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 (95
45
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)
en en en en
ro ^fe^ Wa* '* en -.MtSP®^ cr> -~j oo co CO N> «-; H: CO CD O > «-•- -*»* C3 —^ IM^^Mjjl en ». oo CO ro o ro ro rO eo ro ro en ro cx> •1 CO C•O =3 =3 CQ CO CD CD m ^ O CD =3 _; =3 C=>O CO 96 97 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) -1 -0.5 0 0.5 1 Favours treatment Favours control H H H -H -I CO Weststra t c_ c_ c_ c_ O O 7J anston e CD A A O O b O o O o O O^O^NJ^OJ^J^h^^MWMLOCOWUJlMa^MWMOMr^MLOWWK^WOWMOMh-'OrO Q. • 2 o CD' II ° 2 O o 3 II I I I I I l I I I I I f I l I I I I I I I I I I l I I I I t [ | ] | | | | (O m "SJOOOOOOOOOOOOOOOOOOOOOO!-1OOOOOOOOOOOOOOOOCD OJ 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CD W '—' CH -J '—' Ol CO O Cn Cn '—' '—' Review: Plant sterols 2007 Comparison: 21 Mayonnaise and salad dressing (All doses ) Outcome: 01 LDL-cholesterol mmol/L 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 ) H -i -i H H CO o CO O O 7S 2 CD CD c /ests t H H CO Z Z Z Z Z Z o Q. 20 0 N> Oo CD CD CD -* CD NJ °> °» rat e 01 CO 3 » S o en O o o 01 3 CD cn cn cn » M3 OMM -* 7T 7T 3' _i ' 01 3 co cn cn CD o^ < CD o CD N> § ->• o o o CD CO Si sral l m er a Is** o o o Cg M ^ S" < CD O N1 M o 2 co N3 O O 3 CO ro cn cn o o o O CD CJ O CO o CO CO S> co O _^ O O M OI N) NO IO > S = o (1 0 N3 CO 01 ffi s s ^ 0) C_O* o O 01 O O CO 11) CO N> -N O CD CD no U M CO ect : o eity : 3 |§ S to (« O =* O ror >£< O CD o N 9. 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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). 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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 Page 1 of 19 (page number not for citation purposes) 192 Lipids in Health and Disease 2004, 3 http://www,lipidvyorld.com/content/3/1/5 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 Page 2 of 19 (page number not for citation purposes) 193 Lipids in Health and Disease2004, 3 http:/Awvswlipidvvorld.corn/content/3/1/5 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% Page3of 19 (page number not for citation purposes) 194 Lipids in Health and Disease 2004, 3 http://vwvVv.lipidvvo rld.com/conten t/3/1/5 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 Page 4 of 19 (page numbernot lor citation purposes) 195 Lipids in Health and Disease 2004, 3 http://WwwJipidworld.eom/content/3/1/5 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. Page 5 of 19 (peg* number not for cHalion purposes) 196 Lipids in Health and Disease 2004, 3 http:/foiww.lipidworld.com/conteny3/1/5 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 Page 6 of 19 (page number not tor citation purposes) 197 Lipids in Health and Disease 2004, 3 http7AAMwJipidworld.com/conten t/3/1/5 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 Page; 7 of 19 (pespnumbernot for citation purposes) 198 Lipids in Health and Disease 2004, 3 http:/tovww.lipidwo rld.com/conten t/3/1/5 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 Page 8 of 19 (page number not for cftaftbn purposes) 199 Lipids in Health andDisease 2004, 3 http:/AAAWW.Iipidworld.com/content/3/1/5 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 Page 9 of 19 (page number not for citation purposes) 200 Lipids in Health and Disease 2004, 3 http://www. I i pidworld. com/content/3/115 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 Page 10 of 19 (page number not for citation purposes) 201 Lipids in Health and Disease 2004, 3 http:/AAiww. 1 ipidworld. com/contenr/3/1 /5 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]. Page 11 of19 (page number not lor citation purposes) 202 Lipids in Health and Disease 2004, 3 http:/Awvw. I i pid world, com/content/3/1 /5 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 ^O CO