of ulcer in certain geographical areas, despite a uniformly more, than 521 mg/d reduced risk of ovarian cancer [163]. high prevalence of Helicobacter pylori infection. On the other hand, in a prospective epidemiological study, high dietary intake was not associated with reduced Anti-fungal activity risk of colon and rectal cancers [156]. However, the intake Another area for future investigation is the anti-fungal of plant sterol might reduce the risk of more than one type activity of plant sterols and related trkerpenes [169]; In of cancer. this work, the antifungal activity of triterpenes in the mushroom species Ganoderma annulare was Anti-inflammation activity of plant sterols demonstrated. Bouie [17] and Bpuic etal [1641 have reviewed the possi­ ble roles of phytosterols in the etiology or preventive role Safety of phytosterols in various diseases and conditions, includ­ It has been concluded that plant sterols, within the range ing proliferative responses of lymphocytes, pulmonary that causes desirable reduction in blood levels of total tuberculosis, feline immunodeficiency virus and HIV, cholesterol and LDL-choIesterol, are clinically safe. This stress induced immune suppression, rheumatoid arthritis, conclusion has been reported in short-term studies and allergic rhinitis/sinusitis. The mechanisms by which [ 19,3 9,40,170 ] as well as in long-term study that lasted for plant sterols display their anti-inflammatory activity are 1 year [81]. Since plant sterols decrease the absorption of thought to include inhibition of secretion of inflamma­ cholesterol, they might also affect the absorption of fat- tory mediators such as interleukin-6, and tumor necrosis soluble vitamins. The scientific evidence for the impact of factor-a by monocytes [17]. Most of the work has been phytosterols on carotenoid status and fat soluble vitamins conducted with animals. From these provocative results, it is summarized in Table 3. The effect of plant sterols on the is not unlikely that plant sterols will be further used for blood levels of precursors of fat-soluble vitamins is a con­ purposes related to control of deveopment and spread of troversial issue. In some studies, plant sterols consump­ certain cancers in humans. tion lias been shown to significantly reduce levels of carotenoids [25,37,38,81,170,171], tocopherol [37], and Antl-oxldant activity lycopene [25,38]. Other studies reported that the con­ Another possible effect of plant sterols is their antioxidant sumption of plant sterols does not affect blood levels of activity [165]. It was found that the methanol extract of carotenoids [39,72,104,172], tocopherol [19,39,173|, soybean oil, which has a strong in-vitro protective effect and lycopene [19,173]. against DNA damage in human endothelial cell, contains phytosterols in addition to tocopherols and n-3 polyun­ In a recent trial comparing equal free sterol equivalent saturated fatty acids (PUFA). Results suggest that the anti­ amounts (2.2 g/d) of esterified sterols and free sterols in oxidant activity of soybean oil may be in part related to milk, both forms of sterols decreased the absorption of P- sterol content Moreover, in in-vitro conditions, sitosterol, carotene and a-tocopherol in normocholesterolemk and sitosterol glucoside were found to decrease lipid per­ men. The reduction in P-carotene bioavailability was sig­ oxidation of platelet membranes in the presence of iron nificantly less pronounced with free plant sterols than [18| and in healthy human subjects a 2 and 3-g dose of with plant sterol esters However, there was no difference stanol ester reduced oxidized LDL-C levels [82]. The in cholesterol absorption between the two forms of plant authors suggested that the intake of stanol ester might sterols. [71]. Esters are presumed to have more of an effect protect LDL particles from oxidation. Thus, based on 011 fat soluble vitamins because they partition into die oil results from in vitro studies and on human study, there is phase of the intestine, whereas free sterol would partition a possibility that plant sterols may possess antioxidant into the micellar phase [174].

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Table 3; Summary of scientific evidence for the impact of phytosterols on carotenoid status and fat soluble vitamins

Reference Products Vehicle Subject Mean Condition: Starting Do&egMay Duration of Outcome Age or range mesuiTC mri (range) (free sterol/ study wks stanot equivalents)

Impact of Impact of phytosterols on phytosterols on vitamin A and other fat soluble carotenoid status vitamins

PSl C, DZ D3. Spread Aduits, 18-65 andp

[ I78] DI Spread, Postmenopausal 55 8.0 3.2-DI In Margarine <-» Serum retinol <••> Serum vitamin D Butter women aged spread; 2.4-DI intervention- I a- and p-carotene *•* lipid 50-55 in butter 6 wks; butter concentration* standardized d- interventio-5 tocopherol wks

[I8I] D Spread Adults Moderately *-» retinol «-> vitamin D hypercbolesterolemlc l a- and p-carotenes ^-vratfoof.a- tocopherolto cholesterol

[623 *>l. D2 Spread Adults 5.4-7.5 2.3-Dt; 2.2-D2 8 I p-carotene *-» p-carotene <-* cz-carotene with or wldiout lipid adjustment

[19] CI Spread Adults, aged 19- <7;5 0:9,1,6 and 3.3 3.6 4- a and p carotene and I a-tocopherol 58 lycopene concentrations concentrations reduced reduced *-> tipid-edjusted <-> lipid carotene levels standardized a tocopherol

[41] Spread Adult* Hypercholesterolemic I g 3x/d (2 • vitamin D different formulations) or 2g3x/d

P0] C2.C3 Spread Adults aged 18- <8 4- lycopene 62 «•* o> and p-carotene

[76] CD Spread Adults, mean 4.8-7.0 <-» retinol *-» vitamin D age 49 *••> a carotene or concentration with lycopene or without lipid concentrations, with or adjustment wldiout lipid ad}ustment I p-carotene decreased after each Intervention, but not when lipid adjusted

[961 Spread, Adultsaged 18- <6.5 2 5g/d4unchor 4 <-* retinol i a-tocophoroJ Shortanln 65 In 3 doses of <~» oocarotene after both the Ix 0 4-brealdast, J- lycopene arid (5. and 3x/d regtroe Ix/ 0.8-lunch and carotene after the 3x/d dregime, **tn K3*dfnner regime lipid-adjusted J- P-carotene by Ix/d concentrations regime; ** In any of the lipid- adjusted concentrations

[137] p Spread Children, aged 6 Healthy is 4 P-carotene with and I o^tocopherol but without lipid ** in llpld-adjuited standardization concentration

[134] D Spread 24 children aged Familial 2.2 <-+ retinol 3-13; 4 parents; hyperchoicstoroicmla 4- a and p-carotenes 16 healthy family with and without llpid- members adjustment (measurement* done only in children)

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Table 3: Summary of scientific evidence for trie impact of phytosterols oft carotenoid status and fat soluble vitamins (Continued)

[39] Spread Adults 25-64 >S 8 24 ** retlnol. a-carotene Or P-carOtene

[I70] Spread, Healthy adults Xft 60 or 9.0 8 4- a and trans p- All fat soluble Sobtf carotene reduced in the vitamins remained Dressing 9.0 g/day group, but all within normal range carotenoid values after treatments remained within the normal range

[38] Spread Adults, aged 21- 3.4-5.0 1.1 or 2.2 *-» In serum retlnol, *-+ it- or y~ 75 zeaxanthtnor tocopherol. 25 cryptoxanthln hydroxwitamlJroxyyi n D -1- trans p-orotene, a- or phvflophyfloqulnont e carotene, lycopene and lutein all decreased I lfpld-ad|ustod trahs p- carotene

[40] Yogurt- Adults, aged 33- Modertally I <-» vitamin £, t drink 69 hvperchofesterotemic vitamin D (probably as a result of increased skin nthesis of vitamin g due to the time of year)

[70] C Yogurt Adults Normocholesterolemlc 3 4 I p-carotene t lipid standardized tocopherol

[79] C Spread Healthy adults 1.8 3 «-> vitamin A *-* vitamin E i P-carotono

[173] CD Spread Adults Hyporcholastorolemic !.«: 1.8-D 3 *» <->

[81] C Adults aged 48 Normodiotesterolemic 16 52 illpid-adiusted o-and P- ^tipicUdjustedfat and carotene soluble vitamin hypercholesterolemlc concentration

[71] AZC2 Vegetable Men aged 29 <5,2 2.2 4- (Carotene I ct-tocopheroi oil, partly bioavailability 57% with bioavailability 27% filled milk C2.48* with A2;A with C2, no efffect TRL-reclnyl pa Imitate wlthA2 bioavailability 48% with CZ32%wlthA2No standardization to TAG because TRL-TAG pharmacoicinecks were equivalent In all groups

C«] Vegetable Adults, aged 60 7,0 1.2 or IA 4wk/perlod i c6-andf>r and tocopherol with pVcarotene, hxopene, p* both.doses cryptoxanthln, zeaxanthin with either dose. 4. hpld-ad]usted X change in lutein with 1,6 i

See Table I for abbreviations, TAG, Wacy^lycerol; TRU triacylglycerol-rich lipoprotein.

During plant sterol consumption, increasing the con­ Conclusions sumption of fruits and vegetables to be > fiveserving s and Based on the positive results from studies examining the including one or more carotenoid rich source would be effects of low doses of free plant sterols and sterol esters, enough to avoid reduction in carotenoid levels resulted there is a good likelihood that a minimum dose of 0,8- from plant sterol intake [106]. 1.0 g of free sterol and free sterol equivalents will reduce LDL Cholesterol by 5% or more, and that this reduction in

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LDL cholesterol will correlate with an approximate 6- reduction in LDL cholesterol. Along these lines, it would 10% reduction in coronary heart disease risk at age 70 be fruitful to monitor individuals for markers of choles­ [35,36]. There is also a good likelihood that the reduction terol absorption before recommending consumption of in LDL cholesterol within this same dosage could be enriched plant sterols. Those persons who are poor higher if full compliance of the plant sterol dosage were absorbers of dietary cholesterol (on the basis of lowlevels assured. In studies where subjects were monitored to of serum cholestanol and plant sterols) [175] may not be ensure full compliance, efficacy with a 1.5 to 2 g/d dose ideal candidates for consuming plant sterols to lower Tanged from 12-16%. For maximum efficacy of free plant plasma LDL cholesterol, but may "still benefit from plant sterols, the plant sterols must be administered in a soluble sterols' other positive effects. or microcrystalline form. Efforts must be taken to assure that the free plants sterols remain in this bio-effieadous List of abbreviations form during the shelf life of the product, pur dosage rec­ ABC, ATP-binding cassette; apo, apolipoprotein; LDL, ommendation of 0.8-1.0 g of free sterol and free sterol Low density lipoprotein; PLD, phospholipase D; PUFA, equivalents compares favorably with the FDA interim polyunsaturated fatty acids. final rule 21 CFR 101.83 recommending 0.65 g of sterol esters per serving, twice perdayin spreads, which is equiv­ Authors* contributions alent to 0.8:gof free sterol equivalents per day. As of Jan­ AB completed the first draft of the article and tables. PJHJ uary 2003, the FDArecognized that the scientific literature and SSH then contributed substantially to the text and supports expanding the health claim to include free forms tables, expanding the initial concept, and elaborating and of plant sterols and stanols, and to include a wider range updating specific themes. AB updated the final text. All of products, including low-fat products. The FDA further authors improved overall flow. stated that thescience (as of January 2.003) shows that the lowest effective daily intake of free phytosterols is 800 nig/ Acknowledgements dhttpy/vr«.cfsan,fda,gQy/~dms/cls-ttr30,htnil Tneauthbrs thank Hilary Green of the Nestle Research Center (NRC) in Lausanne^ Switzerland for proof reading and improving the manuscript. AB; In addition to their cholesterol lowering properties, plant completed parts of this review at the NRC, as a Senior Project Leader for sterols have other promising effects, including antican­ plant sterol research. cer, anti-inflammation, anti-atherogenicity; and anti-oxi­ References dation activities. Despite the fact that plant sterols reduce 1. Piironen V, Lindsay DG, Miettinen TA, Toivo J. Lamp! A-M: Plant the carotenoid levels in adults, it seems that an intake of sterols: biosynthesis, biological function and their impor­ plant sterols between 0.8^-1.0 g is essential to prevent tance to human nutrition. ] Scl Food Afrit. 2000, 80:939-966. 2. Moreau R, Whitaker B, Hicks K: Phytosterols, phytostanols, and chronic diseases in adult population. The carotenoid low* their conjugates In foods: structural diversity, quantitative ering effect of plant sterols can be corrected by increasing analysis, and health-promoting uses. Prog Lipid Res 2002,41:457. intake of food that is rich in carotenoids. However, more 3. Ahrens EH Jr, Boucher CA The composition of a simulated American diet J Am Diet Assoc 1978, 73:613-620, studies are needed in pregnant and lactating women as 4. Cerquoira MT, Fry MM. Connor WE The food and nutrient well as on children in order to verify the dose required to intakes of the Tarahumara Indians of Mexico. Am ] CUn Nulr 1979,32:905-915. decrease blood cholesterol without affecting fat-soluble 5. Nair PP, Turjman N, Kessie G, Calkins B, Goodman GT, Davidovitz vitamins and carotenoid sta tus: H, Nimmagadda G: Diet, nutrition intake, and metabolism in populations at high and low risk for colon cancer. Dietary cholesterol, beta-sltosterol, and stfgmasterol. Am } CUn Nulr Plant sterols are naturally occurring molecules that 1984, 40:927-930. humanity has evolved with, which partially counter the 6. Hirai K, Shimazu C, Takezoe R. Ozeki Y: Cholesterol, phytosterol and polyunsaturated fatty acid levels In 1982 and 1957 Japa­ absorption of dietary cholesterol and have other impor­ nese diets. J Nulr Scl Vilaninol (Tokyo) 1986, 32:363-372. tant biological functions described above. The myriad of 7. Miettinen TA, Ttlvis RS, Kesaniemi YA: Serum cholestanol and factors that can affect the efficacy of plant sterols have plant sterol levels In relation to cholesterol metabolism in middle-aged men . Metabolism 1989, 38:136-140. been explored. The resonating conclusion is that properly 8. Morton GM, Lee SM, Buss DH, Uwrancc P: Intakes and major die­ solubilized 4-desmetyl plant sterols, in ester or free form, tary sources of cholesterol and phytosterols in the British in reasonable doses (0.8-1.0 g of equivalents per day) and diet i Hum Nuv Diet 1995, 8:429-440. 9. de Vries JHM, Jansen A Kromhout D, van de Bovenkamp P, van Sta- in various vehicles including natural sources, and as part veren WA, Mensink RP, Katan MB: The fatty acid and sterol con­ of a healthy diet and lifestyle, are important dietary com­ tent of food composites of middle-aged men In seven ponents for maintaining good heart health. Consumption countries. ] Food Comp And 1997, 10:115-141. 10. Jenkins D), Kendall CW, Marchie A, Jenkins AL, Connelly PW, Jones from natural sources should be encouraged for all per­ PJ, Vuksan V: The Garden of Eden-plant based diets, the sons, and consumption of plant sterols in enriched genetic drive to conserve cholesterol and Its implications for heart disease In the 21st century. Comp Biochem Physiol A Mol sources should be encouraged following consultation Imegr Physiol 2003, 136:141-151. with a clinician. The clinician should be assured that the 11. Ostlund R E, Jr, McGill JB, Zeng CM, Covey DF, Steams J, Stenson individual is a responder to plant sterols, and achieves a WF, Spilburg CA: Gastrointestinal absorption and plasma

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kinetics of soy Delta(5)-phytosterols and phytostanols in esters in hypercholesterolaernic subjects . Clin Set 1994, humans. Am ] Physiol Endocrinol Metab 2002, 282:E9I I-E9I6. 87:61-67. 12. Bosner MS, Lange LG, Stenson WF, Ostlund Jr RE: Percent choles­ 33. Pelletier X, Belbraouet S, Mirabel D, Mordret F, Perrin JL, Pages X, terol absorption in normal women and men quantified with Debry G: A diet moderately enriched in phytosterols lowers dual stable isotopic tracers and negative ion mass plasma cholesterol concentrations In normocholesterolemlc spectrometry. J Lipid Res 1999, 40:302-308. humans . Ann Nutr Metab 1995*39:291-295. 13. Miettinen TA, Tilvis RS, Kesaniemi YA Serum plant sterols and 34. Smith SJ, Cooper GR, Myers GL, Sampson EJ: Biological variability cholesterol precursors reflect cholesterol absorption arid in concentrations of serum lipids: sources of variation among synthesis in volunteers Of a randomly selected male results from published studies and composite predicted val­ population. Am] Epidemiol 1990, 131:20-31. ues .On Chan 1993, 39:10.14-1022, 14. Awad AB, Roy R, fink CS: Beta-sitosterol, a plant sterol, 35. Law MR, Wald NJ, Thompson SG: By how much and how quickly Induces apoptosls and activates key caspases in MDA-MB- does reduction in serum cholesterol concentration lower 231 human breast cancer cells. Oncol Rep 2003, 10:497-500. risk of ischaemic heart disease? BM] 1994, 308:367-372. 15. Moghadasian MH, McManus BM, Pritchard PH, Frohlich JJ: "Tall oiT- 36. Stampfer MJ, Sacks FM, Salvini S, Willett WC, Hennekens CH: A pro­ de rived phytosterols reduce atherosclerosis In ApoE-defi- spective study of cholesterol, apollpoproteins, and the risk of eleht mice. Arterioscfer Thnmb Vox Biol 1997, 17:119-126. myocardial Infarction .NEnil] Med 1991, 325:373-381. 16. Moghadasian MH, McManus BM, Godin DV, Rodrigues B, Frohlich J): 37. Hailikainen MA, Sarkkinen ES, Uusitupa ML Plant stanol esters Proatherogenlc and antiatherogenic effects of probucol and affect serum cholesterol concentrations of hypereholestero- phytosterols in apolipoprotein E-deficient mice: possible lemic men and women in a dose-dependent manner, j Nutr mechanisms of action. Circulation 1999, 99:1733-1739. 2000, 130:767-776. 17. Bouic PJ: The role of phytosterols and phytosterolins in 38. Maki KC, Davidson MH, Umporowicz DM, Sdiaefer EJ. Dicklin MR, immune modulation: a review of the past 10 years. Curr Opin Ingram KA, Chen S, McNamsra JR, Gebhart BW, Ribaya-Mercado JD, Clin Nutr Metab Care 2001, 4:471 -475. Perrone G, Robins SJ, Franke WC: Lipid responses to plant- 18. van Rensburg SJ, Daniels WM, van Zyl JM, Taljaard JJ: A compara­ sterol-enriched reduced-fat spreads incorporated Into a tive study of the effects of cholesterol, beta-sitosterol, beta- National Cholesterol Education Program Step I diet Am] CSn sitosterol glucoside, dehydroepiandrosterone sulphate and Nutr 2001, 74:33-43. melatonin on In vitro lipid peroxidation. Metab Brain Dis 2000, 39. Christiansen Li, LShteenmaki PLA, Mannelin MR, Seppa'nen-laakso 15:257-265. TE, Hiltunen RVK, Yliruusi JK: Cholesterol-lowering effect of 19. Hcndriks HF, Weststrate JA van Vliet T, Mcijcr GW: Spreads spreads enriched with microcrystalline plant sterols In enriched with three different levels of vegetable oil sterols hypercholesterolemic subjects. Eur J Nutr 2001, 40:66-73. and the degree of cholesterol lowering in normocholestero- 40. Volpc R, Niitr/nen L Korpela R, Sirtori C, Bucci A Fraonc N, Pazzuc- laemic and mildly hypercholesterolaemlc subjects. Eur J CSn coni F. Effects of yoghurt enriched with plant sterols on Nuu 1999,53:319-327. serum lipids in patients with moderate 20. Sierksma A Weststate JA, Meijer GW: Spreads enriched with hypercholesterolaemia. 6ry Nutr 2001, 86:233-239. plant sterols, either esterified 4,4 dimethylsterols or free 4 41. Nguyen TT, Dale LC, von Bergmarm K, Croghan IT: Cholesterol- desmethylsterols, and plasma total and LDL cholesterol lowering effect of stanol ester In a US population of mildly concentrations. Br] Nuu 1999, 81:273-282. hypercholesterolemic men and women: a randomized con­ 21. Denke MA: Lack of efficacy of low-dose sitostanol therapy as trolled trial. Mayo Clin Proc 1999, 74:1198-2006. an adjunct to a cholesterol-lowering diet in men with mod­ 42. ThomsenAB, Hansen HB, Christiansen C, Green H, Berger A Effect erate hypercholesterolemia. Am] CSn Nuu 1995, 61:392-396. of free plant sterols In low-fat milk on serum lipid profile in 22. Ostlund RE, Spilburg CA, Stenson WF: Sitostanol administered in hypercholesterolemic subjects. Eur] Qin Nutr 2004 in press. lecithin micelles potently reduces cholesterol absorption in 43. Schwab US, Vogel S, Lammi-Keefe Cj, OrdovasJM, Schaefer EJ, Li ZG, humans. Am] CSn Nutr 1999, 70:826-831. Ausman LM, Gualtieri L, Goldin BR, Furr HC, Uchtenstein AH: Var­ 23. Ikeda I, Nakashima-Yoshida K, Sugano M: Effects of cycloartenol ying dietary fat type of reduced-fat diets has little effect on on absorption and serum levels of cholesterol in rats. J Nutr the susceptibility of LDL to oxidative modification in moder­ SdVtawninof 1985, 31:375-384. ately hypercholesterolemic subjects. ] Nutr 1998, 24. Kritchevsky D, Topper SA, Czarneckl SK. Kyle DJ: Effects or 4- 128:1703-1709. methylsterols from algae and of b sitosterol on cholesterol 44. Pouteau E, Monnard I, Piguet-Welsch C, Groux MJA Sagalowicz L metabolism in rats. Nutr Res 1999, 19:1649-1654. Berger A Non-esterified plant sterols solubijized in low fat 25. Wcstratc JA Meijer GW: Plant sterol-enriched margarines and milks inhibit cholesterol absorption: a stable isotope double- reduction of plasma total and LDL-cholesterol concentra­ blind crossover study. £ur/Nutr 2003, 42:154-164. tions in normocholesterolaemlc and midly hypercholestero­ 45. Dcvaraj S, Jialal I, Vega S: Plant sterol-fortlfied orange Juice laemlc subjects. Eur J On Nutr 1998, 52:334-343. effectively lowers cholesterol levels in mildly hypercholeste­ 26. Visscrs MN, Zock PL, Meijer GW, Katan MB: Effect of plant sterols rolemic healthy individuals. Anerioxler Thnmb Vast BM 2004, from rice bran oil and triterpene alcohols from sheanut oil 24:e25. on serum lipoprotein concentrations in humans; Am J Qin Nutr 46. Howard BV, Gidding SS, Liu K: Association of apolipoprotein E 2000, 72:1510-1515. phenotype with plasma lipoproteins in African-American 27. Vanhanen rIT, Miettinen TA: Effects of unsaturated and satu­ and white young adults. The CARDIA Study. Coronary rated dietary plant sterols on their serum contents Artery Risk Development in Young Adults. Am] Epidemiol 1998, Ctn Oka Acta 1992, 205:97-107. 148:859-868. 28. Miettinen TA, Vanhanen H: Dietary sitostanol related to absorp­ 47; Pedersen A Baumstark MW, Mardcmann P, Gylling H, Sandstrom B: tion, synthesis and serum level of cholesterol In different An olive oil-rich diet results In higher concentrations of LDL apolipoprotein E phenotypes. Atherosclerosis 1994, 105:217-226. cholesterol and a higher number of LDL subtraction parti­ 29. Tilvis RS, Miettinen TA- Serum plant sterols and their relation cles than rapeseed oil and sunflower oil diets.} lipid Res 2000, to cholesterol absorption. Am} CSn Nutr 1986,43:92-97. 41:1901-1911. 30. Miettinen TA, Kesaniemi YA Cholesterol absorption: regulation 48. Wagner KH, Tomasdi R, Elmadfa I: Impact of diets containing of cholesterol synthesis and elimination and wlthin-popula- com oil or olive/sunflower oil mixture on the human plasma tion variations of serum cholesterol levels .Am J CSn and lipoprotein lipid metabolism. Eur] Nuu 2001, 40:161-167. Nuu 1989, 49:629-635. 49. Osdund RE, Jr, Racette SB, Okeke A Stenson Wp. Phytosterols 31. Pouteau E, Piguet-Welsdi C, Berger A, Fay LBc Determination of that are naturally present in commercial com oil signifi­ cholesterol absorption in humans: from radiolabel to stable cantly reduce cholesterol absorption in humans. Am] CSn Nuu isotope studies. Isotopes Environ Hearth Stud 2003, 39:247-257. 2002,75:1000-1004. 32. Vanhanen HT, Kajander J, Lehtovirta H, Miettinen TA: Serum lev­ 50. Berger A Gremaud G, Baumgartner M, Rein D, Monnard I, Kratky E, els, absorption efficiency, faecal elimination and synthesis of Geiger W, Burri J, Dionisi F, Allan M, Lambdet P: Cholesterol-low­ cholesterol during Increasing doses of dietary sitostanol ering properties of amaranth grain and oil in hamsters, fnt ] Yitam Nutr Res 2003, 73:39-47.

Page 15 of 19 (pegs number not for citation purposes) 206

Lipids in Health and Disease 2004, 3 http://Wvvw.lipiclvvD rld.com/conten t/3/1/5

51. Berger A. Monnard I, Dionisi F, Gumy D, Lambelet P, Hayes KC: a liquid emulsion on cholesterol absorption and synthesis in Preparation of amaranth flakes, crude oils, and refined oils hypercholesterolemlc men, Eur j Nutr 2002, 41:54-60. for evaluation of cholesterol-lowering properties in hamster. 70. Mehsink RP, Ebbing S, Lindhout M, PlatJ, van Heugten MMA! Effects Food Chem 2003. 81:119-124. of plant stanol esters supplied in low-fat yoghurt on serum 52. Trautwein EA, Schciz C. Rieckhoff D, Kunath-Rau A, Erbersdobler lipids and lipoproteins, non-cholesterol sterols and fat solu­ H F, de Groot WA, Meijer GW: Effect of esterified 4-desmethyl- ble antioxidant concentrations. Atherosclerosis 2002, sterols and -stanols or 4>4'-dlmethylsterols on cholesterol 160:205-213. and bile acid metabolism in hamsters. Br j Nutr 2002, 71. Richelle M, Ensten M, Hager C, Groux M, Tavazzi I. Godin JP, Berger 87:227-238. A Metalron S, Quaile S, Piguet C, Sagalowlcz L Green H, Fay LB: 53. Kritchevsky D, Tepper SA Wright S, Czamecki SK, Wilson TA Nico- Both free and esterified plant sterols decrease the bioavaila­ losl RJ: Cholesterol vehicle in experimental atherosclerosis bility of B-carotene and ^-tocopherol, in normocholestero- 24: avocado oil.;Am Col) Nutr 2003, 22:52-55. lemic humans. Am; CSn Nutr 2004:(ln press). 54. Perona JS, CanizaresJ, Montero E, Sanchez-Dominguez JM, Ruiz-Guti­ 72. Nestel P, Cehun M, Pomeroy S, Abbey M, Weldon G: Cholesterol- errez V: Plasma lipid modifications in elderly people after lowering effects of plant sterol esters and non-esterified administration of two virgin olive oils of the same variety stanols In margarine, butter and low-fat foods. Eur J Ctn Nutr (Olea europaea var. hojiblanca) with different triacylglycerol 2001,55:1084-1090. composition. Br J Nutr 2003, 89:819-826, 73. Matvienko OA, Lewis DS, Swanson M, Amdt B, Rainwater DL Stew­ 55. Garg ML, Blake Rj, Wills RB: Macadamia nut consumption tow­ art J, Alekel DL- A single dally dose of soybean phytosterols In ers plasma total and LDL cholesterol levels in hypercholes- ground beef decreases serum total cholesterol and LDL cho­ terolemic men.; Nutr 2003, I 33:1060-1063. lesterol in young, mildly hypercholesterolemlc men. Am; CJin 56. Khallouki F, Younos Q Soulimani R, Oster T, Charrouf Z, Spiegelhal- Nutr 2002, 76:57-64. der B, Bartsch H, Owen RW: Consumption of argan oil 74. Carr TP, Cornelison RM, lllston BJ, Stuefer-Powell CL, Gallaher DD: (Morocco) with its unique profile of fatty acids, tocopherols, Plant sterols alter bile acid metabolism and reduce choles­ squalene, sterols and phenolic compounds should confer val­ terol absorption in hamsters fed a beer-based diet uable cancer chemopreventive effects. Eur; Cancer Prev 2003, Nutr Res 2002, 22:745-754. 12:67-75. 75. Spilburg CA, Goldberg AC, McGill JB, Stenson WF, Racette SB, Bate- 57. Ostlund R E, Jr, Racette SB, Stenson WF: Inhibition of cholesterol man J, McPherson TB, Osdund R E, Jr: Fat-free foods supple­ absorption by phytosterol-replete wheat germ compared mented with soy stanoMecithin powder reduce cholesterol with phytosterol-depleted wheat germ. Am ; CSn Nutr 2003, absorption and LDL cholesterol. ] Am Diet Assoc 2003, 77H385-I389. 103:577-581. 58. Maki K.C, Davidson MB, Umporowicz PM, Schaefer EJ, Diklin MR, 76. Hallikainen MA, Sarkkinen ES, Gylling H, Erkkila AT, Uusitupa MIJ: Ingram KA, Chen S, McNamaraJrW, Gebhart B, Ribaya-MercadoJP, Comparison of the effects of plant sterol ester and plant Perrone G, Robins SJ, Franke WC: Lipid responses to plant stanol ester-enriched margarines in lowering serum choles­ sterol-enriched reduced-fat spreads incorporated into a terol concentrations In hypercholesterolaemic subjects on a National Cholesterol Education step I diet Am] din Nutr 2001, low-fat diet . Eur; CSn Nutr 2000, S4:715-725. 74:33,43. 77. Jones PJ, Raeini-Sarjaz M, Ntanios FY, Vanstone CA, Feng J, Parsons 59. Mattson FH, Grundy SM, Crouse JR: Optimizing die effect of WE Modulation of plasma lipid levels and cholesterol kinet­ plant sterols on cholesterol absorption in man. Am J Ctn Nutr ics by phytosterol versus phytostanol esters.; lipid Res 2000, 1982, 35:697-700. 41:697-705. 60. Law M: Plant sterol and stanol margarines and health. Sr Med 78. Neil HAW, Meijer GW, Roe LS: Randomised controlled trial of ; 2000, 320:861-864. use by hypercholesterolaemic patients of a vegetable oil 61. Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti ft Effi­ sterol-enriched fat spread. Atherosclerosis 2001, 156:329-337. cacy and safety of plant stanols and sterols In the manage­ 79. Ntanios FY, Homma Y, Ushiro S: A spread enriched with plant ment of blood cholesterol levels. Mayo On Proc 2003, sterol-esters lowers blood cholesterol and lipoproteins with­ 78:965-978. out affecting vitamins A and E in normal and hypercholeste­ 62. Hallikainen MA, Uusitupa MIJ: Effects of 2 low-fat stanol ester- rolemlc Japanese men and women. J Nuu 2002,132:3650-3655. containing margarines on serum cholesterol concentrations 80. Cleghom CL, Skeaff CM, Mann J, Chisholm A: Plant sterol- as part of a low-fat diet in hypercholesterolemlc subjects. Am enriched spread enhances the cholesterol-lowering potential J CSn Nutr 1999, 69:403-410. or a fat-reduced diet. Eur; On Nutr 2003, 57:170-176. 63. Jones PJH, Ntamios FY, Mahmoud Radnl-Sarjaz C: Cholesterol- 81. Hendriks HF, Brink EJ, Meijer GW, Princen HM, Ntanios FY: Safety lowering efficacy of sitostanol-containing phytosterol mix­ of long-term consumption of plant sterol esters-enriched ture with a prudent diet in hyperlipidemic men. Am J On Nutr spread. £ur; an Nutr 2003, 57:681-692. 1999,69:1144-1150. 82. Homma Y, Ikeda I, Ishikawa T, Tateno M, Sugano M, Nakamura H: A 64. van Heyningcn C: Cholesterol lowering margarine may not be randomized, placebo-controlled trial: Decrease In plasma useful in healthy fat modified diet. Brit Med J 1999, 319:186. low-density lipoprotein cholesterol, apolipoproteln B, 65. Mussner MJ, Parhofer KG, Von Bcrgmann K, Schwandt P, Broedl U, cholesteryl ester transTer protein, and oxidized low-density Otto C: Effects of phytosterol ester-enriched margarine on lipoprotein by plant stanol ester-containing spread. Nutrition plasma lipoproteins in mild to moderate hypercholestero­ 2003, l9::369-374. lemia are related to basal cholesterol and fat intake. Metabo­ 83. Jones PJH, Vanstone CA, Raleni-Sarjaz M, St-Onge M-P: Phytoster­ lism 2002, SI: 189-194. ols in low-and non-fat beverages as part or a controlled diet 66. Maki KC, Shinnick F, Seeley MA, Veith PE, Quinn LC, Hallissey PJ. rail to lower plasma lipid levels,; Lipid Res 2003, 44:1713-1719. Temer A, Davidson MH: Food products containing free tall oil- 84; Groux MJA Leser M. Berger A Milk product comprising unes- based phytosterols and oat beta-glucan lower serum total terifled sterol Nesde SA; 2003. (Patent WO03009708 and LDL cholesterol in hypercholesterolemlc adults. J Nutr 85. von BoiwdorfF-Nikander A Karjalainen M, Rantanen J, Christiansen L, 2003, 133:808-813. Yliruusi J: Physical stability or a microcrystalline beta-sito- 67. Judd JT, Baer DJ, Clevidence BA, Muesing RA, Chen SC, Meijer GW: steroI suspension in oil. fur; Phorm Sci 2003, 19:173-179. Dietary fat and ratty acids and plasma lipid modifying effects B6. Jenkins DJ, Kendall CW, Marchie A Faulkner D, Vidgen E, Lapsley KG, of sterol esters. FASEB; 2001, IS:A639, Trautwein EA Parker TL, Josse RG, Leiter LA, Connelly PW: The 68. G remand G, Piguet C, Baumgartner M, Pouteau E, Decarli B, Berger effect of combining plant sterols, soy protein, viscous fibers, A. Fay LBc Simultaneous assessment of cholesterol absorption and almonds In treating hypercholesterolemia. Metabotsm and synthesis in humans using on-line gas chromatography/ 2003,52:1478-1483. combustion and gas chromatography/pyrolysis/isotope-ratio 87. Tikkanen MJ, Hogstrom P, Tuomilehto J, Keinanen-Kiukaanniemi S, mass spectrometry. Rapid Common Mass Spectrum 2001, Sundvall J, Karppanen H: Effect of a diet based on low-rat foods 15:1207-1213. enriched with nonesterified plant sterols and mineral nutri­ 69. Gremaud G, Dalan E, Piguet C, Baumgartner M, Ballabeni P. Decarli ents on serum cholesterol. Am} Cardiol 2001, 88:1157-1162. B, Leser ME Berger A, Fay LB: Effects of non-esterifled stanols in

Page 16 of 19 (peg? number not for citabon purposes) 207

Lipids in Health and Disease 2004, 3 http:/Arvvvw. lipidworld.com/content/3/1 IS

88. Vaskonen T, Mervaala E, Krogerus L, Karppanen H: Supplementa­ 108. Gylling H, Miettinen TA The effects of inhibiting cholesterol tion of plant sterols and minerals benefits obese zucker rats absorption and synthesis on cholesterol and lipoprotein fed an atherogenic diet J Nutr 2002, 132:231-237. metabolism in hypercholesterolemic non-insulin dependent 89. Vaskonen T, al. ct: Effects of calcium and plant sterols on men./Upid/tes 1996,37:1776-1785. serum lipids in obese Zucker rats on a low-fat diet Br J Nutr 109. Igel M, Giesa U, Lutjohann D, von Bergmann K: Comparison of the 2002, 87:239-246. Intestinal uptake of cholesterol, plant sterols, and stanols in 90. Manhas A Farmer JA Hypolipidemic therapy and cholesterol mice. J lipid Res 2003,44:533-538. absorption. Cuir Atherosefer Rep 2004, 6:89-93. 110. Turley SO, Dietschy JM: Sterol absorption by die small 91. Smart EJ, De Rose RA, Farber SA Annexin 2-caveolin I complex intestine. Curr Qpin Upidol 2003, 14:233-240. is a target of ezetimibe and regulates intestinal cholesterol 111. fidd FJ. Bom E. Mathur SN: LXR/RXR ligand activation transport ProcNatl AcadSeiUSA2004, 101:3450-3455. enhances the basolateral efflux of {betaj-sltosterol In CaCo- 92. Cannon CP, Braunwald E, McCabe CH, Rader DJ, Rouleau JL, Belder 2 cells. ] lipid Res 2004, 45:905-913. R, Joyal SV, Hill KA Pfeffer MA, Skene AM: Comparison of Inten­ 112. Mutch DM, Anderle P, Fiaux M, Mansourian R, Vidal K, Wahli W, Wil­ sive and Moderate Lipid Lowering with Statins after Acute liamson G, Roberts MA Regional variations in ABC transporter Coronary Syndromes. NEngl]Med 2004, 350:15. expression along the mouse intestinal tract Physiol Genomics 93. Topol EJ: Intensive Statin Therapy — A Sea Change in Cardi­ 2004, 17:11-20. ovascular Prevention. N Engl) Med 2004, 350:15, 113. Scoggan KA Gruber H, Lariviere K: A mlssense mutation in the 94. FDA Food labeling: health claims; plant sterol/stanol esters AbcgS gene causes phytosterolemia in SHR, stroke-prone and coronary heart disease. Food and Drug Admin Fed (teg 2000, SHR, and WKY nts. J lipid Res 2003, 44:911-916. 65:54686-54739 [http://www.access.gpo,;oY/si> docs/fedreg/ 114. Plat J, Mensink RP: Increased intestinal ABC AI expression con­ aQ.QQ.9Q.8U.tfm!]. tributes to the decrease In cholesterol absorption after plant 95. Australia Now Zealand Food Authority ANZFA: Phytosterol stanol consumption. FASEBJ2002, 16:1248-1253. esters derived from vegetable oils: Final Risk Analysis Report 115. Kaneko E, Matsuda M, Yamada Y, Tachibana Y, Shimomura I, Mak- Application A4I0, 30/5/01. 2001 [http://www.anib.goy.au/doai ishima M: Induction of intestinal ATP-binding cassette trans­ ments/A4IQ IR(FULL\pdfj. porters by a phytosterol-dertved liver X receptor agonist j 96. Plat J, Van Onsden ENM, Van Heugten MMA, Mensink RP: Effects on Biol Chem 2003, 278:36091 -3609a serum lipids, lipoproteins and fat soluble antioxidant concen­ 116. Yu L von Bergmann K, Lutjohann D, Hobbs HH, Cohen JC Selec­ trations of consumption frequency of margarines and short­ tive sterol accumulation in ABCG5/ABCC8 deficient mice, j enings enriched with plant stanol esters . Eur J On Nutr lipid Res 2004, 45:301-307. 2000,54:671-677. 117. Weggemans RM, Zock PL Tai ES, Ordovas JM, Molhuizen HO, Katan 97. Jones PJ, Ntanios FY, Racini-Sarjaz M, Vanstone CA Cholesterol- MB: ATP binding cassette G5 CI950G polymorphism may lowering efficacy of a sitostanol-containlng phytosterol mix­ affect blood cholesterol concentrations in humans. Clin Genet ture with a prudent diet in hyperlipidemic men. Am J din Nutr 2002, 62:226-229. 1999, 69:1144-1150. 118. Berge KE, von Bergmann K, Lutjohann D, Guerra R, Grundy SM, 98. Homma Y, Ishikawa T, Tateno M, Mitaniyama A, Sugano M: Choles­ Hobbs HH, Cohen JC: Herltability of plasma noncholesterol terol and applipoprotein-lowering effect of plant stanol ester sterols and relationship to DNA sequence polymorphism in in healthy Japanese men and women. J Jpn Soc Nutr Food Set ABCG5 and ABCG8. J lipid Res 2002, 43:486-494. 2000,53:155-162. 119. Kesaniemi YA, Ehnholm C, Miettinen TA: Intestinal cholesterol 99. Plat J, Mensink RP: Vegetable oil based versus wood based sta­ absorption efficiency in man is related to apoprotein E nol ester mbctures : effects on serum lipids and hemostatic phenotype. ] Cl/n invest 1987, 80:578-581. factors in hon-hyperehblesterolemle subjects. Atherosclerosis 120. Vanhanen HT, Blomqvist S, Ehnholm C, Hyvonen M, jauhiainen M, 2000,148:101-112. Torstila I, Miettinen TA Serum cholesterol, cholesterol precur­ 100. Vanstone CA Raeini-Sarjaz M, Parsons WE, Jones PJ: Unesterified sors, and plant sterols in hypercholesterolemic subjects with plant sterols and stands lower LOL-cholesterol concentra­ different apoE phenotypes during dietary sitostanol ester tions equivalently in hypercholesterolemic persons. Amy Gin treatment; lipid Res 1993, 34:1535-1544. Nutr 2002, 76:1272-1278. 121. Kramer W, Girbig F, Corsiero D, Burger K, Fahrenholz F, Jung C, 101. Williams CL Bollella MC, Strobino BA, Boccia L, Campanaro L Plant Muller G: intestinal cholesterol absorption: identification of stanol ester and bran fiber in chllhood: effects on lipids, stool different binding proteins for cholesterol and cholesterol weight and stool frequency In preschool children. J Am Cofl absorption inhibitors in the enterocyte brush border Nutr 1999, 18:572-581. membrane. BtoeWm BtophysActa 2003, 1633:13-26. 102. Miettinen TA Puska P, Gylling H, Vanhanen H, Vartiainen E: Reduc­ 122. Harris M, Davis W, Brown WV: Ezetimibe. Drugs Todoy (Bare) 2003, tion of serum cholesterol with sltostanol-ester margarine In 39:229-247. a mildly hypercholesterolemic population. N Engl J Med 1995, 123. Cruz ML Wong WW, Mimouni F, Hachey DL Setchell KD, Klein PD, 333:1308-1312. Tsang RC: Effects of Infant nutrition on cholesterol synthesis 103. Jones PJ, Howdl T, MacDougall DE, Feng JY, Parsons W: Short-term rates. Pedotr Res 1994, 35:135-140. administration of tall oil phytosterols improves plasma lipid 124. Alasmi M, Thorkdson T, Krug-Wispe S, Bayley TM, Jones PJH, Tsang profiles in subjects with different cholesterol levels. Metabo­ RC: Cholesterol supplementation effect on cholesterol syn­ lism 1998,47:751-756. thesis rates (FSR) in Infants. Peofatr Res 1996, 39:116A. 104. Hallikainen MA Sarkldnen ES, Uusitupa MIJ: Effects of low-rat sta­ 125. Bayley TM, Alasmi M, Thorkelson T, Krugwispe S, Jones PJH, Bulani nol ester enriched margarines on concentrations of serum JL Tsang RC: Influence of formula versus breast milk on cho­ carotenoids in subjects with elevated serum cholesterol lesterol synthesis rates In four-month-old infants. Pediair Res concentrations. Eurj CSn Nutr 1999,53:966-969. 1998,44:60-67. 105. De Graaf J, De Sauvage Nolting PR, Van Dam M, Belsey EM, Kastelein 126. Huisman M, van Beusekom CM, Lanting CI, Nijeboer HJ, Muskiet FA JJ, Pritchard PH, Stalenhoef AF: Consumption of tall oil-derived Boersma ER: Triglycerides, fatty acids, sterols, mono- and dis- phytosterols in a chocolate matrix significantly decreases accharides and sugar alcohols In human milk and current plasma total and low-density lipoprotein-cholesterol levels. types of Infant formula milk . Eur J Ctki Nutr 1996, Br J Nutr 2002, 88:479-488. 50:255-260. 106. Noakes M, Clifton P, Ntanios F, Shrapnd W, Record I, Mclnerney J: 127. Fall CH, Barker DJ, Osmond C, Winter PD, Clark PM, Hales CN: An increase in dietary carotenoids when consuming plant Relation of infant feeding to adult serum cholesterol concen­ sterols or stanols Is effective In maintaining plasma caroten- tration and death from ischaemic heart disease. Brit Med j oid concentrations. Am J CSn Nutr 2002, 75:79-86. 1992, 304:801-805. 107. Gylling H, Miettinen TA Serum cholesterol and cholesterol and 128. Kolacek S, Kapetanovic T, Zimolo A Luzar V: Early determinants lipoprotein metabolism In hypercholesterolaemic NIDDM of cardiovascular risk factors in adults. A. Plasma lipids. Acta patients before and during sitostanol ester-margarine Paertatr 1993, 82:699-704. treatment Diobeto/ogia 1994, 37:773-780. 129. Palinsld W, Napoli C: The fetal origins of atherosclerosis: maternal hypercholesterolemia, and cholesterol-lowering or

Page 17 of 19 (page number not for citation purposes) 208

Lipids in Health and Disease 2004, 3 http:/AnrWw.lipidworld,conVconterit/3/1/5

antioxidant treatment during pregnancy influence in utero lemia to a low-fat diet and to a plant sterol ester dietary programming and postnatal susceptibility to atherogenesis. challenge^ UpM Res 2003, 44:1143-1I55. FASEBj 2002, 16:1348-1360. 148. Awad AB;Smith AJ, Fink CS: Plant sterols regulate rat vascular 130. Joki P, Suomalainen H, Jarvinen KM, Juntunen-Backman K, Gyiling H, smooth muscle cell growth and prostacyclin release in Miettinen TA, Ahtikainen M: Cholesterol precursors and plant culture. Prostagfdndins Uukot Essem Faay Adds 2001, 64:323-330. sterols in children with food allergy. Am J On Nutr 2003, 149. Ntanios FY, Jones PJ, Frbhlich JJ: Dietary sitostanol reduces 77:51-5S. plaque formation but not lecithin cholesterol acyl trans­ 13 I. Schlierf G, Vogcl G, Heuck CC, Oster P, Raerxer H, Sdiellenberg B: ferase activity In rabbits. Atherosclerosis 1998. 138:101-110. [Dietary management of familial Hypercholesterolemia in 150. NtaWos FY, van de Kooij AJ, dc Deckerc EA, Duchateau GS, Traut- Children and adolescents - a feasibility study (author's wejri EA Effects of various amounts of dietary plant sterol trans!)]. Monotsxhr Kinderhei/kd 1977, 125:770-773. esters on plasma and hepatic sterol concentration and aortic 132. Becker M, Staab D, Von Bergman K: Long-term treatment of foam cell formation of cholesterol-fed hamsters. Atherosclerosis severe familial hypercholesterolemia In children: effect of 2003,169:4 i-50. sitosterol and bezafibrate. Pediatrics 1992, 89:138-142. 151. Awad AB, Fink CS: phytosterols as anticancer dietary compo­ 133. Gyiling H, Siimes MA, Miettinen TA: Sitostanol ester margarine nents: evidence and mechanism of action . J Nutr 2000, in dietary treatment of children with familial 130:2127-2130. hypercholesterolemia. JUpk/Res 1995, 36:1807-1812. 152. Awad AB, von Holtz RL, Cone JP, Fink CS, Chen YC: beta-sitp- 134. Vuorio AF, Gyiling H, Turtola H, Kontula K, Ketonen P, Miettinen TA: sterol inhibits growth of Ht-19 human colon cancer cells by Stanol ester margarine alone and with simvastatin lowers activating the sphingomyelin cycle. Anticancer Res 1998, serum cholesterol in families with familial hypercholestero­ I ?:471-473; lemia caused by the FH-North Karelia mutation. Arterioscfer 153. Awad AB, Gan Y, Fink CS: Effect of b-sltosteroi, a plant sterol, Thromb Vase Biol 2000,20:500-506. on growth, protein phosphatase 2A, and phbspholipase D in 135. Schlierf G, Oster P, Heuck CC, Raetzer H, Sdidlenberg ft Sito­ LNCaP cells. Nutr Cancer 2000, 36:74-78. sterol in juvenile type II hyperlipoproteinemia . Athero­ 154. Awad AB, Downie A Hnk CS. Kim U: Dietary phytosterol inhibits sclerosis 1978, 30:245-248. the growth and metastasis of MDA-MB-231 human breast 136. Amundsen AL, Ose L Nenseter MS, Ntanios FY: Plantsterol ester- cancer cells grown in SCID mice. Anticancer Res 2000, enriched spread lowers plasma total and LDL cholesterol in 20:821-824. children with familial hypercholesterolemia. Am J Clin Nutr 155. Wilt T, Ishani A, MacDonald R, Stark G, MulrowC, LauJ: Beta-sito- 2002, 76:338-344. sterols for benign prostatic hyperplasia. Cochrane Database Syst 137. Tammi A, Ronncmaa T, Gyiling H, Rask-Nissila L, Viikari J, Tuorninen 2:. Rev 2000: CD001043 Review J, Pulkki K, Simell O: Plant stanol ester margarine lowers serum 156. Normen AL, Brants HA, Voorrips LE, Andersson HA, van den Brandt total and low-density lipoprotein cholesterol concentrations PA Goldbohm RA Plant sterol intakes and colorectal cancer of healthy children: The STRIP project J Pedhtr 2000, risk in the Netherlands Cohort Study on Diet and Cancer. Am 136:503-510. 1dmNutr 2001, 74:141-148. 138. Tammi A, Ronnemaa T, Valsta L, Seppanen R, Rask-Nissila L, Miet­ 157. Quilliot D, Boman F, Creton C, Pelletier X, Floquet J, Debry G: Phy­ tinen TA, Gyiling H, Viikari J, Anttolainen M, Simell O: Dietary plant tosterols have an unfavourable effect on bacterial activity sterols alter the serum plant sterol concentration but not and no evident protective effect on colon carcinogenesis. Eur the cholesterol precursor sterol concentrations in young j Cancer Prev 2001, 10:237-243. children (the STRIP Study). Special Turku Coronary Risk 158. Mendilaharsu M, De Stefan! E, Deneo-Pdlegrini H, CarzoglioJ, Ronco Factor Intervention Project../Nutr 2001, 131:1942-1945. A Phytosterols and risk of lung cancer, a case-control study 139. Mdlies M, Glueck CJ, Sweeney C, Fallat RW, Tsang RC, Ishikawa TT: in Uruguay. Lung Cancer 1998, 21:37-45. Plasma and dietary phytosterols in children . Pediatrics 159. Ronco A De Stefani E, Boffetta P, Deneo-Pellegrini H, Mendilaharsu 1976, 57:60-67. M, Leborgne R Vegetables, fruits, and related nutrients and 140. Kempen HJ, de-Knijff P, Boomsma Dl, Van der.V, Gevers Leuven JA risk of breast cancer: a case-control study in Uruguay. Nutr Havekes L: Plasma levels of lathosterot and phytosterols in Cancer 1999,35:111-119. relation to age, sex, anthropometric parameters, plasma lip­ 160. De Stefani E, Boffetta P, Ronco AL, Brennan P, Deneo-Pellegrini H, ids, and apolipoprotein E phenotype, In 160 Dutch families. Carzoglio JC, Mendilaharsu M: Plant sterols and risk of stomach Metabolism 1991, 40:604-611. cancer, a case-control study in Uruguay. Nutr Cancer 2000, 141. Tammi A, Ronnemaa T, Miettinen TA, Gyiling H, Rask-Nissila L, 37:140-144. Viikari J, Tuorninen J, Mamlemi J, Simell O: Effects of gender, apol­ 161. De Stefani E, Brennan P, Boffetta P, Ronco AL, Mendilaharsu M, ipoprotein E phenotype and cholesterol-lowering by plant Deneo-Pellegrini H: Vegetables, fruits, related dietary antioxi­ stanol esters in children: the STRIP study. Special Turku dants, and risk of squamous cell carcinoma of the esophagus: Coronary Risk Factor Intervention Project Acta Paedatr 2002, a case-control study in Uruguay. Nutr Cancer 2000, 38:23-29. 91:1155-1162. 162. McCann SE, Freudenheim JL, Marshall JR, Brasure JR, Swanson MK, 142. Miettinen TA: Phytosterolaemia, xanthomatosis and prema­ Graham S: Diet in the epidemiology of endometrial cancer in ture atherosclerotic arterial disease: a case with high plant western New York (United States). Cancer Causes Control 2000, sterol absorption, impaired sterol elimination and low cho­ 11:965-974. lesterol synthesis, Eur} Clin Invest 1980, 10:27-35. 163. McCann SE, Freudenheim JL, Marshall JR, Graham S: Risk of human 143. Miettinen HE, Gyiling H, Miettinen TA, Viikari J, Paulin L, Kontula K: ovarian cancer is related to dietary intake of selected nutri­ Apolipoprotein A-IFIn. Dominant!/ Inherited hypoalphalipo- ents, phytochemicals and food groups. J Nutr 2003, proteinemla due to a single base substitution in the apolipo­ 133:1937-1942. protein A-l gene. Arterioxter Thromb Vox Biol 1997, 17:83-90. 164. Bouic PJ, Clark A Brittle W, Lamprecht JH, Freestone M, Uebenberg 144. Patel SB, Salen G, Hidaka H, Kwiterovidi PO, Stalenhoef AF, Miet­ RW: Plant sterol/sterolin supplement use in a cohort of tinen TA, Grundy SM, Lee MH, Rubenstein JS, Polymeropoulos, Mh, South African HIV-infected patients—effects on immunolog­ Brownstein MJ: Mapping a gene Involved in regulating dietary ical and vlrological surrogate markers. S Afr Med J 2001, cholesterol absorption. The sitosterolemla locus Is found at 91:848-850. chromosome 2p2l.) On Invest 1998, 102:1041-1044. 165. Wang T, Hides KB, Moreau R: Antioxidant activity of phytoster­ 145. Patel SB, Honda A, Salen G: Sitosterolemla: exclusion of genes ols, oryzanol, and other phytosterol conjugates. J Am OH Chan involved in reduced cholesterol biosynthesis. J lipid Res 1998, Soc 2002, 79:1201-1206. 39:1055-1061. 166. loscalzo J: Oxidant stress: a key determinant of 146. Stalenhoef AF, Hectors M, Demacker PN: Effect of plant sterol- athe rothrombosls. Biocbem Soc Trans 2003, 31:1059-1061. enrichsd margarine on plasma lipids and sterols in subjects 167. QuongJ, Eppenberger-Castori S, Moore D, 3rd, Scott GK, Birrer MJ, heterozygous for phytosterolaemia. I /ntern Med 2001, Kueng W, Eppenberger U, Benz CC: Age-dependent changes In 249:163-166. breast cancer hormone receptors and oxidant stress 147. Kwiterovich P O, Jr, Chen SC, Virgil DG. Schweitzer A, Arnold DR, markers. Breast Cancer Res Treat 2002, 76:221-236. Kratz LE Response of obligate heterozygotes for phytostero-

Page 18 of 19 (page number not for citation purposes) 209

Lipids in Health and Disease 2004, 3 http:/Awww.lipidworld.corTVcontent/3/1/5

168. Paul Jayaraj A, To/ey Fl, Hobsley M: Duodenal ulcer prevalence: research into the nature of possible protective dietary lipids. Phytotfier Res 2003, 17:391-398. 169. Smania EF, Delle Monache F, Smania A, Jr, Yunes RA, Cuneo RS: Antifungal activity of sterols and triterpenes isolated from Ganoderma annulare. F'ttoterapia 2003, 74:375-377. 170. Davidson MH, Maki KC Umporowici DM, Ingram KA. Dicklin MR. Sehaefcr E, Lane RW, McNamara JR, Ribaya-Mercado JD, Perrone G, Robins SJ, Franks WC: Safety and tolerability of esterified phy- tosterols administered in reduced-fat spread and salad dressing to healthy adult men and women. J Am CM Nun 2001, 20:307-319. 171. Plat J, Mensink RP: Effects of plant stanol esters on LDL recep­ tor protein expression and on LDL receptor and HMG-CoA reductase mRNA expression in mononuclear blood cells of healthy men and women. FASEB; 2002, 16:258-260. 172. Ntanios FY, Duchateau GS; A healthy diet rich In carotenoids is effective In maintaining normal blood carotene Id levels dur­ ing the daily use of plant sterol-enriched spreads. IntJ Yuan Nutr Res 2002, 72:32-39. 173. Raeini-Sarjaz M, Ntahios FY, Vanstone CA, Jones PJ: No changes In serum fat-soluble vitamin and carotenoid concentrations with the intake of plant sterol/stanol esters in the context of a controlled diet. Metabolism 2002, 51:652-656. 174. Nissinen M, Gyliing H, Vuoristo M, Miettinen TA Micellar distribu­ tion of cholesterol and phytosterols after duodenal plant sta­ nol ester infusion. Am J Physiol Casiroihtest Liver Physiol 2002, 282:GI009-I00I5. 175. Miettinen TA, Gyliing H, Lindbohm N, Miettinen TE, Rajaratnam RA, ftelas H: Serum noncholesterol sterols during inhibition of cholesterol synthesis by statins, j Lab Clin Med 2003, 141:131-137. 176. Hememarm T, Leiss O, von Bergniann K: Effect of low-dose sito- stanoi on serum cholesterol in patients with hypercholesterolemia. Atherosclerosis 1986, 61:219-223. 177. Howdl TJ, MacDougall DE, Jones PJ: Phytosterols partially explain differences In cholesterol metabolism caused by corn or olive oil feeding. J Lipid Res 1998, 39:892-900. 178. Gyliing H, Miettinen TA: Cholesterol reduction by different plant stanol mixtures and with variable fat intake. Metabolism 1999, 48:575-580. 179. Nornien L, Dutta P, Lia A, Andersson H: Soy sterol esters and p- sitostanol ester as Inhibitors of cholesterol absorbtion in human small bowel. Am J. CHt\ Nutr 2000, 71:908-913.

180. Hornma Y, Ikeda I, Ishikawa Tt Tateno M, Sugano M, Nakamura H: Decrease In plasma low-density lipoprotein cholesterol, apolipoprotein B,cholesteryl ester transfer protein, and oxi­ dized low-density lipoprotein by plant stanol ester-contain­ ing spread: a randomized, placebo-controlled trial Nutrition 2003, 19:369-374, 181. Gyliing H, Puska P, Vartiairien E, Miettinen TA: Retinol, vitamin D, carotenes and alpha-tocopherol in serum of a moderately hypercholesterolemic population consuming sitostanol ester margarine. Atherosclerosis 1999, 145:279-285.

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Page 19 pf 19 (PA00 nvmbw not tor citation purposes) Appendix 2. Published version of Review Article 3: Physiological and therapeutic factors affecting cholesterol metabolism: does a reciprocal relationship between cholesterol absorption and synthesis really exist? 211

..Vt--',• ;, j Available online at www.sciencedirect.eom

S-Vfc.l *#* ScienGeDirect Life Sciences

ELSEVIER Life Sciences 80 (2007) 505-514 in, www.elsevier.c6Hi/locate/lifiscie Minireview Physiological and therapeutic factors affecting cholesterol metabolism: Does a reciprocal relationship between cholesterol absorption and synthesis really exist?

Sylvia Santosa a, Krista A. Varady a, Suhad AbuMweis a, Peter J.H. Jones b'*

" School of Dietetics caul Human Nutrition, McGill University, Montreal, Quebec, Canada * Richardson Centre for Functional Foods and NutraceuUcals, University of Manitoba, Smartpark, 196 Innovation Xfrive, Winnipeg, Manitoba, Canada R3T 6CS Received 26 May 2006; accepted 10 October 2006

Abstract

Cholesterol absorption and synthesis contribute to maintaining cholesterol homeostasis. Several physiological and therapeutic factors affect cholesterol homeostasis, including: genetics, Orcadian rhythm, body weight, plant sterols, ezetimibe, and statin therapy. The present objective is to determine the main vector, i.e- cholesterol absorption or synthesis, affected by each of these factors, and toexamin e whether an alteration in one vector is linked to a reciprocal change in the other! Current techniques used to assess cholesterol absorption and synthesis are also reviewed. Review of physiological factors affecting cholesterol metabolism suggest a reciprocalrelationshi p between these two vectors. Carriers of'the-£2 isoform of apolipoprotein E and ATP binding cassette (ABC) G8 19H (exon 1 mutation) show a decrease in cholesterol absorption accompanied by a corresponding increase m synthesis, Gircadian rhythm affects cholesterol synthesis, however, its effect on absorption has yet to be established. Obese subjects show an increase in cholesterol synthesis with a subsequent decrease in cholesterol absorption. 'Weight loss down regulates cholesterol synthesis, but has little or.no effect on absorption. In the case of therapeutic factors, plant sterols and stanols inhibit cholesterol absorption, which results in a compensatory increase in synthesis. Ezetimibe also decreases intestinal absorption, while reciprocally increasing synthesis. Statin therapy down regulates synthesis, which is accompanied by a rise in absorption. These findings suggest that a change in one vector, fairly consistently, results in a compensatory and opposing change in the other An understanding of this reciprocal relationship between cholesterol absorption and synthesis may allow for the development of more effective interventions for dyslipidemic disorders. Cs 2006 Elsevier Inc. All rights reserved.

Keywords;. Cholesterol; Absorption; Synthesis; Reciprocal relationship; Physiological factors; Therapeutic factors

Contents

Introduction. , , , ,...... ,..,., 506 Methods of measuring cholesterol absorption and synthesis . . 504 Cholesterol absorption assessment methods ..,.,...,.:...,..,....,.. ...,,., S06 Sterol balance approach...... 506 Plasma isotope ratio technique 508 Plant sterol-torcholesterol ratio determination. , . 5Q7 Cholesterol synthesis assessrhenf methods , , ,.,... , 507 Deuterium incorporation approach . . . , 507 Cholesterol precursor quantification ,:...., .;...... ,../.... 507 Physiological factors affecting cholesterol absorption and synthesis 507 Genetic factors 507

* Cqrrespondingauthor. Tel.r+1 204474 8883; fax:+1 204 474 7552. M-maiiaddress: [email protected] (PJ.H. Tones). 0024-3205/$ -see fitmtmatter-. © 2006 Elsevier Inc. All rights reserved, doi: 10J0J6/JJ6.2006.10.006 212

506 S. Santosa a al /Life Sciences SO (2007) 505-514

Orcadian rhythm 509 Body weight 509 Therapeutic factors affecting cholesterol absorption and synthesis 510 Plant sterol and stanol supplementation , . . - , 510 Ezetimibe treatment , 511 Statin therapy . • - • • 511 Weight loss 511 Smnvnary of findings 512 Conclusion 512 References 512

Introduction Cholesterol absorption assessment methods

Circulating cholesterol levels are a primary target of therapy Sterol balance approach for the prevention and treatment of coronary heart disease The gold standard approach to measuring cholesterol (Anonymous, 2001). Cholesterol absorption and synthesis absorption is the sterol balance method (Grundy and Ahrens, contribute to maintaining cholesterol homeostasis, and thus, 1969; Wilson and Lindsey, 1965). This technique measures the cholesterol levels in the blood. Exogenous cholesterol is mass absorption of cholesterol as the difference between dietary absorbed within the small intestine, whereas endogenous intake of exogenous cholesterol and fecal excretion of cholesterol is produced in both hepatic and peripheral tissues exogenous cholesterol. So as to not underestimate the amount (Dietschy and Siperstein, 1967; Spady and Dietschy, 1983). of exogenous cholesterol absorbed, the quantity of endogenous Cholesterol homeostasis has been shown to be affected by cholesterol excreted in the feces is accounted for by the several key physiological factors, such as: an individual's administration of intravenous radio labeled cholesterol. Thus, genetics, the body's circadian rhythm, and body weight; In the mass absorption (mg/d) of cholesterol is calculated as the addition to these internal modulators of cholesterol metabolism, balance between dietary cholesterol intake and fecal cholesterol certain external therapeutic factors may also affect cholesterol excretion. Although this method is limited in that it is time balance. Examples of commonly implemented therapeutic consuming, this technique allows for the absolute mass options include: plant sterol or stanol supplementation, absorption of cholesterol to be calculated. ezetimibe treatment, statin therapy, and body weight loss. Each of these physiological or therapeutic factors directly Plasma isotope ratio technique affects one of the vectors involved in cholesterol homeostasis, The plasma isotope ratio technique differs from the balance i.e. cholesterol absorption or synthesis. For instance, in the case method in that, instead of quantifying cholesterol absorption, it of ezetimibe therapy, these agents decrease circulating choles­ estimates the fraction of cholesterol absorbed over a defined terol levels by directly inhibiting cholesterol absorption, while period of time (Bosner et al., 1999; Gremaud et al., 2001; Jones having no direct effect on cholesterol synthesis. In view of the et al., 2000). Cholesterol absorption is calculated from the ratio selective effect of these factors either on cholesterol absorption of oral cholesterol tracer enriclvment, taken to represent the or synthesis, certain questions that arise include: Docs an amount of exogenous cholesterol absorbed, to intravenous alteration in one vector result in a reciprocal effect in the other? tracer enrichment, taken to represent the total cholesterol pool in What is the magnitude of the reciproc.il effect? What are current the blood. This method requires that volunteers receive a methodologies implemented in measuring cholesterol absorp­ radioactive or stable isotope labeled cholesterol tracer orally tion and synthesis? Moreover, what are the implications of these (e.g. ''H/'H-cholesterol) while simultaneously receiving a reciprocal effects with regard to cholesterol lowering therapy"? second tracer intravenously (e.g. 13C/14C~cholesterol). Follow­ Thus, in order to address these questions, the objective of this ing isotope administration, blood samples arc collected over paper is to determine the main vector affected by each of the several days. Free cholesterol is isolated from plasma or red physiological and therapeutic factors mentioned above, and to cells, and is subsequently combusted to 2H/3H-labeled- examine whether an alteration in one vector is linked to a I3 l4 hydrogen gas and C/ C-labcled-C02 gas (Jones et al., reciprocal change in the other. In assessing the relationship 2000). Isotopic enrichments: are then analyzed and expressed between cholesterol absorption and synthesis, the current against international standards (Jones et al., 2000). Alternative­ methods used to examine these kinetic parameters will also be ly, on-line GC/combustion and GOpyrolysis/IRMS has been discussed. employed. Once the steady state ratio of the 2H/3H- to UC/14C- cholesterol enrichments are reached, generally within 2 to Methods of measuring cholesterol absorption and synthesis 3 days of isotope administration, the fraction cholesterol absorbed can be determined. The plasma isotope ratio technique is advantageous in clinical settings in that it allows for the direct A variety of techniques exist to measure cholesterol quantification of the percent of cholesterol absorbed. This absorption and synthesis. The following section briefly method, however, may be seen as invasive due to intravenous describes the most common methods currently implemented, isotope administration. and also touches upon the advantages and limitations of each. 213

S. Samosa et al /Life Sciences SO (2007) 505-514 507

Plant sterol-to-cholesterol ratio determination the direct quantification Of the amount of cholesterol synthesized. Evidence suggests that plant sterol metabolism is related to Instead, this method simply indicates whether or not increases or cholesterol absorption. Results of recent human studies indicate decreases in cholesterol precursors occur as a result of specific that circulating plant sterol levels are correlated with cholesterol interventions. absorption rates. More specifically, in a study by Tilvis and Miettinen (1986), it was shown that the ratio of serum Physiological factors affecting cholesterol absorption and campesterol-to-cholesterol was strongly correlated to percent synthesis Cholesterol absorption. In support of these findings, a trial performed in 63 randomly selected Finnish subjects indicated In light of their advantages and disadvantages, the methods that campesterol-to-cholesterol ratios vary directly with percent used in the assessment of cholesterol kinetic parameters should cholesterol absorption (Miettinen et al., 1990). Thus, circulating be considered in reviewing the studies examining these vectors. levels of non-cholesterol sterols may be proportional to the Certain physiological factors, such as genetics, circadian amount of cholesterol absorbed. The use of this method in the rhythm, body weight, have been shown to affect cholesterol assessment of absorption is advantageous; as it is quick and homeostasis by directly modulating either cholesterol absorp­ relatively easy to perform by way of gas-liquid chromatogra­ tion or synthesis. The following section aims to discuss the phy. Thus:, it is feasible for application in large sample primary effect of each of these factors on either one of these populations. This technique, however, is limited in that it only vectors, and attempts to delineate whether a corresponding indicates whether absorption has increased or decreased, and effect between the two vectors exists. A summary of the effects does not provide any indication of the absolute amount of of these physiological factors on cholesterol absorption and cholesterol absorbed. Also, identification of plant sterol peaks is synthesis is displayed in Table 1. sometimes difficult, confounded by the presence of other substances co-eluting off the GC column at the same retention Genetic factors time. Cholesterol metabolism is regulated by a host of different Cholesterol synthesis assessment methods proteins and transporters, many of which have been shown to possess genetic por>morpriisms. The involvement of hereditary Deuterium incorporation approach factors in cholesterol absorption and synthesis is supported by The deuterium incorporation approach determines choles­ human studies showing inter-individual variability in response terol synthesis as the rate of incorporation of deuterium from to cholesterol lowering by statins (Miettinen and Gylling, 2002, body water into red cell membrane free cholesterol over a 24 h 2003a; O'Neill et a]., 2001), heritability of indicators of period (Wang et al., 2004b; Woollett et al., 2003). Employing cholesterol synthesis and absorption (Boonisma et al,, 2003; this technique requires that subjects consume a small amount of Gylling and Miettinen, 2002a), and differences in cholesterol deuterated water following the study intervention. Shortly after absorption between different mouse strains (Carter et al., 1997; ingestion, the deuterated water equilibrates with intra- and Jolley et al., 1999; Schwarz et al., 2001; Wang et al„ 2001). extra-cellular body water pools. In this way, any newly formed Apolipoprotein E (apoE) has been shown to play a major role cholesterol synthesized within 24 h will have a certain number in lipid transport and metabolism. Three common isoforms of of H atoms replaced by deuterium. The fractional cholesterol the apoE gene exist, these being:E2,E3, and E4. Carriers of the synthesis rate (FSR) (pools/d) over a 24 h period is then E2 isoform may have lower rates of cholesterol absorption and calculated as the amount of enriched free cholesterol relative to higher rates of cholesterol synthesis compttred to earners Of the the total cholesterol pool. Although this, method allows for the E3 or E4 isoform (Gylling et ah, 199S; Kesaniemi et al., 1987; direct measuremerit of the cholesterol pool synthesized over a Miettinen et al., 1992). In coijtrast, carriers Of:tfie:E4 isofbrin ,24 h period, the laboratory techniques employed to quantify exhibit increased cholesterol absorption rates^ accompanied by isotope enrichments are quite time^intensive and laborious. a reciprocal decrease in bile acid synthesis (Kesaniemi et al;, 1987). As a result, carriers of the E4 isoform may be better Cholesterol precursor quantification candidates for cholesterol lowering drugs that inhibit absorp­ Previous studies suggest mat circulating cholesterol precursors, tion, i.e. plant sterol's or easetimibe. These effects, however, hay© i.e. lathosterol, lanoslerol, squalene, anddesmosterol levels, may not been cortsistenth/ observed- J*or instance, Miejttincn et al. reflect whole body cholesterol synthesis (Kernpen et al., 1988; (1992) showed that the effect of apoE polymorphism is poorly Pfbhl et al,, 1999). The rationale forusin g precursor levels as an related to cholesterol metabolism at low cholesterol intakes.: in indicator of cholesterol synthesis lies in tlie assumption that these addition, (Jones et al.;, 1993) found no relationship between; compounds leak into plasma lipoproteins at a rate proportional to apoEgenotype and Cholesterol syntliesisrate in subjectson self- that of their formation in the cholesterol synthetic pathway selected low cholesterol arid high cholesterol diets. Therefore;, (Grdoyas arid Tai, 2002). Levels of cholesterol precursors are the effect of the apoE isbfoitas on cholesterol absorption, and deterinined by gas-liquid chromatography from the non-saponi- their: possible reciprocal effect on cholesterol synthesis, may fiable material of serum or plasmalipids. This cholesterol synthesis only be apparent at high intakes of dietary cholesterol. assessment method benefitsfrom being efficient and inexpensive. Sitosterolemia, a rare autosomal recessive disorder characr On the other hand, themethodis limitedin that it does not allow for terized by high absorption and retention of sterols, is caused by 214

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Table 1 also observed lower total cholesterol (TC) levels in carriers of at Physiological, factors that play a role in modulating cholesterol absorption and least one mutant 19H allele (Kajtoiuni et al., 2004). The same synthesis study did not find any differences according to the Q604E on Physiological Cholesterol Cholesterol Reference ABCG5 and T400K polymorphisms (Kajinami et al'., 2004). {actors absorption synthesis Additionally, a novel ABCG8 exon 2 mutation was identified Genetic effects human model on the Island of KoSrae (Sehayek et al, 2004). The carriers of Apolipoprotein the mutant allele had increased plasma plant sterols levels and decreased lathosterol levels, indicating enhanced absorption, Isoform E2 1 T Gylling etal. (1995) Isofoim B4 T | Bile acid Kesaniemi et al. (1987); and a reciprocal decrease in synthesis. These findings indicate synthesis Miettinen et al. (1992) that the existence of a relationship between absorption and ABCG8 synthesis may be mediated by mutations in ABGG8. (allele) Niemann Pick 01 Like 1 (NPCILI), a protein with a sterol- 19H (exon 1 i- T Berge et al. (2002); mutation) Gylling etal. (2004) sensing domain, was recently discovered as the intestinal 19H (exon 2 T I Sehayek et al. (2004) cholesterol transporter (Aitmann et al., 2004). A recent mutation) investigation on the Dallas Heart Study participants by Cohen et al. (2006) resulted in the identification of various mutations in Genetic effects knockouti mouse model NPCILI that were common in individuals with low cholesterol NPCILI 4 •> Aitmann et al. (2002) SREBP-1 o T Shirnano etal. (1997) absorption. Additionally, variant alleles were associated with a. ACAT-2 i ~mKNA Buhman et al. (2000) 10% lower low-density lipoprotein (LDL) eholesterol (Cohen HMG CoA et al., 2006). The variations were found to be eommon in 6% Of reductase w/ 1832 African-American men and women, however, the high choles­ terol diet investigators were unable to determine which alleles may lead Famesoid x- T ? Lambert et al. (2003) to functional impairment of NPCILI (Cohen et al., 2006). receptor Though it is unclear whether individual variations in NPC1 LI Mucin gene 1 I ? Wang et al. (2004a) affect its function, a haplotype common to 1 in 8 subjects was found to be associated with inter-individual variation in the Orcadian rhythm effects human model response of plasma LDL-C to 12 week treatment with 06:00and ? T Cella etal. (1995); 22:00 h Jones and Schoeller (1990) ezetimibe, a drug that inhibits cholesterol absorption (Hegele 14:00-1S:00 h ? I Jones and Schoeller (1990) et al., 2005). Further studies have indicated that feeding a cholesterol/cholate diet to wild type mice results in down Body weight effects regulation of intestinal mRNA expression of NPCILI (Davis Obese subjects i DiBuoiio etal. (1999); etal, 2004). Moreover, mice that were deficient in NPC1 LI had (BMI >30) Miemnen and Gylling (2000); Stahlberg et al. (1997) upregulated intestinal 3-hydroxy-3-methylglutaryl (HMG) CoA synthase mRNA and increased cholesterol synthesis (Davis SREBP-1: Sterol regulatory element binding protein-1, ACAT-2: Acyl CoA: cholesterol acyltransfefase 2, NPCILI: Niemann-Picfc CI Lite 1 protein, BMI: et al., 2004). Thus, NPCILI mutations may play a major role in body mass index. ?: xinstudied effect. the indirect control of cholesterol synthesis through regulation a The effects of these tsolbrms may .only be apparent at higher levels of of cholesterol absorption. cholesterol intake. Studies using knockout mouse models have identified additional geries that express proteins that may affect choles­ imitations in the ATP binding cassette (ABC) transporters, terol absorption and synthesis including sterol regulatory ABCG5 and ABCG8 (Berge et al., 2000; Lee et al, 2001). element binding protein-I (SEEBP4), acyl CoA:ch61estierol ABCG5 and ABCG8 genes express transporters atthe intestine acyltransferase 2 (AGAT2), iarnesoid X-receptor (FXR), and and the liver, which play a key role in regulating cholesterol mucingene 1 (Mucl) (Table 1) (Buhmanetal., 2000; Lambert absorption (Ordovas and Tar, 2002). Although their Specific et al, 2003; Shirnano etal, 1997). Sterol regulatory element mrictiohs are unclear, it has feeeanpostulate d ABCG transporters hiring protem-1 is a membra iftay be responsible for the selective reverse transport of regulates cholesterol sytithesis and uptake from circulating cholesterol from the enlerocyte into the intestinal lumen. LDL, Mice Jacking the SREBP-1 gene had elevated levels of Studies examining non-sitosterolemie individuals were carried mRKAs for HMG CoA synthase and. reductase and squalene out to investigate the effect of sequence variants in ABCG5 or synthase, consequently cholesterol synthesis was elevated ABCGSon cholesterol metabolism. Berge et al. (2002): reported threefold in livers of mese: mice and hepatic cholesterol content that two variants in ABCG8, D19H and T400K, are linked to was increased by 50% (Shirnano et al, 1997). Buhrnatt et al. lower plasma plant sterols concentrations. Further research (2000) showed that ACAT2 is the enzyme responsible for found that the1,9H allele (exon I n»utation) of the ABCG8 gene cholesterol esterifieation in mouse small intestine as well as was associated with low ctelestetol absorption markers and liver. In addition, it was demonstrated that ACAT2-deflcieht high cholesterol synthesis markers (Gylling et al., 2004). An mice had reduced capacity to absorb dietary cholesterol when intervention study that examined, genetic associations in 337 fed a high cholesterol diet but not when fed a chow diet subjects after 52 Weeks of treatment with 10 mg atorvastatin (Buhman et al, 2000). Thus, deficiency of ACAT2 has a 215

S. Samosa et a!. I Life Sciences SO (2007) 505-514 509 protective effect against hypercholesterolemic diets in mice. rhythm of cholesterol synthesis in humans also seems to be Famesoid X-receptor is a nuclear hormone receptor that affected by food intake. regulates many aspects of cholesterol metabolism including Many animals and humans studies have examined circadian reverse cholesterol transport, intestinal cholesterol absorption rhythm in cholesterol synthesis but not circadian rhythm in and enterohepatic circulation of cholesterol. FXR-deficient cholesterol absorption. Nevertheless, the possibility of diurnal mice have been found to have increased intestinal cholesterol variation of cholesterol absorption is supported by data obtained absorption (Lambert et aL, 2003). Recently, it was shown that in rats (Edwards et al., 1972; Ishida et al., 2000). Here, hepatic disruption of Mucl, one of the genes that regulate mucin cholesterol synthesis appeared low at noon, and also indicated secretion in the small intestine, resulted in 50% reduction in that maximal expression and activity of sterol 12a-hydroxylase intestinal cholesterol absorption in mice (Wang et aL, 2004a). (CYP8B) occurred at mid-afternoon in animals subjected to Another regulatory protein, scavenger receptor class B type 1 light/dark cycling (Edwards et aL, 1972; Ishida et aL, 2000). The (SR-BI) which may facilitate cholesterol uptake by enterocytes, CYP8B enzyme is responsible for the synthesis of cholic acid has also been identified (Altmann et al., 2002; Mardones et al., and the determination of the cholic aeid-to-chenodeoxycholie 2001). However, existing evidence indicates that SR-BI may acid ratio (Vlahcevic et al., 2000). In mice lacking the gene that not be necessary for cholesterol absorption (Altmann et al., encodes CYP8B enzyme, cholesterol absorption was reduced 2002; Mardones et al., 2001). Therefore, studies in human are and cholesterol synthesis was upregulated (Murphy et al., 2005). needed to investigate whether genetic variations in genes Seeing as cholic acid facilitates cholesterol absorption in the mentioned above may play a role in explaining absorption and intestine, and since cholic acid supplementation enhanced synthesis responsiveness to various therapeutic factors. Candi­ cholesterol absorption in humans, it can be hypothesized that date genes that play a role in cholesterol absorption and maximal rates of GYP8B results in maximal rates of cholesterol synthesis are potential targets for the development of cholesterol absorption (Chiang, 2004; Woollett et al., 2004). The mecha­ lowering drugs or functional ingredients. nism by which circadian rhythm affects cholesterol synthesis and absorption could be explained at the molecular level. The Circadian rhythm expression of many genes encoding enzymes involved in cholesterol metabolism have demonstrated circadian rhythm, A circadian rhythm of cholesterol biosynthesis has been such as the expression of HMG^CoA (Jurevics et al., 2000) and identified in animals and humans. In studies involving rats and CYP8B enzymes (Ishida et al., 2000). Future research in this hamsters, maximum cholesterol synthesis was shown to occur area should examine the level or the expression of the newly at midnight while minimum synthesis occurs at noon (Edwards discovered proteins that have a role in cholesterol absorption et aL, 1972; Hamprecht et al., 1969; Ho, 1975, 1979; Jurevics such as NPC1L1, as well as ABCG5 and ABCG8 genes. et al., 2000), The peak in synthesis likely corresponds to the nocturnal feeding cycle of rats and hamsters (Edwards et a]., Body weight 1972; Ho, 1979). When rats are fed during the light period, the maximum rate of cholesterol synthesis occurs 9 h after feeding Dyslipidemia is common in individuals who are overweight (Edwards et al., 1972). Another study in hamsters further and obese (Dattilo and Kris-Etherton, 1992). These abnormal indicates that regardless of the lighting period, the peak in fasting lipid profiles may be due to altered cholesterol cholesterol synthesis occurs 4 to 6 h after feeding and the nadir metabolism commonly found in overweight populations (Di occurred at the end of the fasting period (Ho; 1979). In humanSj Buono et al., 1999; Miettmen and Gylling, 2000). Compared to circadian rhythm in cholesterol synthesis has been measured honnd weight individuals, Obese subjects detnonstrate elevated directly by deuterium incorporation and indirectly by blood cholesterol synthesis (Miettinen and Gylling, 2000). Liver coricentratiiaB of ch6tet^lpre

510 S. Santoso a al /life Sciences 80 (2007) 50S-514

Therapeutic factors affecting cholesterol absorption and rise in cholesterol synthesis. A double-blind randomized cross­ synthesis. over study examined the effects of sitostanol ester supplemen­ tation (3 g/d) on cholesterol metabolism in women with In addition to the physiological mediators of cholesterol angiographically documented coronary artery disease (Gylling metabolism mentioned above, certain therapeutic factors have et al, 1997). After 7 weeks of treatment, sitostanol ester also been shown to modulate cholesterol homeostasis; These margarine decreased serum TC and LDL-C by 13% and 20%, fectors include plant sterol and stanol supplementation, respectively (Gylling et al., 1997). Cholesterol absorption Was ezetimibe treatment, statin therapy, and body weight loss. reduced by 45%, while cholesterol synthesis reciprocally Table 2 summarizes the effect of these various therapeutic increased by 39% (Gylling et al., 1997). A similar reciprocal interventions on cholesterol absorption and synthesis. relationship between cholesterol absorption and synthesis was identified by Jones et al. (2000). This study (Jones et al., 200G) Plant sterol and stanol supplementation tested the effect of 1.84 g/d of plant sterol or stanol margarine on cholesterol metabolism in 15 hypercholesterolemic patients The chemical structures of plant sterols and stanols are consuming prepared diets for 21 days. More specifically, the similar to that of cholesterol (Katan et al., 2003; Mel'nikov diets contained either i) margarine (M), ii) phytosterOl ester et al., 2004; Vanstone et al., 2002). Since the structure of plant margarine, or iii) margarine wim phytostanol esters (Jones et al., sterols is analogous to cholesterol, the ABCG5, ABCG8, and 2000). The randomized cross-over designed trial measured NPC1L1 transporters that control cholesterol metabolism also cholesterol absorption and synthesis following each interven­ play a role in the regulation of plant sterol metabolism. tion period by employing the plasma isotope ratio technique Specifically, the ABCG5 and ABCG8 transporters mediate the (Jones et al., 2000). Margarine containing phytosterol and efflux of plant sterols, while the NPC1L1 transporter regulates phytostanol esters decreased TC and LDI>C significantly. their influx (Berge et al., 2000; Sudhop et al., 2002). "This Following sterol and stanol interventions, intestinal absorption structural similarity also causes competition between plant of cholesterol decreased while synthesis increased. Comparable sterols and cholesterol for integration into the micelle, thus results were noted in the trial by Vanstone et al. (2002) that resulting in the displacement of miceliular cholesterol at the included 15 hypercholesterolemic subjects in a randomized level of the small intestine (Mel'nikov et al., 2004; Vanstone cross-over feeding trial which examined the effects of plant et al., 2002). Since brush border cells do not readily take up sterol, plant stanol, or mixed sterol and stanol supplementation plant sterols and stanols, attenuation in cholesterol absorption to a control group consuming cornstarch. Plasma TC and LDL- occurs (Mel'nikov et al., 2004; Vanstone et al., 2002). C concentrations were lowered up to 13 and 16%, respectively, Even though ingestion of plant sterols and stanols effectively in the treatment groups compared to the control group lowers cholesterol absorption, studies indicate a compensatory (Vanstone et al., 2002). Results of this trial (Vanstone et al., 2002) also revealed that absorption diminished while the frac­ tional rate of synthesis rose accordingly in response to 4 weeks Table 2 Therapeutic factors that play a role in modulating cholesterol absorption and of either plant sterol or stanol supplementation (1.84 g/d). synthesis TC and LDL-C both decreased 5% compared to placebo after Therapeutic ekotesterol Cholesterol Reference a 4 week supplementation period of plant sterols in a study factors absorption synthesis conducted by.Takulj etal. (2005). Increased synthesis was found Plant stcroVstanols after participants underwent the 2 g/d treatment with: plant Plant sterol i T Jones et al. (2000); sterols (Jakulj etal, 2005). Collectively, these studies radicate a Vanstone et at. (2002) reciprocal response of cholesterol synthesis to changes in cho­ iHarit stanol I T GyUing et al. (19#7); lesterol absorption. Jones et al. (2000); The mechanism by which a compensatory elevation in Vanstone et al. (2002) Ezetimibe therapy cholesterol synthesis occurs upon abatement of absorption Shoitterra X t Sudhqpetal. (2002) may be, explained through closer examination of the pathway iOjng/d of plasma eholesterol production. In this pathway, the rate- for 2-.weeks limiting enzyme, HMG CoA reductase, is regulated by intracellular eholesterol through negative feedback. More Statin therapy specifically, intracellular cholesterol regulates HMG CoA Short term — 1 Srcith etal. (2000) JOmgffl reductase concentrations through its ability to influence tran­ far 3 weeks scription, mRNA translation, and degradation of the enzyme long-term T I Miettinenetal.pOOO) (Useum, 2002). Suppression in cholesterol absorption by plant 20-40 mg/d sterols results in lower circulating cholesterol t^ncentratipns. for S years As a result, negative feedback Of cholesterol oh HMG CoA Weight loss effects reductase is inhibited, leading to increases in eholesterol 3-8 kg weight ? I DiBupno etal. (1999) synthesis. The reciprocal effect of decreasing cholesterol loss absorption on cholesterol synthesis is therefore, biologically ?: unstudied effect. plausible. 217

S. Santosa et at /life Sciences SO (3007) 505-514 511

Ezetimibe treatment The long-term effect of simvastatin treatment on cholesterol metabolism was studied in a subset of ihe Scandinavian Ezetimibe, also known as SCH 58235* is the first of a series Simvastatin Survival Study (4S) Group (Miettinen et al., of new pharmaceutical agents developed to reduce cholesterol 2000). Eight hundred and sixty seven patients were first divided absorption (Jeu and Gheng, 2003). The inhibition of cholesterol into four quartiles based on cholestanol-to-eholesterol ratios, absorption by ezetimibe is thought to occur through its and were then randomized to receive either placebo or interference with the function of the NPC1L1 transporter simvastatin (20 to 40 mg/d) for five years; The cholestanol-to- (Altmann et aL, 2004; Davies et al., 2005). Ten mg/d of cholesterol ratio served as an indicator of baseline cholesterol ezetimibe may lower total cholesterol up to 15% and LDL- absorption. Results revealed that lower baseline cholesterol cholesterol up to 20% (Jeu and Cheng, 2003). Sudhop et al. absorption was associated with higher baseline synthesis (2002) recently examined the effect of ezetimibe on cholesterol precursor concentrations. After five years, the magnitude by absorption and synthesis in humans. The study was a which simvastatin decreased cholesterol synthesis was posi­ randomized, double-blind, placebo-controlled cross-over trial tively related with initial levels. In turn, cholesterol absorption in individuals with mild to moderate hypercholesterolemia. In parameters rose accordingly. Another study observed that comparing ezetimibe with placebo, researchers found a 54% 6 months of atorvastatin therapy in men with type 2 diabetes decrease in cholesterol absorption, Not surprisingly, the decline led to decreases in serum markers of cholesterol synthesis and in absorption was accompanied by a corresponding rise in an increase in markers of cholesterol absorption (Miettinen and cholesterol synthesis. Thus, as with plant sterols, inhibiting Gylling, 2003b). A trial which also studied the 4S group found a cholesterol absorption through ezetimibe results in compensa­ similar relationship between synthesis and absorption in a tory increases in cholesterol synthesis. double-blinded trial comparing supplementation of atorvastatin and simvastatin (Miettinen et al., 2003). After one year of Statin therapy treatment, those who were taking atorvastatin displayed significantly lower cholesterol synthesis rates than those Who Statins lower circulating levels of cholesterol by inhibiting were taking simvastatin. As a result of this decrease in the action of HMG CoA reductase (Smith et al„ 2000). The synthesis, a reciprocal increase in cholesterol absorption was reduction of HMG CoA reductase at the hepatocytes leads to observed as was seen in other studies. Interestingly, cholesterol increased expression of LDL receptors, which bind circulating absorption was shown to be higher in those taking atorvastatin LDL in turn effecting plasma LDL levels (Lemernas and Fager, compared to those taking simvastatin. This study, therefore, 1997: Ray and Cannon, 2004). As a result of this inhibition of supports the hypothesis that cholesterol absorption responds hepatic cholesterol synthesis, statin therapy has been shown to reciprocally to changes in cholesterol synthesis. It should be reduce cholesterol concentrations by 20-30% (Miettinen etui., noted, however, that the compensatory elevation in cholesterol 2003). Since statin therapy aims to lower cholesterol synthesis, absorption was not equivalent to the decrease in cholesterol the effect of these pharmaceutical agents on overall cholesterol synthesis caused by the drugs. Thus, a beneficial effect on metabolism has been investigated. Smith etal, (2000) conducted circulating cholesterol levels was still observed. a study where 31 gallstone patients participated in a randomized, parallel-arm, placebo-controlled trial examining the effect of Weight loss statin therapy on cholesterol kinetic parameters. Seventeen of these patients received 20 mg/d of simvastatin and the other 14 The assertion that weight loss decreases cholesterol synthesis served as controls. Cholesterol precursor-to-cholesterol ratios is supported by the work of Di Buono et al. (1999) who were used as indicators of hepatic cholesterol synthesis. After showed that a weight loss of 3 to 8 kg by diet in six overweight 3 weeks of treatment, the plasma lathosterol-to-cholesterol ratio and obese men, resulted in a decline in whole body cholesterol decreased, suggesting a decrease in cholesterol synthesis. synthesis. The reciprocal effect of weight loss on cholesterol Despite these reductions, no changes in cholesterol absorption absorption, however, has yet to be clarified. Though the action occurred. More recently, the absorption markers, campesterol, of insulin and glucose on cholesterol metabolism may confound sitosterol and cholestanol, were used to determine the effects of the results (Pihlajamaki et al., 2004), weight loss of approxi­ statin therapy in subjects with high initial cholesterol absorption mately 15 kg in 10 overweight type 2 diabetics decrease TC by versus those with low absorption (Gylling and Miettinen, 21% (Simonen et al., 2002). The decrease in cholesterol was 2002b). After one year of statin treatment, no differences in accompanied by a suppression in cholesterol synthesis and an circulating total cholesterol levels were observed between the elevation cholesterol absorption (Simonen et al., 2002). two groups. Conversely, a greater suppression in synthesis in Although a cause and effect relationship has yet to be estab­ those with low baseline cholesterol absorption levels was noted lished, it can be hypothesized that a synergy exists whereby following treatment. Furthermore, cholesterol absorption was changes in cholesterol synthesis result in an opposite response of shown to increase more in those who had greater declines in cholesterol absorption to maintain cholesterol homeostasis. synthesis. Thus, although the study by Smith et al. (2000) did not Further studies that examine the effects of weight loss on observe an association, this study (Gylling and Miettinen, cholesterol metabolism in normoglycemic individuals are 2002b) indicates that cholesterol absorption responds recipro­ needed to clarify whether this relationship does indeed exist cally to alterations in cholesterol synthesis. Additionally, the question Still remains as to how a decrease in 218

512 S. Santosa a al. / Life Sciences 80 (200?) 50S-514 hepatic synthesis signals anupregulation in intestinal cholesterol certain direct methods are used, such as: 'sterol balance absorption. Future investigation should aim to establish a clear approach, plasma isotope ratio technique, and the deuterium mechanistic cause and effect relationship between synthesis at incorporation method Since these direct methods were not the liver, and absorption at the intestine. always implemented, the magnitude of the reciprocal effect could not be established for certain factors. Summary of findings Conclusion hi sum, cholesterol absorption and synthesis are affected by an array of physiological and therapeutic factors. The purpose In conclusion, findings fromth e current review suggest that a of this paper was to identify the primary vector affected by each change in one vector is generally accompanied by a reciprocal of these factors, and to determine whether a reciprocal effect in and opposing change in die second This relationship however, is the other vector occurs in response to this initial change. Studies not fully compensatory, allowing for the net reduction in body examining the effect of physiological factors on cholesterol pools of cholesterol by therapeutic treatments. Physiological metabolism suggest that a reciprocal relationship does indeed factors that affect cholesterol homeostasis may be implemented exist (Table 1). More specifically, in trials examining human as future targets in the development of effective cholesterol generic polymorphisms, a decrease in cholesterol absorption lowering agents. Furthermore, the presence of such a relation­ was consistently accompanied by an increase in synthesis. Such ship suggests that combination therapies which simultaneously a relationship was shown in carriers of the E2 isoform of apoE, affect both vectors would be more advantageous than therapies and also in those who carry a mutation on exon 1 of the ABCG8 that effect one. 19H allele. In the case of circadian rhythm, though this factor likely affects cholesterol synthesis, its effect on absorption has References yet to be established. With regards to body weight, evidence suggests that the obese state results in an increase in cholesterol Almjann, S.W., Davis Jr., H Jt, Yao.X, Laverty, M, Coinpton, D.S:, Zhu, L.I., synthesis, which is accompanied by a reciprocal decrease in Crona, JJX, Caplen, M.A., Hoos, LJM, Tetdplt G., Priestley, T, Burnett, absorption. On the other hand, a loss of body weight down D.A, Strader, CD, Graziano, MP, 2002. The identification of in­ regulates cholesterol synthesis, while having little or no effect testinal scavenger receptor class B, type I (SR-BJ) by expression cloning and its role in cholesterol absorption. Biochimica et Biophysica Acta 1580 on absorption. Similarly, the majority of studies examining the (1), 77-93. effect of therapeutic interventions on cholesterol metabolism Alttnantii S.W., Davis Jr., Hit., Zhu, U, Yao, X, Hoos, L.M., Tetdoff, G, also support a reciprocal relationship between the two vectors Iyer, S.P, Maguire, M., Golovko, A., Zeng, M., Wang, L„ Murgolo, N, (Table 2). Plant sterols and stanols inhibit cholesterol Graziano, M.P., 2004. Niemann-Pick CI Like 1. protein is critical for absorption, which often results in a compensatory increase in intestinal cholesterol absorption. Science 303 (5661), 1201-1204. synthesis. Ezetimbe also decreases intestinal absorption, while Angel, A., Bray, G.A, 1979. Synthesis of tatty acids and cholesterol by liver, adipose tissue and intestinal mucosa fioin obese and control patients. reciprocally increasing synthesis. Conversely, statin therapy European Journal of Clinical Investigation 9 (5), 355-362. down regulates synthesis, which is frequently accompanied by Angelin, B., Backman, X, Einarsson, K., Eriksson, X., Bwertii, S;, 1982. an increase in cholesterol absorption. In view of these findings, Hepatic cholesterol metabolism in obesity: activity of microsomal 3- it can be concluded that a change in one vector appears to, fairly hydroxy-3-methylglutaryl coenzyme A reductase. Journal of Lipid Research consistently, result in a reciprocal and opposing change in the 23 (5), 770-773. other. Anonymous, 2001. Executive summary of the third report of The National Cholesterol Education Program (NCEP) expert panel on detection, Since the majority of these studies support a reciprocal evaluation, and treatment of high blood cholesterol in adults (adult treatment panel 111). Journal of the American Medical Association 285 (19), relationship between these two vectors, an important question 2486-2497. that arises is the magnitude of this effect Such a question is Berge, KB, Tian, II., Graf, G.A., "Yu, L, Griskin, N.V, Schultz, J, imperative as the reciprocal response of the second vector may Kwiterovich, P., Shan, B., Barnes, R, Hobbs, H.H., 2000. Accumulation completely account for the initial change in the first If such an of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC effect occurred in response to therapy, the effect of the transporters. Science 290 (5497), 177 ]-1775. intervention would be obsolete. The magnitude of the reciprocal Berge, K.E., von Bergmans, K, Lutjohann, D., Guerra, R, Grundy, S.M., Hobbs, HJI., Cohen, J.C, 2002. Heritability of plasma noncbolesterol response was only established in trials examining the effect of sterols and relationship to DNA sequence polymorphism in ABCG5 and plant sterols and statins on cholesterol metabolism. In the case ABCG8. Journal of Lipid Research 4 3 (3), 486-494. of plant sterols, the treatment-induced decrease in absorption Boomstna, D.I., Princen, HJvl, Fruits, R .R, GevcrsLeuvcn, J. A., Kempen, H J., was consistently more pronounced than that of the reciprocal 2003. Genetic analysis of indicators of cholesterol synthesis and absorption: rise in synthesis. As for statins, the drug-induced decrease in lathosterol and phytosterols in Dutch twins and dieirparents. Twin Research synthesis was generally larger than the complementary increase 6 (4), 307-314. Bosner, MS, Lange, L.G, Stensor, WJF, Ostlund Jr., RJB, 1999. Percent in absorption. In this way, the reciprocal change in the second cholesterol absorption in normal women and men quantified with dual stable vector did not fully compensate for the change in the first, thus isotopic tracers and negative ion mass spectrometry. Journal of Lipid allowing for a reduction circulating cholesterol levels to occur. Research 40 (2), 302-308. Establishing the magnitude of response for each factor is not Buhman, K..K, Acead, M, Novak, S, Choi, R.S, Wong, J.S., Hamilton, RX, possible due to the techniques employed in certain studies. The Turley, S, Farese Jr., R.V, 2000. Resistance to diet-induced hypercholes­ terolemia and gallstone formation ui ACAT2-deficieni mice. Nature magnitude of the reciprocal effect can only be established when Medicine 6 (12), 1341-1347. 219

S. Sa*ua et ttl ,'Ufi: Scumaz SO (3007) SttS-lU 513

Outer, CLE, Hawks, P.M, Hoi, RY, 1997. Genetic variation k ofeofcstsirf Ho, K.J, 1^75. tiffed o!ck)ts!5temlfsctlH.goi3 OTcadiaBrbyihiaof beplic and absorption effciaiQ'arosssgtisbw^ straossoftnice. Journal of Nistritioa 127 intestinal elmlesterol biosyntbesia in hamsters. Proceedings of the Society (71 1344-1341 ferEiqxaitnscrstal Biology ait4 Medicate 150 (2\ 271-277. Cells, LJ£, Van Caster, E, SefeoeBw, B.A., 1993. Ettaci of matt timing on Ho, KLJ., IJ79. Ctttiadiais rhythm of dKfatoollJWsyMhais; dietary rt^wtauLw diianal rbyttas sf btjnsac cholesterol syMhcsis. Araertean Journal of IB the liver and snail intestine of harasters, fctrcafonal Jounsat of Ptyiiotagy 20 0 Pt t j, EtTS-Ettt !3mB»ttolo}|)' *. (1), S9-59, Cbing, J. V, MM, Regstation of bit? iacid sjcfessis; pafewtys, Bjslcar fetatk, H, Y-urashtta, C, &inita, Y„ ¥ot*ida, V., Nodstro, M, 2000, Insutin is resciptors, and nrnfenisms. Jatstsai of UgatoSagf 40 (3), 539-551. a doHsa-atst iSKppn^isa of stool 12 alpha-feydioxybse P450 (CV'PiiB) Cote, JJC., PertKasBdis, A., Fataai, S, Estaait §., Vega, OLE., Grassy, SJvl, depression is rat liver possible role at iosuli. in eneadian rhylhn. of Hobbs, HLH,, IWS, Mirttipte rare variants in NBC 1L1 associated with CVP&B, Doraial of BJocteanii*y {TWiyo) 127(0,57-64. isdwced sterol absorption and plasma fow-dsestty lip^aprotcin levels. Jataalj, L, Tiip,MiJ,Sodbo]j,X, wmBeifmiBn, KL,KstefeHt,JJ, VfesaM, Ml», Procesdtogs o f fee National Asasbsy of Seieaess of the United States of 2S;I05. toll ibitifjn of ebolcsletol absorption by the e.nijhirsitic*. of dietary plant Ancnca IBS {«), 1810-1S15. sterols and oetimibe: eifects on plasma lip id Itrveh. Journal of Lipid Reseairh " Dattito. AM, Kiis-Etfctten, P.M., 1992. Effects of weight jedtjeiioc en blood 46(l2),2i»I-2«&, lipids and lipopistems: a taaa-analysisi. Ajaerisan Journal ©f Clinical leu, L, Qssas^, t W, 2B1B, Htsaajjacokgy arod thertfcutics of ejcttmibc ^CH Nutrition 5* C), 320-328. 5S2S5), a iioteslBiiol-ahsorplioia inhibitor. Oieical IbcmpoJties 25 (9), Davics,J.P„ Scott, C, Qtsbi,K., liajiis, A, toannoB, V.A,,3305. Inactivalkm of 2352-2387. NFC1L1 eaiases mulajjlc %od iiairasport defects and prcstects against diet- fatter, CD, DHs%, J.M, Turlty, SD„ 1 W°. Ococtk diffocoecs io ctehsteol ind?K«3 hjp.acfcc4c5tiolcmB- Jsmjial of Biotojjical OrasMUy 280 (13% Asotptiflnta KRSvaisdCSTBlLjSiatcc cfictoa dsofcstetol teapoBMwrsess. 12710-12138. Amoks. Jotitnal of t%aotogy ITS (5 Pt \% 01117-Gl 124. Davis It, H.R., ZbB, L J„ Boos, LM, lWriotT, 0„ Mapire, M, Eiu, J,, Yao, Jones, PJ, Sdiociler, £J.A., Wft IsVidaxe for tlbrnat oaiodMty its tensas X., Iy*i, S.P., Lam, M.H, Load, E.O., Octanes, PA., Orariano, MLR, etetesterot s^yotltests, Joiaffll of Lipid Rests** 31 (4), £67—673. AitasuB, $.W, 2064. Memaim-Fiek CI Lite .1 (NPCILI) is the intasfiisat JOESS, P.J., Main, B.F, UsAtkfe, J A, W%. Rctpasest: of cWisslejot ssyBtbsais to phytasterol aricBoJestcrat transporter atxi a key roadulatssto f «Me4ody ctetestefol fedii^ tn tam •»'..» tSffcn^t apoltj>8piiistsfin E gsB9ly|BS. cholesterol tasjcostasis. Jfoanssl of Biologisat Cfeomstry 219 (32), Metabolism 42 (%% 10t35-1071. 335&f»-33592, Jaots, FJ,KadG,-Sa*ja«, M., l^aoias, ETC, VtestoDi; CA, Fsng, J. YL.PaiaoiB, 01 Btsono, ML, Hannah, IS. Kadzet, LI, Jones, EL, 1W9. Weight hss VrLE, 2WIS. ModolMioi. of plasma lipid levels and cholesteavl kinetics by due to enerig' irauietion sujspKssses eboteterol biosynthesis k over- phytiistefol wrsns phylostanol estere, Jourcal of lipid Research 41 (51, •weij^*, tsBdtjr %pcissholeiteK»kEste ESSE. feKrast of Jtorkten 129 ($}, #7-705, 1545-1548. Jwzwm, H, Itateitkr, J,, BansJt, C, Msrdt, P, TbEws, A.D., 2000. Ditimal and Didsshy, JLM,,SipeeieJn, MB, 1967. EifatisfcksfelejBt feeding aid tasting dietaiy-indiisd ehaoges its eholcsler.il syndesis correlate vnth levels of on Sterol ^ntN^is io ^vsnlx$3s ti^ucs oftt% mt. JmmaH of Lipid Usscai^ I3.RNA fer HMtLX^A re#rctas«s. Joiraat of Lipid Reseifflsb 41 (7\ S CI, 97-lt)4, 164S-I054, Edvsards, P.A, MIMJJ», H., {JswtsS, R.G., 197Z & vivo tosonstiiiisHj of tk: Kajirami, K, BroEseaii, M.E.Orslm'as, J.M., Sdmefer, I'.l.ICCtl. Intera-aions ciRa^ia! thjabm (rtetefcstSK.il hiasyntlKSls is Ih; liveral«i intestin; oi llx btfwsiB rasmj5SOEgeneti c polymotphisaas it ABCG5/CS and CYP7A1 on rat. JoiiBsal of LipM Bctcaieh 13 (1\ 396-iBA. LDL eWe*aol4owsaing nesppnse to artswjt^atra AtteTOscfeasssis 175 (2% GmscomA, CI, Pipa, C, Bmim^rtmi, ML, Pontsao, E,, EteesrB, B., Berpi; A,, 2»7-»3, Fay, LB., 2601. Saa-tossous asas^scoi of cl»fec«crd abssorpljas m4 Katsjis, M.B., Cjnmt% S. M., Ltonis.P, Uw, M, MJettttxis, X, Padetti, R., 2001 ^'rjtl^sisiB l«3n^sisusi]^oivlinep$<^ron!at$)|^i^/0pni^ Hfgf^ry and sat^ty of ^aot sltuso b> sard sterols iir tbe managen^CGl of blood ds-tOBa(ogr^%"pj^y>!fe^fes^-Tatk>i3sais upsaimmVy. Rapid Oonanm- etsotestcrol levels. Mayo CHrae- Ptpcecdhsi^ 7S (% °i.5-#71 ncatioiMio MassS^sMromay 15(14), 1267-1213, Ksrajjeis, H J, Olat^ J.F, Oe*«is l^iwea, JA, vstndtr %or^ HA., Kiiitan, M.B, Ctooi%", S.LM,, AJsreiss Ji„ EH„ 19fi9. Itammts of Elwfcstetol (isrisoya, 19E!.. Serum latboiterol e«»eeDtratk)i, is an indieator of wbole-body ssyt^iesis, ai^l s|)$arotb]% ra mm, catrkstl oqt by isotope idi.0ic ai3£t ^rot dsolesterol syntiscsi^ifihitmans. Joiamatof l,ipidReseaieb291^, 1149—1155. batais»«!ssti.o4i. Lfewnal wf Li»M BsHseands 10(1), §1-107. Kesaiserai, YA„ Irhnholm, C.„ Micttiros, T.A,, 19R7. Intestinal cholesterol Gyllirsg, IL Micrtnos, T.A., ICiC*2a. IrJsjritanci: ot'chdcstaol met^boism of alrsOTptic«cf'ncbKy'inriianisTClatedtoapapotcinlir^ p robands with h igh «tow sbotestcrol absorptiori. Jmiiral of I. ipiii R tscarcl. C&tfcd lisnsaljptitm 10 @\, 578-581, 43 0% W72-I476. I-ansbert, t3L, Ansar, MJ„ doo, (iL, Brewer h„ HLB„ Oonzafcs., F.I-,Shat, CX, Ciytling, H,, MfcttitKn, TA, 21162%, Basttiiw iBtcstiraat ahKirptiffls aisd sya-dhssis 2803, The fLamesoKi X-rceeptor a as essential regrslatar of cholesterol of crsDlotcrol regulate lis Ttsporm to hjpolipidacm ic treatmems in oorocary hoirscostasis. Journal of Biologieal Clxmiitry 271! (4), 250.3-2570. psicrits. AtlscroKfcrosw 160 P), 477-181, hue, M.H, La, K., Hasswi, S-, Yo, H, asntcisin, &, t&Wsa, H„ KojtBsa, HL, Clyllbj.;, H, Konrtib, K,, Mkltocis, 1LA, l'W5, Chokstsrol ahs*irptioi. arid Atikincts, R, Sakiiraa, N„ Pcsoram, R, Srivastava, A,K, Saten, Ci„ Dean, EXtabolkTB ac4 LDL Irinctits in teakby mas uitti ditfercnt apoprotein E M., Patet, S.B., 2001. Mcntifieatioa of a jene, ABt'OS, invportanl in Ike ptenotypes aaj apoprotein B Xba 1 ant! LDL receptor Pvu II genotypes. icgtilatimofeaetaiy'ebotesttrolahTOrpticB.NatiTCCicrjetk^ 79-83. ArteriosdenKts, ITaBmboas m& VaKi-lar Biology IS (2), 20S-211 Leaterras, H„ f ager, 0,1997. Pliarmacodj-ramics and pbarmKoiiiselies of the 6yBiis|, tt, RadhakrisbraE, HL, MitsDracn, T.A., 1S97. Ratetism ef sarms HMG-CoA rcekictase inhibitors. Similarities and difterences. Clinical ebofcsteral m postiiiCTiopausal women with pluvious myoeaidial mfan-tion Phanrneciinctics 32 (5), 403-425. aatl dsisltestoisl malabsorptioi) jsts^(rfbydfe^sitaeito>at«stBfBJttigajhse Usews, L, 2002. to: Vatsec, OM,, Vaisec, J,E (Bds.)t BS»*t3!3tJjJiy of Lipids, w»Hen arstl ilietory alostaool CSnatlatioi. 96 (12), 422S-4231, lipojiolems and Memisanes, 4th cdn. Elsr, icr, Amsterdam, The Nctljei- Oytttug, H^llLifflcsimasv M, Pi&jaHsal^ J.,Agv^J, laalt^M,RiiJ!iamitii^ lasd«,np, 409-431.

R.A„ Raaansaa, R„ Micttiocn, SLA, 4&Si Po^j^i^isBW m tbe AB005 ManioKS, P., Qiiimws, Vn Amigo, I.., Moreno, ML, Micjud, J,K,Schwarz, Ml, and ABD3B giocs assosiatc wife cbokstCKs! absarptkiTi and rnsulir. MM^beo, HE, Trtp«4 B, KA^e^ M, VMPatiUai, S., CJBBM, D.E, aum&oty. Jnwsal rfLiptdRsscEtrch 45 0% lSS0-lfits5- Riifitti, A, 20t>l. Hepatic cholesterol and bile aeid metabolism and HamprscM, B„ Nusssttsr, C-, LycsB, F„ 1S#, Rhylteiac changes of rntestaial cbolestcm! absorption in scavenger reeeprar glass B type l- kyAoBrymjAytgbriaiyl cscatyisae a raJixstata; aeliytly m timas of ifed aisd deficnent mice Jocmal oi Lipid Reacaieh 42 (2i, 170-1 £0. fastearats. FEBSLdissts 4(2\ 117-121. Mel'isto^ S.M., Setjeo *as Hoorn, JLW^ Eptclcnboam, A.P, 2004, EBeet Hsgsfc, JLA, Otsy, J., Bats, M.R., W»g, J„ 28&S, NPC1L1 laptotypc h of phylostaots and phytostaools on the soiibiuLralion of ebolestetol by associated with imer-icdMAra) varfatra- in plasma low-density lipopiriteiB dietarj' mixed Rsiedles: an in vitro study, CLhernislry Mid Physics of Lipids r^p^eos? to tscstimibe; LijiHtfs m H«al^ nos& llis&asc 4,14 127 (2% 121-141. 220

514 X. Santoso a at. / Life Sciences SO (3007) SOS-SU

Miettinen, T.A, 1982. Diurnal variation of cholesterol precursors squalene and Schwarz,M.,Davis, DX., Vtck, BR,Russell, D.W., 2001. Genetic analysis of methyl sterols in human plasma lipoproteins. Journal of Lipid Research 23 intestinal cholesterol, absorption in inbred mice. Journal of Lipid Research (3), 466-473. 42(11), 1801-1811. Miettinen, TA, Gylling, H, 2000. Cholesterol absorption efficiency and sterol Sehayek, E, Yu, HJ, von Bergmann, K, Lutjoharm, D, StoffeL M., Duncan,E-M, metabolism in obesity. Atherosclerosis 153 (1),.241-^248. G^»cia-NavcdaTL,SahvJ.,BhmdelLMX,Friedrr>ari,J.M,Breslow)JX.,2004. MiEttinen. TA, Gylling, H, 2002. Ineffective decrease of serum cholesterol by Phytosterotemia on the island of Kosrae: rounder effect for a novel ABCG8 simvastatin in a subgroup of hypereholesterolemic coronary patients. mutation results in high carrier rate and increased plasma plant sterol levels. Atherosclerosis 164 (1), 147-152. JcmrnalofLrpid Research 45 (9), 1608-1613. Miettinen, T.A., Gylling, H, 2003a. Cholesterol synthesis and absorption in Shimano, H., Snimomura, I, Hammer, R.E, Hens, J., Goldstein, JX, Brown, coronary patients with lipid triad and isolated high LDL cholesterol in a 4S MS,, Horton, J-D, 1997. Elevated levels of SREBP-2 and cholesterol subgroup. Atherosclerosis 168.(2), 343-349. synthesis in livers of mice homozygous for a targeted disruption of the Miettinen, TA., Gylling, H., 2003b. Synthesis and absorption markers of SREBP-1 gene. Journal of Clinical Investigation 100 (8), 2115-2124. Cholesterol in serum and lipoproteins during a large dose of statin treatment. Simonen, P, Gylling, H, Miettinen, TA, 2002. Acute effects of weight European Journal of Clmical. Investigation 33 (11), 976-982. reduction on cholesterol metabolism in obese type 2 diabetes. Clinica Miettinen, T.A., Tilyis, R;S, Kesaniemi, YA, 1990. Serum plant sterols and Chimica Acta 316 (1-2). 55-61. cholesterol precursors reflect cholesterol absorption and synthesis in Smith, JX, Roach, PD, Wittenberg, L.N, Riortot, M, Pillay, SJ?, Nestel, PJ, volunteers of a randomly selected male population. American Journal of Nathanson, L.K, 2000. Effects of simvastatin on hepatic cholesterol Epidemiology 131 (1), 20-31. metabolism, bile lithogenicity arid bile acid hydropliobicity in patients with Miettinen, TA., Gylling, H, Vanhanen, H, Otlus, A, 1992. Cholesterol gallstones. Journal of Gastroenterology and Hepatology 15 (8), 871-879. absorption, elimination, and synthesis related to LDL kinetics during Spady, DX, Dietschy, J.M, 1983. Sterol synthesis in vivo in 18 tissues of the varying fat intake in men with different apoprotein E phenotypes. squirrel monkey, guinea pig, rabbit, hamster, and rat. Journal of Lipid Arteriosclerosis and Thrombosis 12 (9), 1044-1052. Research 24 (3), 303-315. Miettinen, T. A., Strandbefg, TJB., Gyllingj H, 2000. Noncholesterol sterols and Stahlberg, D, Rudling, M, Angelin, B, Bjorkhem, I, Forsell, P, Nttsell, K, cholesterol lowering by long-term simvastatin treatment in coronary Einarsson, K, 1997. Hepatic cholesterol metabolism in human obesity. patients: relation to basal serum cholestanol. Arteriosclerosis. Thrombosis Hepatology 25 (6), 1447-1450. and Vascular B iology 20 (5), 1340-134 6. Sudhop, T, Lutjohann, D, Kodal, A, Igel, M, Tribblc, DX, Shah. S, Miettinen, TA., Gylling, H, Lindbbhm, N, Miettinen, T,E.,.Rajaratnarn, RA, P.erevozskaya,I.i von Bergmann, K,2002. hihibition ofintestinal cholesterol Relas, H, 2003. Serum noncholesterol sterols during inhibition of absorption by ezetiinibe in humans. Circulation 106 (15), 1943^-1948. cholesterol synthesis by statins. The Journal of laboratory and Clinical Tilvis, KS-, Miettinen, TA, 1986. Serum plant sterols and their relation Medicine 141 (2), 131-137. to cholesterol absorption. American Journal of Clinical Nutrition 43 (1), Murphy, C, Parini, P., Wang, J., Bjorkhem, I., Eggertsen, G., GafVels, M., 2005. 92-97. Cholic acid as a key regulator of cholesterol synthesis, intestinal absorption Vanstone, CA, Raeini-Sarjaz, M, Parsons, W.E, Jones, P.J, 2002. Unesterified and hepatic storage in mice. Biochimica etBiophysica Acta 1735,167-175. plant sterols and stanols lower LDL-cholesterol concentrations eriuivalently O'Neill, Fit, PateL DX\, Knight, BX., Neuwirth, C.K, Bourbon, M., Soutar, ui hypereholesterolemic persons. American Journal of Clinical Nutrition 76 A.K, Taylor, G. W, Thompson, G.R, Naoumpva, R.P, 2001. Determinants (6), 1272-1278, of variable response to statin treatment in patients wim refractory rairiilial VTahcevic, Z.R, Eggertsen, G, Bjorkhem, 1, Hylernon, P.B, Redford, K, hypercholesterolemia. Arteriosclerosis, Thrombosis and Vascular Biology Pandak, WM., 2000. Regulation of sterol .12alpha-hydroxylase and cholic 21 (5), 832-837. acid biosynthesis in the rat Gastroenterology 118 (3), 599-607; Ordovas, J.M., Tai, E.S., 2002. The babel of the ABCs: novel transporters Wang, D.Q, Paigen, B, Carey, M.C, 2001. Genetic factors at the enterocyte involved in the regulation of sterol absorption and excretion. Nutrition level account for variations in. intestinal cholesterol absorption efficiency Reviews 60 (I), 30-33. among inbred strains of mice. Journal of Lipid Research 42 (11), Vappu, AS., lllingworth, D.R., 1994. Diurnal variations in the plasma 1820-1830. concentrations of mevalonic acid in patients with abetalipoproteinaemia. Wang, MI, Afdhal, NIL, Gendler, S.J, Wang, D.Q, 2004a. Lack of the European Journal of Clinical investigation24 (Iff), 698-702. intestinal Mucl mucin impairs cholesterol uptake and absorption but not Parker, T.S., McNamara, D.J., Brown, C, Garrigah, O, Kolb, R, Batwin, H., fatty acid uptake in Mucl-/-mice. American Journal of Physiology: Alrrensjr, E.JX, 1982. Mevalonic acid in iumair plasma: relationship of Gastrointestinal and Liver Physiology 287 (3), G547-G554. concentrationarid cu-cadian rlrythmitb cholesterol synthesis rates in man. Wang. Y, Jones, PJ, Austrian, LJvf, Lichtenstcin, A.H, 2004b. Soy protein Proceedings of the National Academy of Sciences of the United States of reduces triglyceride levels and triglyceride tatty acid fractional synthesis rate Arnerica 79 (9), 3037-3041. in hypereholesterolemic subjects. Atherosclerosis 173 (2), 269-275. PfphL M, Sehreiber, I„ Liebich, H.M, Haring, H.U, Hoffmeisler, EM, 1999. Wilson, Ji>, Lindsey Jr., CA, 1965. Studies on the mlluence of dietary lijpregulation of cholesterol synthesis after, acute mybcardiaL irrJarction—ts cholesterol on cholesterol metabolism in the isotopic steady state in man. Cholesterol.apositive acute phase^^reac^nt? Atherosclerosis 142 (2), 389-393. Journal of Clinical Investigation 44 (11), 1805-1814. i%b|amalr4X, (filing,II, Miettinen Woollett,L.A,Buckley,DD,Yao,L,Jcmes,PJ,Gianholm,NA,Tolley,RA, is. associated with iricreased chc&steroi synthesis and decreased cholesterol Heubi, J.E, 2003. Effect of ursodeoxycholic acid on cholesterol absorption absorption in nonnoglyceroic men Journal ofLipi d Research 45 (3),507-5I2. and metabolism in humans. Journal of Lipid Research 44 (5), 935-942. Ray, KiK, Cannon, C.P., 2004. Intensive statin therariy in acute coronary Woollett, LA, Buckley, D X>, Yao, X, Jones, PJ, Granholm, N.A, Tolley, E.A, syndromes: clinical benefits and. vascular biology. Current Opinion in Tso, P, Heubi, J J3, 2004. Cholic acid suppJeinentalion enhances cholesterol Xipidology 15 (6), 637^643; absorption in humans. Gastroenterology 126 (3), 724-731. Appendix 3: Calculations used in the meta-analysis of plant sterols and LDL- cholesterol levels

Effect size:

For parallel trials, endpoint LDL cholesterol in the treatment group was subtracted from endpoint LDL cholesterol in the control group (Deeks et al., 2005). For crossover trials, the LDL cholesterol value at the end of the treatment period was subtracted from the

LDL cholesterol value at the end of the control period (Deeks et al., 2005). Within- individual changes were used when presented; otherwise, group means were used. In symbols, the estimates of effect size (ES) are:

- For parallel trials: ES//= Tf - Cf

Where

ES// = the effect size of a parallel design trial,

Tf = final LDL cholesterol mean in the treatment group

Cf = final LDL cholesterol mean in the control group

- For crossover trials: ESX = T - C

Where

ESX = the effect size of a crossover design trial

T = LDL cholesterol mean at the end of the treatment period

C = LDL cholesterol mean at the end of the control period

Standard Error of effect size

- For parallel trials:

The standard error (SE) of effect size for a parallel study was calculated as follows 2 2 SE// = V (SDT) /nT + (SDc) /nc

where

SE// = standard error of effect size for a parallel study

SDT= standard deviation of LDL-cholesterol endpoints in the treatment group

SDc= standard deviation of LDL-cholesterol endpoints in the control nj= sample size of the treatment group nc= sample size of the control group

Standards deviations (SDs) were extracted from the studies or, if not reported, derived from standards error of mean or confidence interval for group mean, (Deeks et al., 2005) as follows:

- From standard error:

Standard error of group mean = SD/VN

- From confidence interval for group mean:

SD = VN X (upper limit - lower limit) / 2* t (i-confidence level, degree of freedom)

where

t (i-confidence level, degree of freedom)i s the t -value associate with study confidence level,

usually 95%, and sample size of group

- For crossover trials (Deeks et al., 2005; Elbourne et al., 2002):

The standard error (SE) of effect size for a crossover study was calculated as follows

SEx=SD(diff)/Vn 223 where

SE x= standard error of effect size for a crossover study

SD (diff)= Standard deviation of difference between the treatment period and the control period n= sample size

SEX was extracted from reported statistical values in the trial, paired t-value or P-value, confidence interval from a paired analysis or imputed from a number of studies as follows:

- From t or P-value:

t=diff/SEx

where

diff is mean of difference between control period and treatment period

If only the exact P-value or the upper bounded P-value of the paired t-test was

reported, then the correspondent t-value for that P-value was calculated by entering

the P-value and the degree of freedom into a spreadsheet as follows: = tinv(p.Vaiue,

degree of freedom)

- From confidence interval:

SE x = (upper limit- lower limit) / 2* t (I-confidence level, degree of freedom) 224

Where

t (i-confidence level, degree of freedom) is the t -value associate with study confidence level,

usually 95%, and degree of freedom

- From imputed SD (diff):

2 2 Imputed SD (difi) = V(SD T + SD C - (2 * R * SDT * SDC))

Where

SD (diff) = standard deviation of difference between the treatment period and the

control period

SD2x = LDL-cholesterol variance at the end of the treatment period

SD2c = LDL-cholesterol variance at the end of the control period

R= 0.81 which is within-individual correlation between the treatment and control periods that was calculated from a number of studies

Pooled effect size estimate:

Treatment effect size and its standard error were calculated for every trial as described above. To obtain the pooled treatment effect size, the effect size estimates and standards error were entered into RevMan 4.2 under the "Generic inverse variance" outcome. In the inverse variance method the weight given to each study is chosen to be the inverse of the variance of the effect estimate.

A fixed effect meta-analysis using the inverse variance method calculates a weighted average as follows:

Generic inverse variance weighted average = E (ES; / SE,2) 225

I(1/SE,2) where

ES\ is the effect size in study i, SEt is the standard error of that estimate and the summation is across all studies.

Calculation of within-individual correlation between the treatment and control periods for crossover studies

SD (mmol/L) Study ID Control Treatment Difference R AbuMweis 2006 a(AbuMweis et 0.93 1.01 0.51 0.87 al., 2006b) AbuMweis 2006 b (AbuMweis et 0.93 1.06 0.59 0.83 al., 2006b) Noakes 2005 a (Noakes et al., 0.74 0.71 0.32 0.91 2005) Noakes 2005 b (Noakes et al., 0.74 0.76 0.32 0.91 2005) Jones 2003 a (Jones et al., 2003) 0.89 1.08 0.64 0.81 Jones 2003 b (Jones et al., 2003) 0.89 0.81 0.51 0.83 Judd 2002 (Judd et al., 2002) 0.28 0.28 0.28 0.50 Jones 2000 a (Jones et al., 2000) 0.70 0.59 0.40 0.82 Jones 2000 b (Jones et al., 2000) 0.70 0.74 0.39 0.86

Average = 0.81

where

R= within-individual correlation between the treatment and control periods and was calculated as follows:

R= (Control SD)2 +(Treatment SD)2 - (Difference SD)2 2 x (Control SD) x (Treatment SD) 228

McGill Tet.: (514) 398-7842 Scaooi of Dietatlcs and £cole da dWUtique at Fax: (514) 398-7739 Human Nutrition nutrition humalne

Faculty of Agricultural FaeulM da* aeiarwas da 21,111 Lakeshore and Erwironmantal Saianaaa I'agroultura at da I'anvironnamant Ste-Anne-de-Bellavue Quebec. Canada H9X 3V9 McGill University Universile McGill Macdonald Campus Campus Macdonald

Dr Celeste Johnston, Co-Chair "Dt'CLmber 16,2004 Institutional Research Ethics Board •prrraffr McGill University Faculty of Medicine APPROVAL Mclntyre Medical Building # 637-3655 Promenade Sir William Oslfer JAN 10 2005 Montreal, Quebec H3G 1Y6 Tel (514) 398-8302 Faculty of Medicine McGill University

Dear Dr. Johnston.

Thank you for your letter of October 261h indicating acceptance of the research proposal A00-M79-04A entitled "Relative Efficacy of Plant Sterols Given Once or Three Times per Day in Management of Hypercholesterolemia" which was reviewed by your Committee at the September 13th meeting. I would like to request approval of modifications to the protocol which have arisen since the time of original submission on August IS*:

a) The title of the project has been changed to "Efficacy of Plant Sterol Given at Different Times of the Day in Management of Hypercholesterolemia". The new title reflects the slightly refocused aim of looking at plant sterol given at breakfast, as per the previous protocol, against plant sterol given in an evening meal or in a snack before the evening meal. The previous protocol called for comparison of the identical dosage as requested previously, but given either as a single morning dose, or as the same dose divided across the three daily meals.

b) We are asking that the carrier food in which the plant sterols are provided be changed. The plant sterol enriched margarine has been replaced with a plant sterol containing yogurt, which will be supplied by Danone Vitapole Group, Paris, France. The current research project wijl be sponsored by this group.

c) The study design pertaining to the frequency of intake of plant sterols has been changed in order to help test the efficacy of a new plant sterol enriched yogurt product that is being promoted for consumption once a day. In addition, a fourth treatment phase has been added to test if the cholesterol lowering effect of plant 230

Appendix 5. Consent forms

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL

Relative Effect of Different Forms of Plant Sterol Preparations on Plasma Lipid Levels, Cholesterol Absorption and Lipid Metabolism

Pateint Name: Protocol Number

School of Dietetics and Human Nutrition, Macdonald Campus, McGill University Mary Emily Clinical Nutrition Research Unit: (514) 398-7527 Principal Investigator: Dr. Peter Jones, phone: (514) 398-7547, 398-7527 Contact Physician: Dr. William Parsons, phone: (514) 599-4390 (pager)

I, , the undersigned, hereby consent to participate as a subject in the above named research project conducted by McGill University. The nature of the procedure treatment, its risks and/or benefits, and possible alternatives, follow: 1. NATURE AND DURATION OF PROCEDURE: The aim of the study is to examine how the relative effects of different forms of plant sterols added to the diet will affect blood lipid and lipoprotein levels, cholesterol absorption and lipid metabolism in subjects with elevated serum lipid levels. An additional aim will be to determine whether responsiveness of plant sterols is affected by polymorphisms in the cholesterol transporter ABCG5 gene.

As part of a pre-screening visit, a fasting blood sample of 8 ml (less than two teaspoons) will be taken for the laboratory to measure your blood lipid levels and the presence of a specific ABCG5 genotype through a genetic test of your blood cells. Following qualification for the study, you will be invited back for further screening where a fasting blood sample of 9 ml (two teaspoons) will be obtained to measure your lipid profile and complete blood count and to confirm the absence of health abnormalities. Prior to beginning the study, you will be examined by a physician to ensure that you are in good health. An electrocardiogram will be performed at the discretion of the physician in charge.

You will also need to ensure that you REFRAIN from (1) consuming any lipid lowering drugs, including fish oils or probucol within the last 6 months, or (2) having onset of thyroid disease, diabetes mellitus, kidney disease or liver disease within the past 3 months.

Once you start the clinical trial, you will be asked to consume only the test diets provided by the Metabolic kitchen within the Mary Emily Clinical Nutrition Research Unit for five 28 day periods. A period of at least four weeks will separate each diet phase, where you

2002 Page 1 of 3 231

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL will resume consumption of your typical intake. All five diets will contain nutritionally adequate North American foods and be similar in terms of type of food. The diets will be fed to you as three meals per day, where at least two of the meals will be consumed at the Center. No extra foods or beverages, including alcohol, are to be consumed. The variable component of the diets will be tire different forms of plant sterols, material resembling cholesterol obtained from plant sources. These plant sterol esters are tasteless and odorless.

On days 1,2, 3 of each four (4) week treatment phase, a fasting blood sample (20 ml/day) will be obtained for the assessment of lipid, lipoprotein, vitamin, and phyosterol levels. On either day 1, 2 or 3 of each diet phase you will be asked to remain/return to the Research Unit three (3) hours after you consume your normal experimental lunch or dinner so that a blood sample (5 ml) may be obtained in a non-fasting state (after you have eaten). This is only done once each treatment phase to measure how your blood triglycerides respond after eating a meal.

In addition, four (4) days before the end of each dietary phase, you will be given a small amount of tagged cholesterol to eat. The digestion and movement of the tagged cholesterol into your blood stream will permit assessment of how much cholesterol your body absorbs from the diet. On the last day of study, you will also be requested to consume three tablespoons of water tagged with deuterium in order to establish the amount of cholesterol being produced inside your body. All of these tags are non­ radioactive and pose no toxicity hazard. Blood samples (25ml/day) will be collected daily on the mornings of each of the last four (4) days of the study in order to examine the movement of these tags across body compartments as well as to measure your lipid, lipoprotein, vitamin, and phytosterol levels. Also, on the day you are given the cholesterol to eat, an additional blood sample (20 ml) will obtained 12 hours later (which should be around supper time that same day). At the end of each treatment phase, you will again be examined by a physician to ensure that you are in good health

The total amount of blood drawn during each phase of the study will be approximately 202 ml. The total blood volume required for this trial will be 1010 ml. No more than 500 ml will be drawn in any eight (8) week period.

2. POTENTIAL RISKS AND/OR BENEFITS: The phytosterol esters added to the margarines at the proposed level have been shown to have no negative side effects on health in previous animal and human experiments. Bruising and pain can be associated with venipuncture (blood taking). Volunteers may run the risk of becoming anemic, but this will be tested for at the end of each diet phase, and anemic volunteers will receive iron supplementation (ferrous gluconate. 325 mg orally, once a day, for 2 weeks). In case you feel any discomfort during the experimental trial, a physician Dr. Parsons, will be available to contact at any time. Dr. Parsons can be reached at (514) 599-4390 (pager).

2002 Page 2 of3 232

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL

You will be screened for a specific genetic profile which we are seeking for participation in the study. We will need to screen about 200 individuals in order to find the 32 needed for the study, so you have a 1 in 6 chance of being selected should you decide to volunteer for the study. If any health abnormalities are identified in the clinical tests conducted during this experiment, you will be informed of such by the doctor associated with the study.

The substance of the project and procedures associated with it have been fully explained to me, and all experimental procedures have been identified. I have had the opportunity to ask questions concerning any and all aspects of the project and procedures involved. I am aware that I may refuse to participate as well as withdraw my consent at any time. I acknowledge that no guarantee or assurance has been given by anyone as to results to be obtained. Confidentially of records concerning my involvement in this project will be maintained in an appropriate manner. Samples will not be utilized for any additional analyses, nor stored for any prolonged period, nor shared with any other group, other than is indicated in the protocol, without your specific consent.

I, , have read the above description with one of the investigators, , I fully understand that, in compensation for the inconvenience of the study schedule, I will receive $1,500 in five (5) installments of $300.1 will also receive access to my test results when they become available. If I decide to withdraw before completion or should the study be terminated early, I will receive an appropriate pro-rated fraction of this amount.

I acknowledge receiving a copy of this consent form and all appropriate attachments and agree to be contacted by a member of the Research Ethics Committee.

Investigator Signature of Subject

Witness

Date Time

2002 Page 3 of3 SUBJECT CONSENT FORM OF RESEARCH PROTOCOL

September 1,2005

"INVESTIGATING THE EFFICACY OF PLANT STEROL GIVEN ONCE OR THREE TIMES PER DAY IN LOWERING CHOLESTEROL ABSORPTION IN HYPERCHOLESTEROLEMIC INDIVIDUALS"

Commercial Sponsor:

School of Dietetics and Human Nutrition, Macdonald Campus, McGill University Principal Investigator: Dr. Peter Jones Phone: (514)398-7547,398-7527 Contact Physician: Dr. William Parsons Phone: (514)897-5220 1. NATURE AND DURATION OF PROCEDURE:

The objective of the current study is to compare under controlled conditions the efficacy of plant sterol given once in the morning or 3 times through the day in reducing cholesterol absorption. As part of your pre-screening visit, a fasting blood sample of 20ml will be drawn for laboratory analysis in order to confirm the absence of any health abnormalities and to measure your blood lipid levels. An electrocardiogram may be performed at the discretion of the physician in charge. Prior to the beginning of the study, you will be examined by a physician in order to ensure that you are in good health. You will be asked questions concerning your exercise habits and your general health status. This study requires that you (1), have not consumed any lipid lowering drugs, including fish oils or cholesterol lowering medication within the past 6 months, (2) have not developed thyroid disease, diabetes mellitus or kidney, heart or liver disease (3) do not chronically use fiber or stimulant laxatives (greater than 2 doses/wk). Blood samples will be drawn to determine if you are anemic, or suffer from liver or kidney disease. Your blood lipid levels will be measured. The physician may order an electrocardiogram. Once you start the clinical trial, you will be asked to consume only the test diets as instructed by the study coordinators for 4 periods each of 6 days. Each study period will be separated by a two week period when you will consume your regular diet. The study diets will contain nutritionally adequate foods, be typical in terms of type of food and be fed at an energy level which will maintain your body weight. The 4 phases differ in number of plant sterol enriched margarine doses through the day. During the control phase, no plant sterols are consumed. For the single morning dose phase, 1.8 g/d plant sterol dose will be given at breakfast only. During the 3 dose/d phase, 1.8 g/d plant sterol dose will be divided equally among the 3 meals. For the single evening dose phase, 1.8 g/d plant sterol dose will be given in supper only. You will be requested to consume at least 2 of 3 daily meals under supervision at the Mary Emily Clinical Research Unit. 234 Page 2

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL September 1 ,2005

"INVESTIGATING THE EFFICACY OF PLANT STEROL GIVEN ONCE OR THREE TIMES PER DAY IN LOWERING CHOLESTEROL ABSORPTION IN HYPERCHOLESTEROLEMIC INDIVIDUALS"

Blood samples (20 ml) will be collected daily on the morning of the second day until the sixth day of each phase. To measure lipid metabolism, at the second day of each phase you will be requested to consume a small quantity of tagged cholesterol. The movement of this tagged material will permit assessment of how much cholesterol your body absorbs from the diet. On the second to last day of the study, you will also be requested to consume 25ml of water tagged with deuterium in order to establish the amount of cholesterol being produced inside your body. All of these tags are non­ radioactive and pose no toxicity hazard. Blood samples (20 ml) will be collected daily on the mornings of each of the last 5 days of each phase in order to examine the movement of these tags across body compartments. The total amount of blood drawn during each phase will be 110 ml. If anemia develops you will be given iron supplements to correct the anemia. Subjects with too high cholesterol levels will be referred for treatment after finishing the study.

2. POTENTIAL RISKS AND BENEFITS:

This is not a treatment study, but rather a study to obtain more information on the potential benefit of taking added plant sterols. You will be making a contribution to scientific knowledge which may help others in the future.

There is no risk associated with taking plant sterols. There may be bruising and pain from the blood taking. You may develop mild anemia as previously mentioned, which will be treated with iron supplements.

3. CONFIDENTIALITY:

Confidentiality of records concerning your involvement in this project will be carefully maintained in such a way that your identity and your results will not be disclosed to anyone other than the Principal Investigator and appointed associates. In addition, the McGill Faculty of Medicine Institutional Review Board may have access to these records. Samples will not be utilized for any other analyses, not stored for a long period, nor shared with any other group other than the Principal Investigator and appointed associates, without your specific consent. 235 Page 3 of3 SUBJECT CONSENT FORM OF RESEARCH PROTOCOL September 1 ,2005

"INVESTIGATING THE EFFICACY OF PLANT STEROL GIVEN ONCE OR THREE TIMES PER DAY IN LOWERING CHOLESTEROL ABSORPTION IN HYPERCHOLESTEROLEMIC INDIVIDUALS"

4. CONSENT:

The substance of the study and procedures associated with it has been fully explained to me, and all experimental procedures have been identified. I have had the opportunity to ask questions concerning any and all aspects of the study and procedures involved. I am aware that I may refuse to participate as well as withdraw my consent at any time. I acknowledge that no guarantee or assurance has been given by anyone as to results to be obtained. Confidentiality of records concerning my involvement in this study will be carefully maintained in such a way that my identity and my results will not be disclosed to anyone other than the Principal Investigator and appointed associates and the McGill Faculty of Medicine Institutional Review Board. Samples will not be utilized for any additional analyses, nor stored for any prolonged period, nor shared with any other group, other than the Principal Investigator and appointed associates, without my specific consent.

I, , have read the above description with one of the investigators, I comprehend the procedures, advantages and disadvantages of the study, which have been explained to me. I understand that, in compensation for the inconvenience of the study schedule, I will receive $ 350.1 will also receive access to my test results when they become available. If I decide to withdraw before completion or should the study be terminated early, I will receive an appropriate pro-rated faction of this amount.

I, , the undersigned, hereby consent to participate as a subject in the above named research study conducted by McGill University.

I acknowledge receiving a copy of this consent form and all appropriate attachments and agree to be contacted by a member of the McGill Faculty of Medicine Institutional Review Board, if requested.

Investigator Signature of Subject

Witness

Date Time 236

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL

February 22th, 2005

"Efficacy of Plant Sterol Given at Different Times of the Day in Management of Hypercholesterolemia"

Patient Name: Protocol Number: Commercial Sponsor: Danone Vitapole Group

School of Dietetics and Human Nutrition, Macdonald Campus, McGill University Principal Investigator: Dr. Peter Jones Phone: (514) 398-7547, 398-7527 Contact Physician: Dr. Eric Lillie Phone: (514)457-9355 1. NATURE AND DURATION OF PROCEDURE:

The objective of the current study is to compare the blood cholesterol-lowering efficacy of plant sterols given at different times of the day. Plant sterols are compounds similar to cholesterol; however, unlike cholesterol they are derived from plants.

As part of your screening visit, a fasting blood sample of 20ml will be drawn for laboratory analysis in order to confirm the absence of any health abnormalities and to measure your blood lipid levels.

Prior to the beginning of the study, you will be examined by a physician in order to ensure that you are in good health. You will be asked questions concerning your exercise habits and your general health status. This study requires that you (1) have not consumed any lipid lowering drugs, including fish oils or cholesterol lowering medication within the past 6 months, (2) have not developed thyroid disease, diabetes mellitus or kidney, heart or liver disease (3) do not chronically use fiber or stimulant laxatives (greater than 2 doses/wk) (4) are not allergic to soya. Blood samples will be drawn to determine if you are anemic, or suffer from liver or kidney disease. Your blood lipid levels will be measured. The physician may order an electrocardiogram. 237

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL

February 22th, 2005

"Efficacy of Plant Sterol Given at Different Times of the Day in Management of Hypercholesterolemia"

Once you start the clinical trial, you will be asked to consume only the test meals as instructed by the study coordinators for 4 one month periods, each period separated by one month periods when you will consume your regular diet. The total duration of the study is 7 months. The study meals will contain nutritionally adequate foods, be typical in terms of type of food and be fed at an energy level which will maintain your body weight. The 4 phases (A, B, C and D) differ in time of consuming plant sterol enriched yogurt. Your phase sequence assignment will be randomly assigned. During the control phase, no plant sterols are consumed and yogurt without plant sterols will be provided at breakfast and supper. For the single morning dose phase : 1.6 g/d plant sterol dose will be given in yogurt only consumed with breakfast and a yogurt without plant sterols will be provided at supper. For the single evening dose phase: 1.6 g/d plant sterol dose will be given in yogurt only consumed with the supper and a yogurt without plant sterols will be provided at breakfast. During the afternoon snack dose phase : 1.6 g/d plant sterol dose will be given in yogurt only consumed as a snack 2 hrs prior to supper and a yogurt without plant sterols will be provided at breakfast.

On the first day of each phase you will provide a 20 ml blood sample. To measure lipid metabolism, five days before the end of each phase, you will be requested to consume a small quantity of tagged cholesterol. The movement of this tagged material will permit assessment of how much cholesterol your body absorbs from the diet. On the second to last day of study, you will also be requested to consume 25ml of water tagged with deuterium in order to establish the amount of cholesterol being produced inside your body. All of these tags are non-radioactive and pose no toxicity hazard. Blood samples (20 ml) will be collected daily on the mornings of each of the last 5 days of each phase in order to examine the movement of these tags across body compartments. The total amount of blood drawn during each phase will be 140 ml. In total this will amount to about the same amount as drawn in a blood donation. If anemia develops you will be given iron supplements to correct the anemia.

2. POTENTIAL RISKS AND BENEFITS:

This is not a treatment study, but rather a study to obtain more information on the potential benefit of taking added plant sterols. You will be making a contribution to scientific knowledge which may help others in the future. 238

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL

February 22th, 2005

"Efficacy of Plant Sterol Given at Different Times of the Day in Management of Hypercholesterolemia"

There is no risk associated with taking plant sterols. There may be bruising and pain from the blood taking. You may develop mild anemia as previously mentioned, which will be treated with iron supplements. If during the study you note any unusual symptoms or disturbances, you should inform the study physician, Dr. Eric Lillie or the study principal investigator Dr. Peter Jones.

3. CONFIDENTIALITY:

Confidentiality of records (including computer data) concerning your involvement in this project will be carefully maintained in such a way that your identity and your results will not be disclosed to anyone other than the Principal Investigator and appointed associates. Samples will not be utilized for any other analyses, not stored for a long period, nor shared with any other group than indicated in the protocol, without your specific consent.

4. CONSENT:

The substance of the project and procedures associated with it have been fully explained to me, and all experimental procedures have been identified. I have had the opportunity to ask questions concerning any and all aspects of the project and procedures involved. I am aware that I may refuse to participate as well as withdraw my consent at any time. I acknowledge that no guarantee or assurance has been given by anyone as to results to be obtained. Confidentiality of records concerning my involvement in this project will be carefully maintained in such a way that my identity nor my results will not be disclosed to anyone other than the Principal Investigator and appointed associates. Samples will not be utilized for any additional analyses, nor stored for any prolonged period, nor shared with any other group, other than is indicated in the protocol, without your specific consent.

I, , have read the above description with one of the investigators, I fully understand the procedures, advantages and disadvantages of the study, which have been explained to me. I understand that, in compensation for the inconvenience of the study schedule, I will receive $ 1,500. I will also receive access to my test results when they become available. If I decide to withdraw before completion or should the study be terminated early, I will receive an appropriate pro-rated faction of this amount. 239

SUBJECT CONSENT FORM OF RESEARCH PROTOCOL February 22th, 2005

"Efficacy of Plant Sterol Given at Different Times of the Day in Management of Hypercholesterolemia"

I, , the undersigned, hereby consent to participate as a subject in the above named research project conducted by McGill University.

I agree not to take part in any other studies during the 15 days following completion of the present study.

I acknowledge receiving a copy of this consent form and all appropriate attachments and agree to be contacted by a member of the McGill Faculty of Medicine Institutional Review Board, if requested.

Investigator Signature of Subject

Witness

Date Time Appendix 6.1 Journal waiver for Manuscript 1

.IasonMurrav_LI.M_iirr;iiuf_ifis org] Sunt. Thu 6/28/2007 3:46 AM To: Suhiid Ahiiitwuifr. Mi- Cc: Subject- RR: Permission Attachments:

Dear Suhad

Apologies for the late reply, we've been having a few email problems recently here. I hereby grant you permission to reproduce your article from the Food Science and Technology Bulletin in your doctoral thesis, providing you supply the appropriate citation regarding where it was initially published {Food Science and Technology Bulletin: Functional Foods 2(9): 101—110).

All the best and good luck with your thesis!

Jason

Jason Murray

Web Editor, Food Science Central

Journal Production Editor

Food Manufacturing Efficiency/Food Science & Technology Bulletin

Direct line: +44 (0) 118 984 0439

International Food Information Service (IFIS Publishing),

Lane End House, Shinfield Road, Shinfield, Reading, RG2 9BB, UK.

Telephone: +44 (0) 118 988 3895. Fax +44 (0) 118 988 5065

World Wide Web http://www.foodsciencecentral.com 242

Appendix 6.3 Journal waiver for Review Article 2

Page 1'of 2

Suhad Abumweis, Ms

To... Undurtl Das

Cc... Bcc... Subject: RE: Permission Attachments:

Frtrm Undurti Das [mailto:undutU®lipiawor1d.com] Sent::Tue#/27/2007 M:36 AM .'•"".• To: Subad'AburriwSfe, Ms Subject: .Rs::RerTDisslon

You rr^treproduiftttearticle.h^ RleasenotethaKhisreph^uetl^ permitted only fora^

UndurtiNDas :

On 3/27/05!, Suhad Abumweis, Ms :wote-. : Dear Sir/Madam,

1 am a PhD studentand I amiVJritingtoaskfor-p^rmissibn ^it^^u^UVe^tern^J^'.ar^^-^^wssfiwbJIsh^d^in' Lipids in Health and Disease fbrindusiprim levels and whole body lipid melabolisrri" that willbe published in June 2007,

Contact information:

Suhad AbuMvyeis

: PhD Candidate

v-SshMl4W!#

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https://exchaiige.mcgi]l.ca/exchangG''[email protected]/Drafta'RB:%20Pcr... 3/27/2007 243

Appendix 6.4 Journal waiver for Review Article 3

Page 2 of2

Suhad Abumweis, Ms

To... Downing, Hannah (EL5-OXF)

Cc... Bcc... Subject: RE: Obtain Permission Attachments:

From: Downing, Hannah (ELS-OXF) [mailto:[email protected]] Sent: Thu 5/24/2007 10:23 AM To: Suhad Abumweis, Ms Subject: RE: Obtain Permission

Dear Suhad AbuMweis

We hereby grant you permission to reprint the material detailed below at no Charge in your thesis subject to the following conditions:

1. If any part of the material to be used (for example, figures) has appeared in our publication with credit or acknowledgement to another source, permission must also be sought from that source. If such permission is not obtained then that material may not be included in your publication/copies.

2. Suitable acknowledgment to the source must be made, either as a footnote or in a reference list at the end of your publication, as follows:

"This article was published in Publication title, Vol number, Author(s), Title of article, Page Nos, Copyright Elsevier (or appropriate Society name) (Year)."

3. Your thesis may be submitted to your institution in either print or electronic form.

tafo^fcaflnan^ 3//ZMMXH1 245

Page 3 of3

H3H2N9 ":•.-•• MontreL/QC : Canada-

Contact Details: Telephone: 514932 9353 . : Fax::r»...... V 5143#7?#: -:„: :y,:;=, . Emailirtaress: ; :;gu^

Touse the following1material: ISSN/ISBN: Tltle:;;V "LifeSciences - AUthopfs): ; Sylvia SaHtosa, Krista:A Varady; Suhad^ibuMweis Volume: :-•-: 80 .'" Issuer':." 6;: fear: -.;.: 2007 Pages:::,: 505-S14:; Article title:;. 'Physiological and therapeutic factors affecting

How much of the 'requestedmaterial is to be used::

Are you the author:. Yes AUthorat institute: Yes

How/wherewillthe requested material be used: [how_usedJ

Details:

amaPhp student'andjj am;yyrttin^ in LifeS#nces;fc^:lrkjusion;;in rhyd preparations on plasma lipid levels and whole body; lipldmetabolism" that will be published in June 2007

Additional Info:

-end - '-."'•:

ftrfurther info; regarding: this automatic email, please contact: WEB AiRPLICATloNS TEAiH ( esweb.admin@elsevier,co.uK)

https://exchange.mcgill.ca/cxchange/[email protected]/Drafts/R.E:%20Obta... 5/24/2007