regulation of lipoprotein transport in the metabolic syndrome · structure, function and...
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Regulation of Lipoprotein Transport in the
Metabolic Syndrome:
Impact of Statin Therapy
Esther M. M. Ooi
Bachelor of Science (Hons)
This thesis is presented for the degree of
Doctor of Philosophy of Medicine
of the University of Western Australia
School of Medicine and Pharmacology
Royal Perth Hospital
Medicine
2007
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ABSTRACT
The metabolic syndrome is characterized by cardiovascular risk factors including
dyslipidemia, insulin resistance, visceral obesity, hypertension and diabetes. The
dyslipidemia of the metabolic syndrome includes elevated plasma triglyceride and
apolipoprotein (apo) B levels, accumulation of small, dense low-density lipoprotein
(LDL) particles and low high-density lipoprotein (HDL) cholesterol concentration.
However, the precise mechanisms for this dyslipoproteinemia, specifically low plasma
HDL cholesterol, are not well understood. This thesis therefore, focuses on HDL, its
structure, function and metabolism. However, lipoprotein metabolism is a complex
interconnected system, which includes forward and reverse cholesterol transport
pathways. Hence, this thesis also examines and discusses the metabolism of apoB-
containing lipoproteins.
This thesis tests the general hypothesis that apolipoprotein kinetics are altered in the
metabolic syndrome, and that lipid regulating therapies can improve these kinetic
abnormalities. The aims were first, to compare and establish the clinical, metabolic and
kinetic differences between metabolic syndrome and lean subjects; and second, to
determine the regulatory effects of statin therapy, specifically, rosuvastatin on
lipoprotein transport in the metabolic syndrome. Five observation statements were
derived from the general hypothesis and examined in the studies described below. The
findings are presented separately as a series of original publications.
Study 1 Twelve men with the metabolic syndrome and ten lean men were studied in a
case-control setting. The kinetics apoB-100 and HDL apoA-I and apoA-II were
measured using D3-leucine, gas chromatography-mass spectrometry and
multicompartmental modeling. The kinetics of HDL subpopulations LpA-I and LpA-
I:A-II were also examined using a new compartment model. Compared with lean men,
men with the metabolic syndrome had higher concentrations of very-low-density
lipoprotein (VLDL), intermediate-density lipoprotein (IDL) and LDL-apoB (+78%,
+57% and +59%, respectively, p
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-17%, respectively, p
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dose-dependent reductions in LpA-I FCR, with no changes in LpA-I PR. These findings
explain the HDL raising effects of rosuvastatin in the metabolic syndrome.
Collectively, these studies suggest that the dyslipidemia of the metabolic syndrome
results from increased production rates of VLDL and LDL particles, reduced fractional
catabolic rates of these lipoproteins, together with accelerated catabolism of HDL
particles. Treatment with rosuvastatin increases the catabolic rates of all apoB-
containing lipoproteins and at a higher dose, decreases LDL apoB production. These
effects are consistent with inhibition of cholesterol synthesis leading to an upregulation
of LDL receptors. Rosuvastatin decreases the fractional catabolism of HDL particles.
The effects of rosuvastatin on HDL kinetics may be related to a reduction in triglyceride
concentration and cholesterol ester transfer protein activity. These findings are
consistent with the general hypothesis that apolipoprotein kinetics are altered in the
metabolic syndrome, and that statin therapy improves these kinetic abnormalities.
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TABLE OF CONTENT
ABSTRACT 2
TABLE OF CONTENTS 5
LIST OF TABLES 14
LIST OF FIGURES 17
LIST OF ABBREVIATIONS 21
PERSONAL CONTRIBUTION BY THE CANDIDATE 25
PUBLICATIONS AND COMMUNICATIONS 28
ACKNOWLEDGMENTS 33
CHAPTER 1 LITERATURE REVIEW 35
1.1 Overview of the Metabolic Syndrome 36
1.1.1 Definition of the metabolic syndrome 36
1.1.2 Prevalence of the metabolic syndrome 37
1.1.3 Components of the metabolic syndrome 40
1.1.3.1 Insulin resistance 40
1.1.3.2 Obesity and insulin resistance 41
1.1.3.3 Dyslipidemia and insulin resistance 42
1.1.3.4 Hypertension and insulin resistance 42
1.1.3.5 Inflammation, prothrombosis and insulin resistance 43
1.1.4 Significance of the metabolic syndrome 44
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1.1.4.1 Association between the metabolic syndrome and cardiovascular
disease 44
1.1.4.2 Association between the metabolic syndrome and diabetes 46
1.1.5 Prevention and management of the metabolic syndrome 46
1.1.5.1 Therapeutic lifestyle intervention 47
1.1.5.2 Pharmacotherapy 47
1.2 Lipoprotein metabolism 50
1.2.1 Definition and classification of lipids 50
1.2.2 Lipoprotein metabolism 50
1.2.3 The lipoproteins 52
1.2.3.1 Chylomicrons 52
1.2.3.2 Very low density lipoprotein 52
1.2.3.3 Intermediate density lipoprotein 54
1.2.4.4 Low density lipoprotein 54
1.2.2.5 High density lipoprotein 56
1.2.2.6 Lipoprotein (a) 60
1.2.4 Apolipoproteins 62
1.2.4.1 Apolipoprotein A-I 62
1.2.4.1 a Structure and composition 62
1.2.4.1 b Metabolism 63
1.2.4.1 c Biological role and significance 64
1.2.4.1 d Genetics 65
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1.2.4.2 Apolipoprotein A-II 66
1.2.4.2 a Structure and composition 66
1.2.4.2 b Metabolism 67
1.2.4.2 c Biological role and significance 67
1.2.4.2 d Genetics 68
1.2.4.3 Apolipoprotein A-IV 69
1.2.4.4 Apolipoprotein A-V 69
1.2.4.5 Apolipoprotein B 70
1.2.4.5 a Structure and composition 70
1.2.4.5 b Metabolism 72
1.2.4.5 c Biological role and significance 73
1.2.4.5 d Genetics 74
1.2.4.6 Apolipoprotein C 75
1.2.4.6 a ApoC-I 75
1.2.4.6 b ApoC-II 75
1.2.4.6 c ApoC-III 76
1.2.4.7 Apolipoprotein E 77
1.2.4.8 Apolipoprotein (a) 78
1.2.5 Receptors, transport proteins, enzymes and transfer proteins 81
1.2.5.1 Receptors and transport proteins 81
1.2.5.1 a LDL receptor 81
1.2.5.1 b LDL receptor related protein 82
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1.2.5.1 c Scavenger Receptor Class B Type 1 83
1.2.5.1 d ATP binding cassette transporters 83
1.2.5.2 Enzymes 84
1.2.5.2 a Lipoprotein lipase 84
1.2.5.2 b Hepatic lipase 85
1.2.5.3 c Endothelial lipase 86
1.2.5.3 Cholesterol esterification and lipid transfer protein 86
1.2.5.3 a Lecithin-cholesterol acyltransferase 86
1.2.5.3 b Cholesteryl ester transfer protein 87
1.2.5.3 c Phospholipid transfer protein 88
1.3 Management of dyslipidemia 90
1.3.1 Lipid regulating drugs 91
1.3.2 HMG CoA reductase inhibitors 93
1.3.2.1 Overview 93
1.3.2.2 Rosuvastatin 95
1.4 Kinetic Studies and Lipoprotein Metabolism 103
1.4.1 Background 103
1.4.2 Principles of stable isotope methodology 104
1.4.3 Laboratory and mathematical methods of stable isotope studies 106
1.4.3.1 Tracer administration 106
1.4.3.2 Laboratory methodology 106
1.4.3.3 Compartmental modeling 106
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1.4.3.4 Definitions in kinetic analysis 108
1.4.4 Overview of lipoprotein kinetic studies 109
1.4.4.1 Kinetic studies of apoB 109
1.4.4.1 a Obesity and apoB kinetics 109
1.4.4.1 b Nutritional interventions on apoB kinetics 111
1.4.4.1.c Effects of statins on apoB kinetics 111
1.4.4.2 Kinetic studies of apoA-I and apoA-II 115
1.4.4.2 a ApoA-I and apoA-II kinetics in normolipidemic and
insulin resistant subjects 115
1.4.4.2 b Genetic disease and apoA-I and apoA-II kinetics 116
1.4.4.2 c Nutritional and hormonal interventions on apoA-I and
apoA-II kinetics 119
1.4.4.2 d Effect of statins on apoA-I and apoA-II kinetics 119
1.4.4.3 Kinetic studies of other apolipoproteins and lipids 122
1.4.4.3 a Kinetic studies of other apolipoproteins 122
1.4.4.3 b Kinetic studies of triglycerides and cholesterol 122
CHAPTER 2 HYPOTHESIS, AIMS AND STUDY DESIGN 124
2.1 Philosophical Perspective 125
2.2 Hypothesis 125
2.3 General Hypothesis 127
2.3.1 Study 1: Apolipoprotein B-100 and A-I Kinetics in the Metabolic Syndrome
127
2.3.2 Study 2: High-Density Lipoprotein Apolipoprotein A-I Kinetics in Obesity
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128
2.3.3 Study 3: Plasma Phospholipid Transfer Protein Activity, a Determinant of
HDL Kinetics In Vivo 128
2.3.4 Study 4: Dose-ranging Effect of Rosuvastatin on Apolipoprotein B-100
Kinetics in the Metabolic Syndrome 129
2.3.5 Study 5: Dose-Dependent Improvement in High-Density Lipoprotein
Metabolism with Rosuvastatin in the Metabolic Syndrome 130
CHAPTER 3 STUDY DESIGN, SAMPLE SIZE AND POWER CALCULATIONS
131
3.1 Overview of Studies 132
3.2 Studies Details 132
3.3 Sample Size and Power Calculations 134
CHAPTER 4 MATERIALS AND METHODS 143
4.1 Subjects 144
4.2 Method of recruitment 147
4.3 Protocol for screening 148
4.4 Anthropometric measurements 149
4.5 Blood pressure 149
4.6 Urinalysis 150
4.7 Assessment of nutrient intake 150
4.8 Protocol for blood sampling 150
4.9 Preparation of D3-leucine solution 151
4.10 Stable Isotope Injection Protocol 151
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4.11 Collection and storage of buffy coat (white cells) protocol 152
4.12 Collection and Storage of Cholesterol Efflux and Preβ 152
4.13 Collection of samples for genetic analysis 153
4.14 Biochemical assays 153
4.15 Kinetic Analyses 162
4.15.1 Isolation and Measurement of Isotopic Enrichment of Apolipoproteins
and Plasma Leucine 162
4.15.1.1 ApoB-100 165
4.15.1.2 Apolipoprotein A-I and Apolipoprotein A-II 166
4.15.1.3 Plasma Leucine (Oxazolinone Method) 167
4.15.2 Derivatization of leucine 167
VLDL, IDL, LDL-apoB, HDL-apoA-I, apoA-II and plasma leucine eluate
4.16 Mathematical modeling 168
4.16.1 Model for VLDL, IDL and LDL-apoB 168
4.16.2 Model for apoA-I in HDL LpA-I and LpA-I:A-II particles and apoA-II
173
4.17 Statistical analyses 178
4.17.1 Measures of variability 178
4.17.2 Coefficient of variation 178
4.17.3 Distribution of data 179
4.17.4 Independent t-test 179
4.17.5 Correlational analyses 180
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4.17.6 Regression analyses 181
4.17.7 The mixed model: an extension of the general linear model 184
CHAPTER 5 Apolipoprotein B-100 and A-I Kinetics in the Metabolic Syndrome 187
CHAPTER 6 High-Density Lipoprotein Apolipoprotein A-I Kinetics in Obesity 203
CHAPTER 7 Plasma Phospholipid Transfer Protein Activity, a Determinant of HDL
Kinetics In Vivo 213
CHAPTER 8 Dose-ranging Effect of Rosuvastatin on Apolipoprotein B-100 Kinetics in
the Metabolic Syndrome 222
CHAPTER 9 Dose-Dependent Improvement in High-Density Lipoprotein Metabolism
with Rosuvastatin in the Metabolic Syndrome 245
CHAPTER 10 Conclusion: Overview, Limitations, Implications and Future Research
267
10.1 Overview 268
10.2 Study Limitations 274
10.3 Implications 276
10.3.1 Dyslipidemia, metabolic syndrome and cardiovascular disease 276
10.3.2 Management and treatment of dyslipidemia in the metabolic syndrome
277
10.4 Synthesis: Perspectives for Future Research 278
10.4.1 Study population 278
10.4.2 Lipid substrate availability 278
10.4.3 VLDL and LDL subspecies 279
10.4.4 HDL subspecies 279
10.4.5 Cholesterol efflux 279
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10.4.6 ApoC-III, apoA-IV, apoA-V and apoE 280
10.4.7 Genetic polymorphisms 280
10.4.8 Other interventions 280
10.5 Conclusion 286
REFERENCES 287
APPENDICES
Appendix 1 High-Density Lipoprotein Apolipoprotein A-I Kinetics: Comparison of
Radioactive and Stable Isotope Studies 344
Appendix 2 High-Density Lipoprotein (HDL) Transport in the Metabolic Syndrome:
Application of a New Model for HDL Particle Kinetics 352
Appendix 3 Relationships between Plasma Lipids Transfer Proteins and Apolipoprotein B-
100 Kinetics during Fenofibrate Treatment in the Metabolic Syndrome 360
Appendix 4 Fish oils, phytosterols and weight loss in the regulation of lipoprotein transport
in the metabolic syndrome: lessons from stable isotope tracer studies 368
Appendix 5 Dietary Plant Sterols Supplementation Does Not Alter Lipoprotein Kinetics in
Men with the Metabolic Syndrome 375
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LIST OF TABLES
Chapter 1
Table 1.1 Clinical definitions of the metabolic syndrome 38
Table 1.2 Therapeutic interventions in the metabolic syndrome 49
Table 1.3 Classification and properties of major human plasma
lipoproteins
61
Table 1.4 Characteristics of the major apolipoproteins 79
Table 1.5 Lipid regulating drugs 92
Table 1.6 LDL reduction by percentage change according to statin and
daily dose (summary estimates from 164 randomized placebo
controlled trials)
94
Table 1.7 Summary of clinical efficacy studies with rosuvastatin 98
Table 1.8 Studies of apoB kinetics in normolipidemic and obese subjects 110
Table 1.9 Effects of statins on apoB kinetics 113
Table 1.10 Studies of apoA-I and apoA-II kinetics in normolipidemic and
insulin resistant subjects
117
Table 1.11 Effects of statins on apoA-I and apoA-II kinetics 121
Chapter 5
Table 1 Clinical and biochemical characteristics of the subjects with the
metabolic syndrome and the lean subjects
200
Table 2 Kinetic characteristics of VLDL, IDL and LDL-apoB in
subjects with the metabolic syndrome and lean subjects
201
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Table 3 Kinetic characteristics of LpA-I, LpA-I:A-II, apoA-II and
plasma apoA-I in subjects with the metabolic syndrome and
lean subjects
202
Chapter 6
Table 1 Stable isotope studies included in the summary analysis 206
Table 2 Clinical and biochemical characteristics of lean and
overweight-obese subjects
206
Table 3 Associations of HDL apoA-I FCR and PR with other variables
in lean subjects
207
Table 4 Associations of HDL apoA-I FCR and PR with other variables
in overweight-obese subjects
208
Table 5 Associations of HDL apoA-I FCR and PR with other variables
from data pooled from both lean and overweight-obese group
209
Table 6 Multiple regression model of age, sex, BMI, triglycerides, and
HOMA score as predictors of HDL apoA-I FCR in pooled data
from lean and overweight-obese subjects
209
Table 7 Multiple regression model of age, sex, BMI, triglycerides, and
HOMA score as predictors of HDL apoA-I PR in pooled data
from lean and overweight-obese subjects
210
Chapter 7
Table 1 Clinical and biochemical characteristics of participants studied 216
Table 2 HDL parameters of the participants studied 217
Table 3 Multivariate regression analysis of the relationship between
HOMA score and plasma PLTP activity adjusting for age,
217
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triglycerides, waist circumference and CETP activity
Table 4 Associations (Pearson correlation coefficients) of plasma PLTP
activity with clinical and biochemical characteristics of
participants studied
217
Table 5 Associations between plasma PLTP activity and HDL
parameters
217
Table 6 Multivariate regression analyses of the relationship between
plasma PLTP activity and LpA-I concentration, LpA-I
production rate and LpA-I fractional catabolic rate, adjusting
for HOMA score, triglycerides and CETP activity
218
Chapter 8
Table 1 Plasma lipids, lipoproteins and apolipoproteins in placebo, 10
mg/day rosuvastatin and 40 mg/day rosuvastatin
239
Table 2 Body weight, blood pressure, measure of insulin resistance and
nutrient intake on placebo and rosuvastatin treatments
240
Table 3 VLDL, IDL and LDL apoB-100 kinetics in placebo, 10 mg/day
rosuvastatin and 40 mg/day rosuvastatin
241
Chapter 9
Table 1 Plasma lipids and apolipoproteins during treatment with
placebo, 10 mg/day rosuvastatin and 40 mg/day rosuvastatin
261
Table 2 ApoA-I, apoA-II, LpA-I and LpA-I:A-II kinetic parameters
during placebo and rosuvastatin treatments
262
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LIST OF FIGURES
Chapter 1
Figure 1.1 Insulin resistance, the metabolic syndrome, and cardiovascular
disease risk.
44
Figure 1.2 Typical Structure of a lipoprotein particle 51
Figure 1.3 Schematic diagram of lipid-free apoA-I 62
Figure 1.4 Role of apoA-I in the reverse cholesterol transport pathway 65
Figure 1.5 Schematic representation of the apo A-II gene, APOA2 68
Figure 1.6 Schematic diagram of the structure of apoB-100 on the surface
of LDL.
71
Figure 1.7 Schematic diagram of the pentapartite structural model, NH3-
ß 1-ß1- 2-ß2- 3-COOH, for apoB-100
71
Figure 1.8 Schematic representation of the pathway for assembly and
secretion of hepatic apoB containing lipoproteins
73
Figure 1.9 Independent role of apoB and apoA-I in predicting fatal
myocardial infarction in men.
74
Figure 1.10 Model of human apoE 77
Figure 1.11 Overview of lipoprotein metabolism 80
Figure 1.12 Schematic illustrating the uptake of lipoprotein particles by the
LDLR
82
Figure 1.13 A schematic representation of the structural model of human
PLTP
89
Figure 1.14 Cholesterol biosynthesis pathway 93
Figure 1.15 Molecular structure of rosuvastatin 95
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Figure 1.16 The balancing feedback loop 103
Figure 1.17 Principle of tracer methodology 105
Chapter 2
Figure 2.1 Philosophical construct employed in this thesis 126
Chapter 4
Figure 4.1 Quantification of LpA-I concentration by differential
electroimmunoassay using Hydragel LpA-I kit
159
Figure 4.2 Derivatization of leucine with TFA/TFAA 168
Figure 4.3 Compartmental model describing apoB tracer kinetics 172
Figure 4.4 Isotopic enrichment of VLDL, IDL and LDL-apoB in a
representative metabolic syndrome subject
173
Figure 4.5 Compartment model describing apoA-I in LpA-I and LpA-I:A-
II particles, apoA-II and apoA-I tracer kinetics.
176
Figure 4.6 Isotopic enrichment for apoA-I in LpA-I and LpA-I:A-II
particles, apoA-II and apoA-I with D3-leucine in a
representative subject.
177
Chapter 6
Figure 1 Association between HDL apoA-I FCR with apoA-I
concentration in lean and overweight-obese subjects
208
Figure 2 Association between HDL apoA-I PR with apoA-I
concentration in lean and overweight-obese subjects
208
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Figure 3 Association between HDL apoA-I FCR and estimated HDL
particle size (HDL cholesterol:apoA-I ratio) for data pooled
from lean and overweight-obese groups
209
Chapter 7
Figure 1 Compartmental model describing apoA-I in LpA-I and LpA-
I:A-II particles, apoA-II and apoA-I tracer kinetics
216
Figure 2 Associations between plasma PLTP activity and (a) LpA-I
concentration, (b) LpA-I production rate, and (c) LpA-I
fractional catabolic rate
218
Figure 3 A proposed model of the relationships between insulin
resistance, PLTP and LpA-I kinetics in vivo
219
Chapter 8
Figure 1 Isotopic enrichment for VLDL (A), IDL (B) and LDL (C) apoB
with D3-leucine in a representative subject on placebo,
rosuvastatin 10 mg/day and rosuvastatin 40 mg/day
242
Figure 2 Fractional catabolic rates of VLDL (A), IDL (B) and LDL (C)
apoB on placebo, 10 mg rosuvastatin and 40 mg rosuvastatin
243
Figure 3 Associations between changes (Δ) in VLDL apoB FCR and
apoC-III concentration (A) and LDL apoB FCR and
lathosterol:cholesterol ratio (B) on 40mg/day rosuvastatin
relative to placebo
244
Chapter 9
Figure 1 Compartment model describing apoA-I in LpA-I and LpA-I:A-
II particles, apoA-II and apoA-I tracer kinetics.
264
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Figure 2 Isotopic enrichment for HDL apoA-I (A) and apoA-II (B) with
D3-leucine in a representative subject on placebo, rosuvastatin
10 mg/day and rosuvastatin 40 mg/day
265
Figure 3 HDL particles fractional catabolic rate (FCR) (A) and
production rate (PR) (B) on placebo, 10 mg rosuvastatin and 40
mg rosuvastatin treatment
266
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS
The following abbreviations and special terms are used in this thesis Abbreviation or special term Definition
ABC ATP-binding-cassette transporter
ACAT Acyl:cholesterol acyltransferase
ACEI Angiotensin Converting Enzyme Inhibitor
AF/Tex CAPS Ait Force/Texas Coronary Atheroma Prevention Study
ALT Alanine aminotrasferase
AP Alkaline phosphatase
Apo Apolipoprotein
AST Aspartate aminotransferase
ATP Adenosine triphosphate
ATP III Adult Treatment Panel III
ANOVA Analysis of variance
BMI Body mass index
BP Blood pressure
CAD Coronary artery disease
CARE Cholesterol and Recurrent Events
CB1 Cannabinoid 1
CE Cholesteryl ester
CETP Cholesteryl ester transfer protein
CK Creatine kinase
CHD Coronary heart disease
CI Confidence Interval
CM Chylomicrons
CR Chylomicron remnants
CRP C-reactive protein
CV Coefficient of variation
CVD Cardiovascular disease
DECODE European Diabetes Epidemiology Group
DGAT Diacylglycerol acyl transferase
ECG Electrocardiogram
EDTA Ethylene diamine tetra acetic acid
EL Endothelial Lipase
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Abbreviation or special term Definition
ER Endoplasmic reticulum
FBE Full blood examination
FC Free cholesterol
FCR Fractional Catabolic Rate
FDB Familial defective apoB-100
FFA Free fatty acids
FH Familial hypercholesterolemia
FHA Familial hyperalphalipoproteinemia
FM Fat mass
FFM Free fat mass
FSR Fractional secretion or synthesis rate
GCMS Gas Chromatography - Mass Spectrometry
GLM General Linear Modeling
HBL Hypobetalipoproteinemia
HDL High-density lipoprotein
HL Hepatic lipase
HMG-CoA 3-Hydroxy-3-methylglutaryl-coenzyme A
HOMA Homeostasis model assessment
HPS Heart Protection Study
HSPG Heparan sulphate proteoglycans
IDF International Diabetes Federation
IDL Intermediate-density lipoprotein
IFG Impaired fasting glucose
IGT Impaired glucose tolerance
IL Interleukin
LCAT Lecithin:cholesterol acyltransferase
LDL Low-density lipoprotein
LDLR Low-density lipoprotein receptor
LFT Liver function test
LIPID Lipid Intervention with Pravastatin in Ischemic Disease
Lp Lipoprotein
LPL Lipoprotein lipase
LRP LDL receptor-related protein
LXR-α Liver X receptor
mg Milligram
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Abbreviation or special term Definition
MIXED Linear mixed-effects model
ML Maximum likelihood
mmol/L Millimoles per litre
mRNA Messenger ribonucleic acid
MTP Microsomal triglyceride transfer protein
NATA National Association of Testing Authorities
NCEP National Cholesterol Education Program
NCI Negative ion chemical ionisation
NEFA Nonesterified fatty acids
NHMRC National Health and Medical Research Council
NIDDM Non-insulin dependent diabetes mellitus
NPC1L1 Niemann-Pick C1 Like 1
PAI Plasminogen activator inhibitor
PBMC Peripheral blood mononuclear cells
PLTP Phospholipid transfer protein
PPAR Peroxisome proliferator-activated receptor
PR Production rate
preβ HDL Preβ subfraction of HDL (high density lipoprotein)
PRIME Prospective Epidemiological Study of Myocardial Infraction
PVD Peripheral vascular disease
RAP Receptor associated protein
RCT Reverse cholesterol transport
REML Restricted maximum likelihood
RIO Rimonabant in Obesity
rpm Revolutions per minute
RT Residence time
SAAM II Simulation, Analysis and Modelling Software II
SAE Serious adverse event
SD Standard deviation
SEM Standard error of the mean
4S Scandinavian Simvastatin Survival Study
SR Secretion or synthesis rate
SR-B1 Scavenger Receptor B1
SREBP Sterol regulatory element binding proteins
T4 Thyroxine
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Abbreviation or special term Definition
TC Total cholesterol
TFA Trifluoroacetic acid
TFAA Trifluoroacetic anhydride
TFT Thyroid function test
TG Triglyceride
TNF-α Tumour necrosis factor alpha
TNT Treat to New Targets
TRL Triglyceride-rich lipoprotein
TSH Thyroid stimulating hormone
TZD Thiazolidinedione
UKPDS UK Prospective Diabetes Study
ULN Upper limit of normal
VA-HIT Veterans Administration-High-Density Lipoprotein Intervention Trial
VLDL Very-low-density lipoprotein
WHO World Health Organisation
WOSCOPS West of Scotland Coronary Prevention Study
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PERSONAL CONTRIBUTION BY THE CANDIDATE
My personal contributions to the thesis are outlined as follows. Contributions of other
individuals and organizations to the thesis are also acknowledged.
Clinical
I had the pleasure of performing the following aspects of the studies:
• Ethics preparation and write-up
• Preparation of Patient Information Sheet and Consent Form
• Clinical Study Protocol Crestor Trial 4522AS/0004
(with Dr Fiona Dunagan and Ms JoAnn Lyons of AstraZeneca Pty Ltd and
Professor Gerald Watts of School of Medicine and Pharmacology, Royal Perth
Hospital)
• Volunteer Recruitment
(with Research nurses, Ms Mary-Ann Powell, Ms Clare Haworth, Ms Sandy
Hamilton, Ms Estelle Zecchin, Ms Michelle Murphy and Professor Gerald
Watts)
• Participated in screening visits
• Participated in stable isotope studies
• Maintained patient records
• Analysis of food and exercise diaries
(with dietician, Ms Nicky Campbell, Ms Clare Jurczyk and Ms Amina
Currimbhoy)
Research nurses, Ms Mary-Ann Powell, Ms Clare Haworth, Ms Sandra Hamilton, Ms
Estelle Zecchin and Ms Michelle Murphy assisted in the recruitment of volunteers, the
measurement of anthropometric parameters (weight, height and waist circumference)
and provided excellent clinical expertise during the stable isotope injection visits. They
also counseled and informed subjects on health and weight reduction. All clinical
examinations, patient reviews and follow-up were performed by Professor Gerald Watts
and Dr Gerard Chew. Associate Professor Frank van Bockxmeer (PathWest Laboratory
Medicine, Royal Perth Hospital) performed the apoE genotyping for the determination
of the apoE2/E2 genotype. The Department of Pharmacy (Royal Perth Hospital)
prepared and dispensed D3-leucine and study medications.
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Laboratory Analyses
I was responsible in carrying-out the following laboratory procedures:
• Collection and separation of plasma from subjects by centrifugation
• Isolation and storage of buffy coat from subjects
• Isolation of lipoprotein fractions VLDL, IDL, LDL and HDL by
ultracentrifugation (density gradient or use of heparin manganese treated
plasma)
• Delipidation and hydrolysis of apoB-100
• Isolation of HDL apoA-I and apoA-II by polyacrylamide gel and western
blotting
• Hydrolysis of HDL apoA-I and apoA-II
• Extraction of leucine by ion exchange chromatography
• Measuring plasma leucine, apoB-100, apo A-I and apo A-II enrichment with gas
chromatography-mass spectrometry (GCMS)
• Quantification of VLDL, IDL and LDL-apoB using the Lowry Method
• Quantification of LpA-I concentration by differential electroimmunoassay
• Isolation and snap-freezing of streptokinase treated plasma for cholesterol efflux
and preβ-HDL
Mr Kevin Dwyer, with the assistance of Dr Dick Chan, set-up the method for the
measurement of isotopic enrichment by GCMS at the School of Medicine and
Pharmacology, Royal Perth Hospital and verified (QC) all data acquired to ensure
authenticity. Ms Jock Ian Foo and Dr Juying Ji assisted with laboratory procedures of
studies. All biochemical analyses were carried out by NATA accredited laboratories at
the Division of Laboratory Medicine (PathWest Laboratory Medicine, Royal Perth
Hospital). Lipid transfer protein measurements were carried out by Professor Paul
Nestel, Dr Dmitri Sviridov and Ms Anh Hoang at the Baker Heart Research Institute,
Melbourne and Associate Professor Kerry-Anne Rye at the Heart Research Institute,
Sydney. I am familiar with the techniques applied in each these various procedures.
Data collection
Drs Maryam Farvid, Simon Zilko and Michael Allen collected data for the pooled
analyses in Chapter 6 and Appendix 1.
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Kinetic Analyses
I was responsible for the determination of the kinetics (tracer/tracee ratio, pool size and
production and fractional catabolic rates) for VLDL, IDL and LDL-apoB-100 and
apoA-I in LpA-I and LpA-I:A-II and apoA-II using multicompartmental modeling.
Professor P Hugh R Barrett (School of Medicine and Pharmacology, Royal Perth
Hospital) advised on the modeling of the kinetic data.
Database
I am personally responsible for the management of all laboratory databases. All
databases were password coded and accessible by authorized persons only. The research
nurse attached to the study and I, were responsible for the management of patient
database (personal details and patient records).
Statistical Analyses
I performed all statistical analyses using the statistical package SPSS 12.0 (Chicago,
Illinois, USA). Expert statistical advice and statistical verification (QC) was provided
by Dr Valerie Burke (Senior Research Officer, School of Medicine and Pharmacology,
Royal Perth Hospital). Statistical analyses using SAS (SAS Proc Mixed, SAS Institute)
were carried out by Dr Valerie Burke.
Publications
I conducted literature reviews and drafted all publications presented in this thesis
(Chapters 5, 6, 7, 8 and 9 and Appendix 1 and 5) including write-up, data presentation
of tables and production of figures. Revisions of drafts were performed following
comments and suggestions from co-authors. In the manuscripts included appendices 2, 3
and 4, I contributed to the clinical, laboratory and statistical analyses of the data.
Written declaration to support these contributions can be provided if required.
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PUBLICATIONS AND COMMUNICATIONS
Publications arising from research conducted for this thesis at the time of submission
include:
Papers
1. Ooi EMM, Watts GF, Farvid MS, Chan DC, Allen MC, Zilko SR, Barrett PHR.
High-Density Lipoprotein Apolipoprotein A-I Kinetics in Obesity. Obesity
Research 2005; 13: 1008 – 1016 (Chapter 6)
2. Ooi EMM, Watts GF, Ji J, Rye KA, Johnson AG, Chan DC, Barrett PHR.
Phospholipid Transfer Protein Activity, a Determinant of HDL Kinetics In Vivo.
Clinical Endocrinology 2006; 65: 752 – 759 (Chapter 7)
3. Ooi EMM, Watts GF, Farvid MS, Chan DC, Allen MC, Zilko SR, Barrett PHR.
High-Density Lipoprotein Apolipoprotein A-I Kinetics: Comparison of
Radioactive and Stable Isotope Studies. European Journal of Clinical
investigation 2006; 36: 626 – 632 (Appendix 1)
4. Ji J, Watts GF, Johnson AG, Chan DC, Ooi EMM, Rye KA, Serone AP, Barrett
PHR. High-Density Lipoprotein (HDL) Transport in the Metabolic Syndrome:
Application of a New Model for HDL Particle Kinetics. Journal of Clinical
Endocrinology and Metabolism 2006; 91: 973 – 979 (Appendix 2)
5. Watts GF, Ji J, Chan DC, Ooi EMM, Johnson AG, Rye KA, Barrett PHR.
Relationships between Plasma Lipids Transfer Proteins and Apolipoprotein B-
100 Kinetics during Fenofibrate Treatment in the Metabolic Syndrome. Clinical
Science 2006; 111: 193 – 199 (Appendix 3)
6. Watts GF, Chan DC, Ooi EMM, Nestel PJ, Beilin LJ and Barrett PHR. Fish oils,
phytosterols and weight loss in the regulation of lipoprotein transport in the
metabolic syndrome: lessons from stable isotope tracer studies. Clinical and
Experimental Pharmacology and Physiology 2006; 33: 877 -882 (Appendix 4)
7. Ooi EMM, Barrett PHR, Chan DC, Nestel PN, Watts GF. Dose-Dependent
Effect of Rosuvastatin on Apolipoprotein B-100 Kinetics in the Metabolic
Syndrome (Chapter 8, accepted for publication in Atherosclerosis)
8. Ooi EMM, Watts GF, Barrett PHR, Chan DC, Ji J, Clifton PM, Nestel PJ.
Dietary Plant Sterols Supplementation Does Not Alter Lipoprotein Kinetics in
28
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Men with the Metabolic Syndrome (Appendix 5, accepted for publication in
Asia Pacific Journal of Clinical Nutrition)
Manuscripts submitted
9. Ooi EMM, Watts GF, Nestel PN, Sviridov D, Hoang A, Barrett PHR. Dose-
Dependent Improvement of High Density Lipoprotein Metabolism with
Rosuvastatin in the Metabolic Syndrome (Chapter 9)
Published Abstracts
1. Ooi EMM, Watts GF, Nestel PN, Sviridov D, Barrett PHR. Dose-Dependent
Effect of Rosuvastatin on High Density Lipoprotein Kinetics in the Metabolic
Syndrome (American College of Cardiology 56th Annual Scientific Session,
New Orleans, Louisiana, USA)
2. Ooi EMM, Watts GF, Ji J, Rye KA, Johnson AG, Chan DC, Barrett PHR Role
of Plasma Phospholipid Transfer Protein Activity in Determining HDL Kinetics
In Vivo, XIV International Symposia of Atherosclerosis, Rome Italy, 2006
(Selected for Young Investigators Award)
3. Ooi EMM, Ji J, Watts GF, Rye KA, Johnson AG, Chan DC, Barrett PHR
Balancing Feedback and Lipoprotein Metabolism in the Metabolic Syndrome,
XIV International Symposia of Atherosclerosis, Rome Italy, 2006 (Selected for
Young Investigators Award)
4. Ji J, Watts GF, Johnson AG, Chan DC, Ooi EMM, Rye KA, Barrett PHR. High-
density lipoprotein transport in the metabolic syndrome: application of a new
model for HDL particle kinetics, XIV International Symposia of
Atherosclerosis, Rome Italy, 2006
5. Ooi EMM, Watts GF, Chan DC, Barrett PHR. HDL kinetics in overweight-
obese subjects with stable isotopy: Relative significance of catabolism and
production in determining apolipoprotein AI plasma concentration. Arterio
Thromb Vasc Biol 2005; 25: e74.
6. Ooi EMM, Watts GF, Barrett PHR, Clifton PM, Nestel PJ. Effects of
phytosterols on lipoprotein metabolism in subjects with the metabolic syndrome.
Arterio Thromb Vasc Biol 2005; 25: e75.
7. Ooi EMM, Watts GF, Chan DC, Barrett PHR. HDL kinetics in overweight-
obese subjects with stable isotopy: Relative significance of catabolism and
production in determining apolipoprotein AI plasma concentration.
29
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Atherosclerosis Supplement 23-26 April 2005, 6(1S) (Selected for Young
Investigators)
8. Ooi EMM, Watts GF, Barrett PHR, Clifton PM, Nestel PJ. Effects of
phytosterols on lipoprotein metabolism in subjects with the metabolic syndrome.
Atherosclerosis Supplement 23-26 April 2005, 6(1S)
Conference Abstracts
1. Ooi EMM, Barrett PHR, Chan DC, Nestel PN, Watts GF. Dose-Dependent
Effect of Rosuvastatin on Apolipoprotein B Kinetics in the Metabolic
Syndrome, Australian Atherosclerosis Society Meeting, Couran Cove,
Queensland (2006)
2. Ooi EMM, Watts GF, Nestel PN, Sviridov D, Barrett PHR. Dose-Dependent
Effect of Rosuvastatin on High Density Lipoprotein Kinetics in the Metabolic
Syndrome, Australian Atherosclerosis Society Meeting, Couran Cove,
Queensland (2006)
3. Ooi EMM, Barrett PHR, Chan DC, Nestel PN, Watts GF. Dose-Dependent
Effect of Rosuvastatin (CRESTOR™) on Apolipoprotein B Kinetics in the
Metabolic Syndrome, School of Medicine and Pharmacology Research
Showcase, University Club (2006)
4. Ooi EMM, Watts GF, Ji J, Rye KA, Johnson AG, Chan DC, Barrett PHR. PLTP
Activity: A determinant of LpA-I Kinetics (Selected for Young Investigators
Competition), Australian Atherosclerosis Society Meeting, Darwin, Northern
Territory (2005)
5. Ooi EMM, Watts GF, Barrett PHR, Clifton PM, Nestel PJ. Effects of
Phytosterols on Lipoprotein Metabolism in Subjects with the Metabolic
Syndrome, School of Medicine and Pharmacology Research Showcase,
University Club, UWA (2005)
6. Ooi EMM, Watts GF, Chan DC, Barrett PHR. Kinetic Determinants of HDL
Apolipoprotein A-I in Lean and Overweight Subjects: Summary Analysis of
Stable Isotope Studies, Australian Atherosclerosis Society Meeting (2004)
7. Ooi EMM, Watts GF, Barrett PHR, Clifton PM, Nestel PJ. Effects of
Phytosterols on HDL kinetics in Overweight Subjects, Australian
Atherosclerosis Society Meeting (2004)
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8. Ooi EMM, Watts GF, Barrett PHR, Clifton PM, Nestel PJ. Effects of
Phytosterols on HDL Kinetics in Overweight Subjects School of Medicine and
Pharmacology Annual Meeting, QEII, WA (2004)
Communications
1. Dose-Dependent Effect of Rosuvastatin on Apolipoprotein B Kinetics in the
Metabolic Syndrome, Australian Atherosclerosis Society Meeting, Couran
Cove, Queensland (2006) (Winner of the Young Investigator Award)
2. Dose-Dependent Effect of a Novel HMG CoA Reductase Inhibitor on
Apolipoprotein B-100 Kinetics in the Metabolic Syndrome, Clinical Pathology
and Biochemistry PathWest Laboratory Medicine, Royal Perth Hospital (2006)
3. Dose-Dependent Effect of Rosuvastatin (CRESTOR™) on Apolipoprotein B
Kinetics in the Metabolic Syndrome, School of Medicine and Pharmacology
Research Showcase 2006, UWA, Perth, WA Australia (2006)
4. Student perceptions of the effectiveness of writing medical prescriptions using
the national prescribing service case based education package, Teaching &
Learning Forum, UWA, Perth, WA, Australia (2006)
5. Effects of phytosterols on lipoprotein metabolism in subjects with the metabolic
syndrome, Satellite Symposium on Kinetics and Kinetic Modeling of
Lipoprotein, Lipid, and Sterol Metabolism in Systems Ranging from Human
Subjects to Cultured Cells, Washington D.C., USA (2005)
6. PLTP Activity: A determinant of LpA-I Kinetics (Selected for Young
Investigators Competition), Australian Atherosclerosis Society Meeting,
Darwin, NT, Australia (2005)
7. Effects of Phytosterols on HDL Kinetics in Overweight Subjects, Merck Sharp
& Dohme Young Investigators Day, Royal Perth Hospital, WA, Australia (2004)
8. Kinetics of Lipoprotein Metabolism in Chronic Renal Failure, Department of
Nephrology, Royal Perth Hospital, WA, Australia (2003)
Publications arising from work not related to this thesis
1. Ooi EMM, Arena G, Lake F, Joyce DA, Ilett KF. Evaluation of Student
Perceptions of Teaching Medical Prescription Writing Using a Web-based
Education Package.
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Awards
1. Young Investigator Award, Australian Atherosclerosis Society Meeting, Couran
Cove, Queensland, Australia, 2006
2. Convocation Postgraduate Research Travel Award for 2007, UWA Graduates
Association, 2006
3. Japan Atherosclerosis Society Educational Travel Grant, XIV International
Symposia of Atherosclerosis (ISA), Rome, Italy, 2006
4. Australian Atherosclerosis Travel Grant, XIV International Symposia of
Atherosclerosis (ISA), Rome, Italy, 2006
5. UWA Postgraduate Student Association Conference Travel Award, XIV
International Symposia of Atherosclerosis (ISA), Rome, Italy, 2006
6. Australian Atherosclerosis Society Student Travel Award, Australian
Atherosclerosis Society (AAS) Meeting, Couran Cove, Queensland, Australia,
2006
7. Australian Atherosclerosis Society Student Travel Awards, European
Atherosclerosis Society (EAS), Prague, Czech Republic & 6th Annual
Atherosclerosis Thrombosis and Vascular Biology Meetings (ATVB),
Washington D.C., USA, 2005
8. National Heart Foundation Travel Grant, European Atherosclerosis Society
(EAS), Prague, Czech Republic & 6th Annual Atherosclerosis Thrombosis and
Vascular Biology Meetings (ATVB), Washington D.C., USA, 2005
9. University of Western Australia Graduate Research Student Travel Awards
European Atherosclerosis Society (EAS), Prague, Czech Republic & 6th Annual
Atherosclerosis Thrombosis and Vascular Biology Meetings (ATVB),
Washington D.C., USA, 2005
10. Australian Atherosclerosis Society Student Travel Award, Australian
Atherosclerosis Society (AAS) Meeting, Darwin, NT, Australia, 2005
11. National Heart Foundation Travel Grant Australian Atherosclerosis Society
(AAS) Meeting, Darwin, NT, Australia, 2005
12. Australian Atherosclerosis Society Student Travel Award, Australian
Atherosclerosis Society (AAS) Meeting, Barossa Valley, SA, Australia, 2004
13. National Heart Foundation Travel Grant, Australian Atherosclerosis Society
(AAS) Meeting, Barossa Valley, SA, Australia, 2004
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ACKNOWLEDGEMENTS
First, I thank my principal supervisor Professor P Hugh R Barrett for his continuous
support in my research. Thank you for encouraging me to ask questions and to express
my ideas; for showing me different ways to approach a problem; for availing time to
instruct, listen and discuss; and for instilling a spirit of excellence towards research and
academia. Your mentorship truly encouraged creativity, built confidence and guided me
to discover how to learn on my own. I would also like to acknowledge your amazing
patience and fortitude in enduring daily “accidental” interruptions from me for the four
long years!
I would also like thank my co-supervisor, Professor Gerald Watts for his significant
contribution to my research. Thank you for emphasizing and imparting the value of
critical thinking, sound rationalization and self-reflection. Your creativity and intellect
continues to inspire learning and research.
I also thank Dr Dick Chan, our senior research fellow, for being an outstanding role
model and providing fantastic support. It is an honor to learn from you. I would also like
to acknowledge Professor Paul Nestel (Baker Heart Research Institute, Melbourne) for
his intellectual input and role in securing a research grant for the Crestor Study.
I am grateful to the nurses, Mary-Ann Powell, Sandy Hamilton, Claire Haworth, Estelle
Zecchin and Michelle Murphy, at the School of Medicine and Pharmacology for their
nursing assistance and Dr Gerard Chew for his clinical expertise.
I would also like to thank the laboratory staff at the Metabolic Research Centre, for their
assistance with laboratory analyses. A special thanks to Ms Jock Ian Foo, Mr Kevin
Dwyer and Dr Juying Ji for their excellent technical advice and constant support. Thank
you also to Dr Valerie Burke for invaluable statistical advice provided.
I wish to thank the Royal Perth Hospital Pharmacy for dispensing the isotope and study
medications, and PathWest Laboratory Medicine for routine analyses and apoE
genotyping.
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I am grateful for the willing participation of the volunteers who undertook the stable
isotope studies.
I would also like to acknowledge the National Health and Medical Research Council of
Australia, the National Heart Foundation of Australia, the UWA Postgraduate
Association, AstraZeneca Pty. Ltd., the UWA Graduate Association, and the University
of Western Australia for financial support offered. In particular, I would like to
acknowledge Ms JoAnn Lyons from AstraZeneca (Australia), the study monitor
involved in the Crestor intervention study for her proficiency and encouragement.
I thank all my friends for their support, good cheer and constant encouragement.
Friendship adds a brighter radiance to prosperity and lightens the burden of adversity by
dividing and sharing it. A few special thanks: Alastair, the “big brother”, thank you for
tolerating the messy desk next to you the last 3 years; Lydia, my best friend, thank you
for your prayers, laughter and advice; and Doris and Wai, fellow PhD students, partners
in bubble tea and shopping, thank you for cheering me on through every challenge, for
sharing the good times, and laughing hysterically through the down times, and for
believing in me when I doubted myself.
To my family, thank you for your prayers, support, love and understanding; and finally,
to Sheng, thank you for your enduring patience, your steadfast prayers, your daily
encouragement, for all the calm you bring, and for your unconditional love that is all
sustaining. It must have been hard living with a high-strung and loud wife the past year.
Congratulations! You survived part one!
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CHAPTER 1
Literature Review
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1.1 OVERVIEW OF THE METABOLIC SYNDROME
1.1.1 Definition of the metabolic syndrome
The metabolic syndrome is characterized by a constellation of abnormalities that
includes glucose intolerance, insulin resistance, obesity, dyslipidemia, and hypertension.
These pathologies contribute to an increased risk of cardiovascular disease (CVD) and
type 2 diabetes. The clustering of metabolic abnormalities was first recognized in the
early 1920s by the Swedish physician Eskil Kylin. He defined this multifactorial disease
to include hypertension, hyperuricemia and hyperglycemia (Kylin 1923). A quarter
century later, Jean Vague, a professor at the University de Marseille established an
association between abdominal obesity with the risk of diabetes and CVD (Vague
1947). In the 1960s, Pietro Avogaro and Gaetano Crepaldi described the metabolic
syndrome as being accompanied by hyperlipidemia due to increased triglycerides (TG),
obesity, diabetes, hypertension, and a high risk of coronary artery disease (CAD)
(Avogaro et al 1965). 1n 1988, Gerald Reaven characterized the lipoprotein
abnormalities associated with the metabolic syndrome, and proposed the new term
“syndrome X”. This was followed by terms including “deadly quartet” (Kaplan 1989)
and “insulin resistance syndrome” (Ferrannini et al 1991). In 1998, the World Health
Organization (WHO) coined the term “metabolic syndrome”, which is the most widely
used description for this metabolic disorder.
The WHO definition of the metabolic syndrome includes insulin resistance, defined by
glucose intolerance, impaired fasting glucose or type 2 diabetes accompanied by at least
two of the following; hypertension, elevated TG, decreased high-density lipoprotein
(HDL) cholesterol, high body mass index (BMI) or microalbuminuria. The United
States National Cholesterol Education Program’s Adult Treatment Panel III (NCEP
ATP III) proposed a slightly different criteria that includes three out of five of the
following risk factors; abdominal obesity, elevated TG, decreased HDL cholesterol,
hypertension and elevated glucose (NCEP ATP III 2002) to define the metabolic
syndrome. The International Diabetes Federation (IDF) has since modified the NCEP
ATP III criteria to include central obesity as the major feature and fulfillment of two of
the other four factors. It also recommends an oral glucose tolerance test when the upper
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limit of glucose concentration is exceeded and the waist circumference criterion comes
with cutpoints appropriate for different ethnic groups (Table 1.1).
1.1.2 Prevalence of the metabolic syndrome
Comparisons between published data on the prevalence of the metabolic syndrome are
confounded by the use of different definitions. Furthermore, the prevalence of the
metabolic syndrome differs according to age, sex and ethnicity. For example, the
prevalence of the metabolic syndrome is less than 10% for men and women in the 20 –
29 years age group, rising to 38% and 67% in the 60 – 69 years age group, respectively
(Azizi et al 2003). In addition, the National Heath and Nutrition Examination Survey
(NHANES III) reported that the prevalence of the metabolic syndrome increased from
7% in participants aged 20 – 29 years to 44% and 42% in the 60 – 60 years and 70 years
and over groups, respectively (Ford et al 2002).
Large epidemiological surveys have shown that the metabolic syndrome is common.
The NHANES III 1999 – 2002 estimated the age-adjusted prevalence of the metabolic
syndrome in the United States, aged 20 years and over to be between 34.6% (NCEP
ATP III) and 39.1% (IDF) (Ford 2005a). In the Australian population, data from the
AusDiab study between 1999 and 2000 demonstrated that the adjusted estimated
metabolic syndrome prevalence to be between 23.9% (NCEP ATP III) and 26.0%
(WHO) (Adams 2005).
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Table 1.1 Clinical definitions of the metabolic syndrome
World Health Organization (WHO)
National Cholesterol Education Program (NCEP) (Adult Treatment Panel III)
International Diabetes Federation (IDF)
Criteria required
Hyperglycemia/insulin resistance plus two or more of four other criteria
Three or more of five criteria
Central obesity plus two or more of four other criteria
Central obesity
Waist/hip ratio > 0.90 (men), > 0.85 (women) and/or body mass index > 30 kg/m2
Waist circumference: Caucasian: ≥ 102 cm (men), ≥ 88 cm (women) Asian: ≥ 90 cm (men), ≥ 80 cm (women) Consider lower cut-offs (≥ 94 cm [men], ≥ 80 cm [women]) for some non-Asian adults with strong genetic predisposition to insulin resistance
Waist circumference (ethnic-specific): Europid, Sub-Saharan African, Eastern Mediterranean and Middle Eastern (Arab): ≥ 94 cm (men), ≥ 80 cm (women) South Asian, Chinese, South/Central American: ≥ 90 cm (men), ≥ 80 cm (women) Japanese: ≥ 85 cm (men), ≥ 90 cm (women)
Hyperglycemia Insulin resistance: diabetes, impaired fasting glucose, impaired glucose tolerance or hyperinsulinemic euglycemic clamp glucose uptake in lowest 25% of the population
Fasting plasma glucose level ≥ 5.6 mmol/L or current drug treatment for elevated glucose level
Fasting plasma glucose level ≥ 5.6 mmol/L or previous diagnosis of type 2 diabetes
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Triglyceride levels ≥ 1.7 mmol/L or current drug treatment for hypertriglyceridemia
Triglyceride levels ≥ 1.7 mmol/L or current drug treatment for hypertriglyceridemia
Dyslipidemia† Triglyceride levels ≥ 1.7 mmol/L and/or HDL-cholesterol level < 0.9 mmol/L (men), < 1.0 mmol/L (women)
HDL-cholesterol level < 1.0 mmol/L (men), < 1.3 mmol/L (women) or current drug treatment for low HDL-cholesterol level
HDL-cholesterol level < 1.0 mmol/L (men), < 1.3 mmol/L (women) or current drug treatment for low HDL-cholesterol level
Elevated blood pressure
Blood pressure ≥ 140/90 mmHg
Blood pressure ≥ 130/85 mmHg, or current drug therapy for known hypertension
Blood pressure ≥ 130/85 mmHg, or current drug therapy for known hypertension
Other Microalbuminuria: urinary albumin excretion rate > 20 μg/min or Urinary albumin/creatinine ratio > 3.5 mg/mmol
• † Elevated triglycerides and low HDL cholesterol are considered separate criteria
in the NCEP and IDF definitions.
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1.1.3 Components of the metabolic syndrome
The pathogenesis of the metabolic syndrome is still unclear, although it is evidently
multifactorial and influenced by environmental, genetic and epigenetic causes
(Magliano et al 2006). The following section discusses the various components of the
metabolic syndrome.
1.1.3.1 Insulin resistance
Insulin resistance underpins the spectrum of abnormalities of the metabolic syndrome. It
is classically defined as impaired insulin mediated glucose uptake by hepatic and
peripheral tissues (DeFronzo et al 1983). In the early stage of insulin resistance, a
compensatory increase in insulin concentration is observed. As a consequence,
hyperinsulinemia may result in overexpression of insulin action in tissues with normal
or minimally impaired insulin sensitivity. In the insulin resistant state, glucose tolerance
is impaired by several mechanisms including resistance to peripheral glucose uptake
and utilization, impaired hepatic glycogen synthesis, and increased hepatic
gluconeogenesis (Lewis et al 1997). Persistent stimulation of insulin secretion leads to
pancreatic β-cell failure and eventually the development of type 2 diabetes (Reaven
1988). Thus, the accentuation of some insulin actions and resistance to others, give rise
to the diverse clinical manifestations of the insulin resistance syndrome (McFarlane et al
2001).
A major contributor to the development of insulin resistance is free fatty acid (FFA).
FFA are derived from adipose tissue TG stores released via the action of the cyclic
adenosine monophosphate (cAMP) dependent enzyme hormone sensitive lipase (HSL),
and the lipolysis of TG-rich lipoproteins in tissues through the action of lipoprotein
lipase (LPL). Insulin stimulates postprandial uptake of FFA, inhibits HSL and
upregulates LPL. In insulin resistant states, there is an increase in FFA release from
adipose tissue concomitant with a decrease in FFA uptake by muscle tissues (Arner
2002). The vicious cycle may result in attenuated insulin signaling in these tissues,
increased FFA flux to the liver and exacerbation of insulin resistance (Dresner et al
1999, Shulman 1999). In addition, insulin controls the hepatic sterol regulatory element
binding protein (SREBP) expression, the key transcription factor in fatty acid regulation
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and cholesterol biosynthesis. Chronic hyperinsulinemia may increase hepatic secretion
of SREBP-1c and hepatic lipogenic enzymes, leading to increased lipogenesis and
particle secretion of VLDL.
1.1.3.2 Obesity and insulin resistance
Obesity is a key component of the metabolic syndrome (Kahn and Flier 2000) and a
powerful risk factor for CVD and type 2 diabetes. It is a preventable condition with
multifactorial etiology, and defined as an accumulation of excess fat tissues from an
imbalance in energy intake and expenditure (WHO Expert Committee 2000). Several
methods can be applied to measure obesity including body mass index (BMI), waist
circumference, waist-to-hip ratio, underwater-weighing, bioelectrical impedance, dual
energy e-ray absorptiometry, and magnetic resonance imaging (MRI). The prevalence
of obesity (BMI >30kg/m2) is increasing in both developed and developing nations. In
the United States, the prevalence of obesity is 50.5% (Flegal et al 2002) while in
Australia, the prevalence for men and women is 27% and 39%, respectively (Cameron
et al 2003).
Obesity is strongly associated with insulin resistance, with studies reporting a positive
correlation between body fat mass and fasting or postprandial insulin concentration.
Furthermore, in most obese individuals, whole body insulin sensitivity is reduced
(Tappy et al 1991, Woo et al 2003). In particular, visceral obesity is highly associated
with insulin resistance (Despres and Lemieux 2006). Visceral fat accumulation,
increased expression of adrenergic receptors, increased catecholamine-mediated
lipolysis, and reduced insulin mediated antilypolysis contribute ultimately to increased
release of FFA. Visceral adipose tissues deliver FFA to the liver via the portal vein,
thereby inducing hepatic insulin resistance (Wajchenberg 2000, McGarry 2002).
Adipose tissues also secrete a diverse array of bioactive molecules known as
adipokines, including tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), visfatin,
leptin, adiponectin, and resistin. These adipokines through autocrine, paracrine, or
endocrine mechanisms, regulate energy metabolism in adipose tissue, and may play
important roles in the development of the metabolic syndrome (Ruan & Lodish 2004).
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1.1.3.3 Dyslipidemia and insulin resistance
Atherogenic dyslipidemia is a cardinal feature of the metabolic syndrome and
characterized by increased fasting plasma TG, reduced HDL cholesterol (in particular
HDL2 cholesterol), elevated apolipoprotein (apo) B levels and the predominance of
small, dense low-density lipoprotein (LDL) particles. Plasma LDL cholesterol is usually
raised. These abnormalities are frequently associated with insulin resistance (Ginsberg
2000).
In insulin resistant states, there is increased release of FFA from peripheral fat tissue
that subsequently stimulates hepatic synthesis of VLDL particles. Insulin resistance also
decreases sensitivity to the inhibitory effects of insulin on apoB synthesis, the main
structural protein of VLDL. The availability of lipid substrates within the endoplasmic
reticulum (ER) lumen further stabilizes newly synthesized apoB that are normally
degraded by proteasomal and non-proteasomal pathways in a lipid poor state
(Sniderman and Cianflone 1993, Yao et al 1997). These, together with the upregulation
of microsomal triglyceride protein (MTP) and SREBP-1c expression contribute to an
enhanced VLDL-TG pool. In the presence an expanded VLDL-TG pool, plasma HDL
cholesterol concentration is decreased, as a result of increased neutral lipid exchange of
cholesterol esters for TG with VLDL, a process facilitated by cholesteryl ester transfer
protein (CETP) (Barter et al 2003a). The consequences of cholesterol ester depletion
and TG enrichment are increased hydrolysis of HDL particles through hepatic lipase
(HL) activity resulting in dissociation of apoA-I from HDL and hypercatabolism of
apoA-I by the kidney, and an increased proportion of small, dense HDL particles
(Barter et al 2003a). By a similar mechanism, LDL particles become TG-enriched and
are subject to further lipolysis by HL to form small, dense LDL particles (Baynes et al
1991). Hence, the dyslipidemia associated with insulin resistance is highly atherogenic
and can contribute to increased CVD risk in subjects with metabolic syndrome (Grundy
2006, Ninomiya et al 2004).
1.1.3.4 Hypertension and insulin resistance
Hypertension occurs in a third of those with metabolic syndrome and present in those
with evidence of insulin resistance (Ferranini et al 1987, Natali et al 1997). Insulin
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resistance itself has been directly linked with hypertension (Ferranini et al 1987). The
possible mechanisms include impaired response to insulin-mediated vasodilation
(Steinberg et al 1994); impaired endothelial nitric oxide production (Montagnani et al
2002); increased sympathetic nervous system activity (Anderson et al 1991); sodium
retention (Natali et al 1993, Hall 1997); enhanced growth factor production and
activation, leading to proliferation of smooth muscle cells in the vessel wall, and
increased rates of intimal expansion (Trovati and Anfossi 2002); and more recently,
activation of the endothelin system (Sarafidis and Bakris 2006) and elevated
nonesterified free fatty acid (NEFA) concentration (Sarafidis and Bakris 2007).
1.1.3.5 Inflammation, pro-thrombosis and insulin resistance
Inflammation is an important feature of the metabolic syndrome. Evidence suggests that
insulin resistance is associated with chronic subclinical inflammation such as increased
C-reactive protein (CRP) concentration (Haffner 2006). Prospective studies showed that
CRP is also associated with increased risk of developing CVD and type 2 diabetes
(Ridker et al 2003, Santos et al 2005, Soinio et al 2006). Other studies further
demonstrated that elevated CRP is an independent predictor of atherosclerosis and that
other markers of the metabolic syndrome are significant determinants of CRP levels in
this population (Blackburn et al 2001, Linnemann et al 2006). Hence, it was suggested
that CRP should be included in future definitions of the metabolic syndrome, with a
CRP cutoff of 3 mg/L, a value thought to provide further prognostic information (Sattar
et al 2003).
A prothrombotic state is also frequently associated with the metabolic syndrome.
Plasminogen-activator inhibitor 1 (PAI-1) is synthesized in the liver and in adipose
tissues, and regulates thrombus formation by inhibiting the activity of tissue-type
plasminogen activator, an anticlotting factor. Hyperinsulinemia increases PAI-1 and
impairs fibrinolysis in normal human subjects, leading to increased risk of abnormal
coagulation and thrombosis (Calles-Escandon et al 1998). Insulin resistance is also
associated with increased platelet aggregation that may in part explain an altered
intracellular environment with elevated cytosolic Ca2+, enhanced thromboxane A2
synthesis, an increased number and/or function of complexes on platelet membranes,
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and oxidative stress (Anfossi and Trovati 2006). These aberrations predispose to
atherogenesis and increase CVD risk in the metabolic syndrome.
Figure 1.1 Insulin resistance, the metabolic syndrome, and cardiovascular disease
risk.
Adapted from Avramoglu et al 2006
1.1.4 Significance of the metabolic syndrome
1.1.4.1 Association between the metabolic syndrome and CVD
The metabolic syndrome raises CVD risk at any given LDL cholesterol level. The
NCEP ATP III recognizes the metabolic syndrome as a secondary target, after LDL
cholesterol, for risk-reduction therapy for CVD. Several key studies have examined the
incidences of atherosclerosis and CVD in subjects with or without the metabolic
syndrome.
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In the Kuopio Ischemic Heart Disease Risk Factor Study in Finland, (11 year follow up,
1209 Finnish men), middle-aged men with the metabolic syndrome as defined by the
WHO and NCEP had increased cardiovascular and overall mortality; this was
independent of the presence of CVD and diabetes at baseline, and also after adjustment
for conventional cardiovascular risk factors including smoking, alcohol consumption,
and serum LDL cholesterol levels (Lakka et al 2002). A larger study involving 4,483
subjects aged 35-70 years (the Botnia Study), showed that the risk for CVD and stroke
was increased three-fold and cardiovascular mortality six-fold in subjects with
metabolic syndrome (Isomaa et al 2001). Italian Bruneck Study, a prospective
population-based survey examining subjects aged 40-79 years, demonstrated that
subjects with the metabolic syndrome, as defined using WHO and NCEP criteria had
increased incidence of CHD during the five-year follow-up study (Bonora et al 2003).
The West of Scotland Coronary Prevention Study (WOSCOPS) reported that men with
four or five features of the metabolic syndrome had a 3.7-fold increase risk of CVD
(Sattar et al 2003). In the Turkish Adult Risk Factor Study (2398 men and women,
follow up three years), the metabolic syndrome was the major determinant of CVD
events, with the relative risk increased by approximately 70% (Onat et al 2002). In a
large multi-ethnic population study in Canada, individuals with metabolic syndrome had
a greater prevalence of CVD compared with those without (Anand et al 2003).
The DECODE Study examined the metabolic syndrome and its association with all-
cause and cardiovascular mortality in nondiabetic European men and women. The study
was based on 11 prospective European cohort studies comprising 6156 men and 5356
women without diabetes, aged 30-89 years, with a median follow up of 8.8 years. The
overall hazard ratios for all-cause and cardiovascular mortality in subjects with the
metabolic syndrome, compared with those without, were 1.44 (95% confidence interval
[CI], 1.17-1.84) and 2.26 (95% CI, 1.61-3.17) in men and 1.38 (95% CI, 1.02-1.87) and
2.78 (95% CI, 1.57-4.94) in women after adjustment for age, blood cholesterol levels,
and smoking. Nondiabetic subjects with the metabolic syndrome have an increased risk
of death from all causes and CVD (Hu et al 2004). The Atherosclerosis Risk in
Communities Study (ARIC) in the United States (14,502 black and white middle-age
subjects) showed that CHD prevalence was 7.4% among those with the metabolic
syndrome (NCEP) compared with 3.6% in control subjects. After adjustment for
established risk factors, subjects who had the metabolic syndrome were two times more
likely to have CHD than were those who did not (McNeill 2004).
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Other studies that have examined the incidence of atherosclerosis and CVD in subjects
with the metabolic syndrome include the Women’s Health Study (WHS), the 4S Study,
the San Antonio Heart Study, the Air Force/Texas Coronary Atherosclerosis Prevention
Study (AFCAPS/TexCAPS) and the Framingham Offspring Study. As the studies
discussed previously, these studies confirmed that the metabolic syndrome increased
CVD risk and identified of a larger number of subjects at high risk of atherosclerosis
and cardiovascular events independent of cholesterol levels.
1.1.4.2 Association between the metabolic syndrome and diabetes
Several concurrent features of metabolic syndrome, including insulin resistance, are
observed in subjects with impaired glucose tolerance, impaired fasting glucose and type
2 diabetes (Haffner et al 2000). In the metabolic syndrome, insulin resistance leads to a
compensatory increase in insulin secretion; in those with inadequate pancreatic β-cell
insulin response, insulin resistance results in glucose intolerance and subsequently, the
development of type 2 diabetes (Kendall et al 2002). The associations between the
metabolic syndrome and diabetes are well established. In a population based cohort
study, middle-aged Finnish men with the metabolic syndrome, as defined by NCEP and
WHO have five to nine-fold (odds ratios = 5.0-8.8) increased likelihood of developing
diabetes (Laaksonen et al 2002). A summary analysis further reported that the metabolic
syndrome increased the risk of diabetes by 30-52%, which is higher than that for all-
cause mortality (6-7%) and CVD (12-17%) (Ford 2005b).
1.1.5 Prevention and management of the metabolic syndrome
First-line therapies for all lipid and non-lipid risk factors associated with the metabolic
syndrome are therapeutic lifestyle interventions, including weight reduction, increased
physical activity and dietary modification. Pharmacotherapies to improve insulin
sensitivity, dyslipidemia and hypertension are effective second-line strategies.
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1.1.5.1 Therapeutic lifestyle intervention
Therapeutic lifestyle interventions play important roles in managing excess body
weight, insulin resistance, dyslipidemia, hypertension and hyperglycemia seen in the
metabolic syndrome. Two studies reported that weight reduction in obese subjects, with
or without diabetes, is associated with reduced incidence of CVD; in those with IGT, it
is associated with decreased progression to type 2 diabetes mellitus (Tuomilehto et al
2001, Knowler et al 2002). Dietary modification, in particular, restricted carbohydrate
intake may lower blood glucose and TG levels, and increase insulin sensitivity (Volek
and Feinman 2005). Fish consumption, a rich source of n-3 fatty acids, was shown to
effectively raise HDL cholesterol and reduce TG in overweight hypertensive individuals
(Mori et al 2000). Additionally, moderate alcohol intake was associated with increased
HDL cholesterol and reduced CVD risk (Pearson 1996). Available evidence suggests
that increased physical activity is associated with decreased plasma TG, increased HDL
cholesterol levels, increased insulin sensitivity and decreased blood pressure (Carroll
and Dudfield 2004). These lifestyle modifications need to be promoted as first-line
therapies to subjects with the metabolic syndrome. More effort to integrate educational
and lifestyle interventions into the regular care of subjects with the metabolic syndrome
is essential. Success with such interventions will limit the need for pharmacotherapy,
and may provide added benefits if drug therapies are employed.
1.1.5.2 Pharmacotherapy
Pharmacological management currently treats individual components of the metabolic
syndrome including abnormal glucose tolerance, dyslipidemia, hypertension and
thrombosis (Table 1.2). In those with established diabetes or CVD, intensive glycemic
control is associated with reduced risk of microvascular disease, and to a lesser extent,
macrovascular complications (Jenkins et al 2004). Given that insulin resistance is an
etiologic factor, insulin sensitizers such as thiazolidinediones (TZD) and metformin
may be important treatment options (Colca 2006, Bhatia and Viswanathan 2006). These
interventions were associated with greater than 50% reduced risk of diabetes
(Tuomilehto et al 2001, Buchanan et al 2002) and hence, have significant potential to
limit CVD risk in the metabolic syndrome. Furthermore, metformin was shown to
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prevent vascular complications in those with type 2 diabetes (UK prospective Diabetes
Study, UKPDS, 1998).
Although there are no guidelines on the appropriate LDL cholesterol treatment target in
the metabolic syndrome, it is likely that the presence of additional traditional risk
factors (family history or smoking) or newer risk factors (elevated CRP levels), may
represent a CVD risk equivalent. Hence, the target of LDL cholesterol < 2.6 mmol/L
should be recommended and statin therapy is a appropriate choice (NCEP 2001). To
treat the atherogenic dyslipidemia (high TG, low HDL cholesterol), a fibrate or niacin
may be an suitable option. Combination therapy with statin-fibrate, statin-niacin, statin-
ezetimibe or ezetimibe-fibrate may further optimize the lipid profile of subjects with the
metabolic syndrome. In addition, a combination of fibrates with either metformin or
TZD may be beneficial as it would simultaneously address both insulin resistance and
dyslipidemia. Combined α and γ peroxisome proliferator-activated receptor (PPAR)
agonists that can simultaneously improve insulin resistance, glucose intolerance,
elevated TG and low HDL cholesterol levels, are also potential options (Pourcet et al
2006).
As inflammation and pro-thrombosis are major components of the metabolic syndrome,
anti-inflammatory and anti-thrombotic treatments would be beneficial. In particular,
aspirin has anti-inflammatory properties and an established role in preventing
atherothrombotic complications of CHD (Fuster et al 1993, Patrono et al 2005). Low-
dose aspirin administration for the prevention of ischemic events in CHD subjects is
now considered routine practice (Chapman 2006). Combination therapy with statin and
aspirin may be an effective and cost efficient secondary preventative measure to avoid
large numbers of premature deaths and cardiovascular events (Chapman 2006).
Angiotensin Converting Enzyme Inhibitors (ACEI) therapy may also be considered for
metabolic syndrome subjects with hypertension. Both β–blockers and angiotensin-2
receptor blockers can be considered appropriate alternatives to ACEI therapy in high-
risk patients (Komers and Komersova 2000). In addition, insulin sensitizers are also
beneficial for the management of hypertension observed in the metabolic syndrome
(Kurtz 2006).
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http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBG-4KWT6BY-1&_coverDate=09%2F15%2F2006&_alid=511648540&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5142&_sort=d&view=c&_acct=C000028118&_version=1&_urlVersion=0&_userid=554529&md5=9b3f8def7294322307d20cc3e6b93b4f#bib24#bib24http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBG-4KWT6BY-1&_coverDate=09%2F15%2F2006&_alid=511648540&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5142&_sort=d&view=c&_acct=C000028118&_version=1&_urlVersion=0&_userid=554529&md5=9b3f8def7294322307d20cc3e6b93b4f#bib57#bib57
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Table 1.2 Therapeutic interventions in the metabolic syndrome
Lifestyle interventions Weight loss
Physical exercise
Diet modifications in macro- and micro-nutrients
Insulin sensitisers Glitazones/ Thiazolidionediones
Metformines
Lipid modifiers Statins
Fibrates
Niacin
Ezetimibe
Anti-platelet/anti-inflammatory Aspirin
Anti-hypertensive Angiotensin converting enzyme inhibitors
Angiotensin receptor antagonists
Anti-obesity Sibutramine
Orlistat
Rimonabant
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1.2 LIPOPROTEIN METABOLISM
Dyslipidemia is an important feature of the metabolic syndrome. A better understanding
of lipoprotein metabolism may provide insight into the underlying mechanisms that
impact the development of the metabolic syndrome, and hence, improve management of
the syndrome.
1.2.1 Definition and Classification of Lipids
Lipids refer to a heterogeneous group of compounds that have ready solubility in
organic solvents and low solubility in water. Chemically, lipids are noted to be
compounds that yield fatty acids on hydrolysis or complex alcohols that couple with
fatty acids to form esters. The definition may include compounds related closely to fatty
acid derivatives through biosynthetic pathways (e.g. prostanoids, aliphatic ethers or
alcohols) or by their biochemical or functional properties (e.g. cholesterol). Complex
lipids contain additional non-fatty acid groups, which include amino acids, sulphates,
phosphoryl or sialic groups that increase lipid solubility in polar solvents.
The major lipids found in human plasma are triglycerides, phospholipids, fatty acids,
cholesterol, cholesterol esters and glycolipids. They have important physiological roles,
which include energy production, substrate storage and body absorption of fat-soluble
vitamins. Lipids are also major constituents of cells. Many structural components of the
cell membrane are lipids such as phospholipids and cholesterol. In addition, lipids such
as cholesterol are precursors to steroid hormones and bile acids.
1.2.2 Lipoprotein Metabolism
All lipids, with the exception of free fatty acids (FFA) are transported in plasma in the
form of lipoproteins. Lipoproteins are complex macromolecules of lipid and protein. All
lipoproteins consist of a non-polar lipid core, mainly triglyceride (TG) and cholesterol
esters (CE), surrounded by a polar monolayer of phospholipids, heads of free
cholesterol and apolipoproteins, with protruding hydroxide groups. Although they are
structurally similar, lipoproteins differ in the content of their non-polar lipid core, the
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proportion of the lipids within the core, and proteins found on their surface.
Lipoproteins also differ in their metabolic pathway, which is determined in part by the
apolipoproteins embedded in the surface monolayer. These apolipoproteins modulate
the activation of lypolytic enzymes and serve as ligands in receptor-mediated processes
(Kwiterovich 2000). Figure 1.2 illustrates a typical lipoprotein particle with a
hydrophobic core surrounded by a hydrophilic outer shell.
Lipoproteins are classified according to their density, electrophoretic mobility, particle
size, buoyancy (floatation rate) and chemical composition. Of the various means of
classification, electrophoretic mobility and density are the most widely used as the basis
for identification and isolation of lipoproteins. The five major classes of lipoproteins
according to increasing density isolated are: chylomicron (CM), very-low-density
lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein
(LDL) and high-density lipoprotein (HDL). CM are noted to be the largest particles that
contain the highest proportion of TG and lowest proportion of protein, while HDL are
the smallest, have the lowest proportion of lipids and are the most protein dense
lipoproteins.
Figure 1.2 Typical Structure of a lipoprotein particle
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1.2.3 The Lipoproteins
1.2.3.1 Chylomicrons (CM)
Chylomicrons (CM) are TG-rich lipoproteins, ranging between 100 to 1000nm with one
molecule of apoB-48 per particle (Young et al 1990). Following intestinal absorption,
dietary fat and cholesterol are esterified to form TG and CE within the enterocytes.
These lipids are packaged together with apoB-48, phospholipids, free cholesterol, apoE
and apoC to form nascent CM. CM enter the circulation via the thoracic lymph. TG in
CM are hydrolyzed by lipoprotein lipase (LPL), and FFA are subsequently released for
energy production and storage (Mahley et al 1984). As TG are removed, CM become
smaller CM and are removed from the circulation by hepatic receptors including LDL
receptors (LDLR) or the LDL receptor related protein (LRP). This cascade from dietary
lipids to removal of the remnants is known as the exogenous pathway (Figure 1.11).
Abnormalities in CM metabolism have been shown to be associated with increased risk
of coronary disease in subjects with insulin resistance (Ginsberg and Illingworth 2001).
Increased postprandial levels of TG and apoB-48, as well as abnormal retinyl palmitate
dynamics (a measure of CM metabolism), were found to be associated with increased
presence of CAD (Karpe et al 1994, Mero et al 2000). These abnormalities may be
related to decreased hepatic receptor activity and/or decreased LPL activity (Mamo et al
2001, Panarotto et al 2002). The insulin resistant state also results in increased levels of
apoC-III, an inhibitor of LPL, and impaired apoE-mediated receptor uptake of CM and
its remnants. Moreover, increased secretion of cytokines such as TNF-α and IL-6
inhibit LPL activity, this, contributing further to postprandial chylomicronemia (Kern et
al 1995, Yudkin et al 2000). Animal studies also support that the accumulation of CM
remnants in the metabolic syndrome may be due to oversecretion of intestinally derived
apoB-48 containing particles (Haidari et al 2002).
1.2.3.2 Very-low-density lipoprotein (VLDL)
Very-low density lipoprotein (VLDL) accounts for most of the TG in plasma and is an
energy source for extrahepatic tissues. It is synthesized in the liver and structurally
similar to CM. In the endogenous pathway, fatty acids that are returned to the liver from
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CM metabolism are re-esterified to form TG. These TG are then packaged together with
cholesterol, CE, phospholipids, apoB-100, apoC and apoE into nascent VLDL, which
are then secreted into the bloodstream (Bjorkegren et al 1998). Depending upon the
availability of TG, VLDL may differ in size and flotation rate. Compared with large
VLDL (Sf 60 to 400), smaller VLDL particles (Sf 20 to 60) are enriched in CE, depleted
in TG and have a low ratio of apoE and apoC to apoB. Large TG-rich VLDL are
secreted in situations where excess TG are synthesized, such as obesity (Egusa et al
1985). By contrast, small VLDL, and possibly IDL and LDL-like particles, are secreted
when TG availability is reduced (Ginsberg et al 1985).
T