i
Studies of the metabolism of lipoprotein(a) in humans
Louis Lok Shun Ma
Bachelor of Science (Medicine and Pharmacology) (Honours)
This thesis is presented for the degree of
Doctor of Philosophy
of The University of Western Australia
School of Medicine
Faculty of Health and Medical Sciences
2020
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THESIS DECLARATION
I, Louis Ma, certify that:
This thesis has been substantially accomplished during enrolment in this degree.
This thesis does not contain material which has been submitted for the award of any other degree or diploma in my name, in any university or other tertiary institution.
In the future, no part of this thesis will be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint-award of this degree.
This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text and, where relevant, in the Authorship Declaration that follows.
This thesis does not violate or infringe any copyright, trademark, patent, or other rights whatsoever of any person.
The research involving human data reported in this thesis was assessed and approved by The University of Western Australia Human Research Ethics Committee. Approval #: RA/4/1/6730, RA/4/20/4900 and RA/4/1/9310. Written patient consent has been received and archived for the research involving patient data reported in this thesis.
Third party editorial assistance was provided in preparation of this thesis by Drs ID and L Mackinnon.
This thesis contains published work and/or work prepared for publication, some of which has been co-authored.
Signature:
Date: 19th February 2020
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ABSTRACT
Lipoprotein(a) [Lp(a)] is a low-density lipoprotein (LDL)-like particle comprising one
molecule of apolipoprotein(a) [apo(a)] covalently bound to apoB-100 (apoB) through a
disulphide bond. Epidemiological and genetic studies suggest that an elevated plasma
concentration of Lp(a) is a strong, causal risk factor for atherosclerotic cardiovascular
disease (ASCVD). Large clinical trials have consistently shown that elevated Lp(a)
concentration remains a risk factor despite reduction of LDL-cholesterol with statins.
However, the metabolism of Lp(a) particles remains poorly understood. Understanding
Lp(a) metabolism will provide mechanistic insights that can contribute to the development
of new therapies that target Lp(a).
This thesis focused on the metabolism of Lp(a), specifically the coupling of apo(a) and
apoB-100, production of Lp(a) in relation to elevated and normal Lp(a) plasma
concentration, and kinetic differences in Lp(a) and LDL-apoB following apheresis in
subjects with elevated Lp(a) concentration.
Study 1 compared the validity of a new liquid chromatography-mass spectrometry (LC-
MS) method with the well established Northwest Lipid Metabolism and Diabetes Research
Laboratories (NLMDRL) method for measuring apo(a) concentration in human plasma.
The apo(a) concentration measured by the LC-MS method correlated highly with the
NLMDRL method (r=0.977, y=1.10x – 0.11, P<0.001). There was substantial overall
agreement for apo(a) concentration measurement between the LC-MS and NLMDRL
methods, as shown by Lin’s concordance coefficient (rc) of 0.958 (95% confidence interval
(CI) 0.940–0.971. This study concluded that apo(a) concentration measured using an LC-
MS method correlates strongly with that measured using the NLMDRL method.
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Study 2 investigated the fractional turnover of apo(a) and apoB within plasma Lp(a)
particles in statin-treated patients with elevated and with normal plasma Lp(a)
concentration. The fractional catabolic rates (FCRs) of Lp(a)–apo(a) and Lp(a)–apoB were
similar in patients with elevated Lp(a) concentration: 0.46 (95% CI 0.37–0.57 pools/day) vs
0.46 (95% CI 0.37–0.58 pools/day] (P=0.861). Similarly, the FCRs for Lp(a)–apo(a) and
Lp(a)–apoB did not differ significantly in patients with normal Lp(a) concentration: 0.47
(95% CI 0.38–0.58] pools/day vs 0.48 (95% CI 0.39–0.60] pools/day (P=0.373). The FCRs
for Lp(a)–apo(a) and Lp(a)–apoB correlated strongly in patients with elevated and normal
Lp(a) concentrations (r=0.970 and 0.979, respectively, P<0.001 for both). There was
substantial agreement between the FCRs for Lp(a)–apo(a) and Lp(a)–apoB in patients
with elevated and normal Lp(a) concentrations (rc=0.978 and rc=0.966, respectively). This
study concluded that Lp(a)–apo(a) and Lp(a)–apoB have similar FCRs and are therefore
tightly coupled as an Lp(a) holoparticle.
Study 3 examined whether the hepatic production of apo(a) is increased in patients with
elevated Lp(a) concentration taking statins and the association between apo(a) isoforms
and Lp(a) kinetics. Apo(a) production rate (PR) was significantly higher in patients with
elevated Lp(a) concentration than those with normal Lp(a) concentration: 3.8 (2.6, 5.6] vs
0.24 (0.14, 0.40] nmol/kg/day (P<0.001). Plasma apo(a) concentration was significantly
and positively associated with the PR of apo(a) in patients with elevated Lp(a)
concentration (r=0.699, P=0.005). Apo(a) concentration also correlated significantly with
the PR in patients with normal Lp(a) concentration (r=0.949, P=0.001). Apo(a) isoform size
correlated significantly and inversely with the concentration and PR of apo(a) (r=–0.741
and r=–0.708, respectively, P<0.0001 for both). This study concluded that elevated plasma
Lp(a) concentration is a consequence of increased hepatic production of Lp(a) particles.
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Study 4 investigated the kinetics of Lp(a) and LDL–apoB particles in patients with elevated
Lp(a) concentration and coronary artery disease (CAD) undergoing apheresis. The FCR
was significantly lower for Lp(a) than for LDL–apoB: 0.39 (0.31, 0.49] vs 0.57 (0.46, 0.71]
pools/day (P=0.03). The estimated concentration was significantly higher for Lp(a) than for
LDL–apoB: 853 (780, 932] vs 614 (551, 685] mg/L (P=0.01). The PR of Lp(a) did not differ
significantly from that of LDL-apoB PR: 14.8 (11.3, 19.3) vs 14.6 (11.2, 19.1) mg/kg/day
(P=0.77). This study concluded that the FCR is lower for Lp(a) than for LDL–apoB
particles, which implies that different metabolic pathways are involved in the catabolism of
these lipoproteins.
The studies in this thesis support the notion that Lp(a) metabolism is driven primarily by
production and that catabolism plays a smaller role. The FCRs for the apo(a) and apoB
proteins within Lp(a) particles seem to be tightly coupled. In addition, the FCR is slower for
Lp(a) than for LDL–apoB following apheresis, which suggests that the LDL receptor does
not play a major role in Lp(a) catabolism. These findings should be extended by
investigating the kinetic associations between Lp(a) and other atherogenic lipoproteins and
the mechanisms of actions of newer therapies especially apo(a) RNA based therapies.
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TABLE OF CONTENTS
Page
Thesis Declaration ii
Thesis Abstract iii
Table of Contents vi
Acknowledgements xiv
Authorship Declaration xvi
Personal Contribution by the Candidate xx
Publications, Presentations and Awards xxvi
List of Tables xxviii
List of Figures xxx
List of Abbreviations xxxiii
CHAPTER 1 Literature Review
1.1 General introduction to the role of lipoprotein(a) in human health and
diseases
2
1.2 Molecular aspects of lipoprotein(a) 3
1.2.1 Structure and function 3
1.2.2 Genetics 7
1.2.3 Metabolism 9
1.3 Epidemiology of lipoprotein(a) 17
1.3.1 Lipoprotein(a) distribution and reference values 17
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1.3.2 Geographic variation in lipoprotein(a) concentration 19
1.3.3 Genetic epidemiology 19
1.3.4 Physiological determinants 19
1.4 Pathobiology of lipoprotein(a) 22
1.4.1 Thrombosis 23
1.4.2 Fibrinolysis 24
1.4.3 Oxidative Stress 24
1.4.4 Inflammation 25
1.5 Significance of lipoprotein(a) in clinical medicine 25
1.5.1 Atherosclerotic cardiovascular disease 25
1.5.2 Aortic valve stenosis 28
1.5.3 Other medical conditions 29
1.6 Analytical biochemistry 31
1.6.1 Principle of the assays 32
1.6.2 Description of the assays 33
1.6.3 Evaluation and comparison of assays 34
1.6.4 Standardisation and precision 35
1.6.5 Analytical issues and limitations 36
1.7 Treatments for elevated lipoprotein(a) concentration 36
1.7.1 Standard/older lipid-lowering pharmacotherapies 37
1.7.2 Lipoprotein apheresis 40
1.7.3 Newer therapies 43
1.8 Tracer kinetic methods in human lipoprotein research 47
1.8.1 Principles 48
1.8.2 Definition of terms 49
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1.8.3 Isotope labelling methods 50
1.8.4 Clinical protocols 50
1.8.5 Analytical techniques 51
1.8.6 Analytical aspects of kinetic studies 51
CHAPTER 2 Anatomy of the Thesis
2.1 Study 1: Comparison of methods for measuring plasma lipoprotein(a)
particle concentration
56
2.1.1 Rationale 56
2.1.2 Hypothesis 56
2.1.3 Aim 57
2.1.4 Study design 57
2.1.5 Statistical analyses 57
2.2 Study 2: Apolipoprotein(a) and apolipoprotein B-100 kinetics within
lipoprotein(a) particles
58
2.2.1 Rationale 58
2.2.2 Hypothesis 58
2.2.3 Aim 58
2.2.4 Study design 59
2.2.5 Statistical analyses 59
2.3 Study 3: Apolipoprotein(a) kinetics in statin-treated patients with
elevated plasma lipoprotein(a) concentration
59
2.3.1 Rationale 59
2.3.2 Hypothesis 60
2.3.3 Aim 60
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2.3.4 Study design 60
2.3.5 Statistical analyses 61
2.4 Study 4: Lipoprotein(a) and low-density lipoprotein–apolipoprotein-B
metabolism following apheresis in patients with elevated lipoprotein(a)
concentration and coronary artery disease
61
2.4.1 Rationale 61
2.4.2 Hypothesis 62
2.4.3 Aim 62
2.4.4 Study design 62
2.4.5 Statistical analyses 62
2.5 Conclusion, overall discussion, and future directions 63
CHAPTER 3 Subjects, Materials and Methods
3.1 Subjects 65
3.2 Inclusion criteria 66
3.2.1 Study 1 66
3.2.2 Studies 2 and 3 66
3.2.3 Study 4 66
3.3 Exclusion criteria 67
3.3.1 Studies 1-3 67
3.3.2 Study 4 67
3.4 Protocol for screening 67
3.5 Anthropometric measurements 68
3.6 Blood pressure 68
3.7 Protocol for blood measurements 69
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3.8 Protocol for stable isotope infusion 69
3.8.1 Preparation of leucine infusion 69
3.8.2 Stable isotope infusion 70
3.9 Protocol for apheresis 70
3.10 Laboratory methods 71
3.10.1 Total cholesterol 71
3.10.2 Triglycerides 72
3.10.3 HDL-cholesterol 72
3.10.4 LDL-cholesterol 73
3.10.5 Total apoB 73
3.10.6 LDL–apoB 74
3.10.7 Glucose 74
3.10.8 Insulin 75
3.10.9 Insulin resistance 75
3.10.10 Dutch Lipid Clinic Network Score 75
3.11 Measurements of lipoprotein(a) concentration 75
3.11.1 NLMDRL method 76
3.11.2 LC-MS method 77
3.11.3 Abbott Quantia method 79
3.11.4 DiaSys 21FS method 79
3.12 Determination of apolipoprotein(a) isoform size 79
3.13 Analytical methods for isotopic enrichment 81
3.13.1 Extraction of free leucine from plasma 81
3.13.2 Derivatisation of leucine 82
3.13.3 Isolation and measurement of the isotopic enrichment of 82
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lipoprotein(a) in whole plasma
3.13.4 Isolation and measurement of isotopic enrichment of
lipoprotein(a) in LDL/HDL fractions
84
3.14 Kinetic analyses and mathematical modelling 84
3.14.1 Study 2 84
3.14.2 Study 3 85
3.14.3 Study 4 86
3.15 Statistical Analyses 87
3.15.1 Descriptive statistics 88
3.15.2 Dispersion statistics 88
3.15.3 Distribution of data 89
3.15.4 Independent-samples t test 89
3.15.5 Paired- samples t test 90
3.15.6 Correlational analysis 90
3.15.7 Linear regression 91
3.15.8 Bland–Altman analysis 91
3.15.9 Lin’s concordance correlation 92
3.16 Sample size and power calculations 92
CHAPTER 4 Study 1: Comparison of methods for measuring plasma
lipoprotein(a) particle concentration
4.1 Introduction 97
4.2 Methods 99
4.3 Results 101
4.4 Discussion 103
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CHAPTER 5 Study 2: Apolipoprotein(a) and apolipoproteinB-100
kinetics within the lipoprotein(a) particle
110
CHAPTER 6 Study 3: Lipoprotein(a) kinetics in statin-treated patients
with and without elevated plasma concentration of
lipoprotein(a)
115
CHAPTER 7 Study 4: Lipoprotein(a) and LDL particle metabolism
following apheresis
125
CHAPTER 8 Conclusions, Overall Discussion and Future Directions
8.1 Overview and principal findings 136
8.2 Study limitations 139
8.3 Implications of the research findings 141
8.4 Suggestions for future studies 143
8.4.1 Study population 143
8.4.2 Integration of other lipoproteins and kinetic parameters 143
8.4.3 Measurement of lipoprotein(a) concentration 144
8.4.4 Molecular, biological and genetic studies of lipoprotein(a)
metabolism
144
8.4.5 Treatment modalities 145
8.4.6 Guidelines 148
8.5 Conclusion 149
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REFERENCES 151
APPENDICES 189
Appendix I PCSK9 Inhibition with alirocumab increases the catabolism of
lipoprotein(a) particles in statin-treated patients with elevated
lipoprotein(a)
190
Appendix II Plasma concentration measured by NLMDRL method and
production rate of apo(a) in patients with elevated and normal
plasma Lp(a) concentrations
233
Appendix III Royal Perth Hospital Ethics Approval Form 235
Appendix IV Patient Information Sheet and Informed Consent 272
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ACKNOWLEDGEMENTS
I am grateful to Winthrop Professor Gerald Watts, my primary supervisor. Thank you for
your support, supervision and expertise throughout the course of my PhD.
I am in debt to Dr. Dick Chan for his time and effort guiding me through my PhD. I cannot
thank you enough for the opportunities you have given me to learn and ask questions.
I thank Dr. Esther Ooi for her time supporting me during the start of my PhD when there
were many uncertainties about what I should do.
I thank Professor Hugh Barrett for his patience, guidance and time helping me with the
modelling of the kinetic studies.
I appreciate the help of nurses Sayeh Nejad and Sharon Radtke, for their hard work
conducting the clinical trials at the Medical Research Foundation.
I am grateful for the willing participation of the volunteers who participated in the studies.
I also acknowledge the funding bodies, the National Health and Medical Research Council
of Australia and Sanofi.
I also acknowledge the financial assistance provided by the University of Western
Australia, Ad-Hoc Postgraduate Research Scholarship. This research was also supported
by an Australian Government Research Training Program (RTP) scholarship.
The interactions I had with all PhD students cannot be forgotten. A special thanks to Nicky
Bondonno, Denise Demmer, Lauren Blekkenhorst, Anindhita Chakraborty and Sunil Bhat
for the great memories.
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I also thank Julie Le for her endless support throughout the years. She has helped me
through tough times and has always reassured me when I was in doubt about my
decisions.
Lastly, I thank my family for always being supportive of the decisions I have made
throughout the years. I dedicate this PhD to them. Thank you.
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AUTHORSHIP DECLARATION: CO-AUTHORED PUBLICATIONS
This thesis contains work that has been published.
Details of the work: Fractional turnover of apolipoprotein(a) and apolipoprotein B-100 within plasma lipoprotein(a) particles in statin-treated patients with elevated and normal Lp(a) concentration. Location in thesis: Chapter 5, page 108 Student contribution to work: Clinical component
• Participated in screening visits • Participated in stable isotope studies and collecting plasma samples • Maintained patient records
Laboratory component • Collection and separation of plasma by centrifugation from participants • Isolation and storage of buffy coats • Isolation of lipoprotein fractions LDL and HDL by ultracentrifugation • Extraction of leucine by ion-exchange chromatography • Development of the method for Lp(a) isolation • Isolation of Lp(a) • Separation of Lp(a)-apo(a) and Lp(a)-apoB using SDS-PAGE and Western blot
analysis Writing component
• Write-up • Data presentation in tables and production of figures
Co-author signatures and dates:
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Details of the work: Apolipoprotein(a) kinetics in statin-treated patients with elevated plasma lipoprotein(a) concentration. Location in thesis: Chapter 6, page 113 Student contribution to work: Clinical component
• Participated in screening visits • Participated in stable isotope studies and collecting plasma samples • Maintained patient records
Laboratory component • Collection and separation of plasma by centrifugation from participants • Isolation and storage of buffy coats • Isolation of lipoprotein fractions LDL and HDL by ultracentrifugation • Extraction of leucine by ion-exchange chromatography • Development of the method for Lp(a) isolation • Isolation of Lp(a) • Separation of Lp(a)-apo(a) and Lp(a)-apoB using SDS-PAGE and Western blot
analysis Writing component
• Write-up • Data presentation in tables and production of figures
Co-author signatures and dates:
xviii
Details of the work: Lipoprotein(a) and low-density lipoprotein apolipoprotein B metabolism following apheresis in patients with elevated lipoprotein(a) and coronary artery disease. Location in thesis: Chapter 7, page 123 Student contribution to work: Kinetic analyses I was responsible for the determination of the kinetics for Lp(a) and LDL-apoB using a single compartment model. Database I was responsible for the management of the database Statistical analyses I performed all statistical analyses using the statistical package IBM SPSS Statistics (version 21.0; IBM Corp., Armonk, NY, USA). Co-author signatures and dates:
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Student signature: Date: 24th Feb 2020 I, Gerald Watts certify that the student’s statements regarding their contribution to each of the works listed above are correct. I have the permission of all other co-authors to include any published works in the thesis by email.
Coordinating supervisor signature: Date: 24th Feb 2020
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PERSONAL CONTRIBUTIONS BY THE CANDIDATE
My personal contributions are outlined below. I have acknowledged the assistance of other
individuals and institutions.
Clinical (Studies 2–3)
I performed the following aspects of the studies:
• Participated in screening visits
• Participated in stable isotope studies and collecting plasma samples
• Maintained patient records
Gerald Watts, Jing Pang and the nursing staff were responsible for patient screening and
recruitment. PathWest facilitated the biochemical measurements, and Royal Perth Hospital
Pharmacy prepared and dispensed D3 leucine.
Laboratory analyses (Studies 2–3)
I was responsible for performing the following laboratory procedures:
• Collection and separation of plasma by centrifugation from participants
• Isolation and storage of buffy coats
• Isolation of lipoprotein fractions LDL and HDL by ultracentrifugation
• Extraction of leucine by ion-exchange chromatography
• Development of the method for Lp(a) isolation
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• Isolation of Lp(a)
• Separation of Lp(a)–apo(a) and Lp(a)–apoB using SDS-PAGE and Western blot
analysis.
Dick Chan and Jock Ian Foo contributed to the development of the method for Lp(a)
isolation.
Kinetic analyses (Studies 2–4)
I was responsible for the determination of the kinetics (tracer/tracee ratio, pool size and
production, and fractional catabolic rates) for Lp(a)–apo(a) and Lp(a)–apoB using
multicompartmental modelling. Professor P Hugh R Barrett (School of Biomedical
Sciences, The University of Western Australia) advised on the modelling of the kinetic data.
Databases (Studies 1–4)
I was personally responsible for the management of all laboratory databases. All
databases were password coded and accessible by authorised persons only. The
research nurses (Sayeh Nejad and Sharon Radtzke) and I, were responsible for the
management of the patient databases (personal details and patient records).
Statistical analyses (Studies 1–4)
I performed all statistical analyses using the statistical package IBM SPSS Statistics
(version 21.0; IBM Corp., Armonk, NY, USA). Expert statistical advice was provided by Dr
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Sally Burrows (Senior Research Fellow, Faculty of Health and Medical Sciences, Royal
Perth Hospital).
Publications
I conducted the literature reviews and drafted all publications presented in this thesis
(Chapters 5, 6 and 7), including the write-up, data presentation in tables and production of
figures. Drafts were revised after comments and suggestions from co-authors.
The contributions for each chapter are presented in the following tables.
Chapter 4 Comparison of methods to measure apo(a) concentration
Contribution percentage
Study concept and design Candidate
Other authors
70
30
Conduct of the study Candidate
Other authors
70
30
Collection of blood samples Candidate
Other authors
70
30
Statistical analyses Candidate
Other authors
80
20
Data interpretation Candidate
Other authors
70
30
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Chapter 5 Apolipoprotein(a) and apolipoprotein B-100 turnover within lipoprotein(a)
particles
Published as Ma L, Chan DC, Ooi EMM, Barrett PHR, Watts GF. Fractional turnover of
apolipoprotein(a) and apolipoprotein B-100 within plasma lipoprotein(a) particles in statin-
treated patients with elevated and normal Lp(a) concentration. Metabolism 2019:96, 443–
454.
Contribution percentage
Study concept and design Candidate
Other authors
40
60
Conduct of the study Candidate 100
Collection of blood samples Candidate 100
Laboratory analyses Candidate 100
Statistical analyses Candidate
Other authors
70
30
Kinetics analyses Candidate 100
Data interpretation Candidate
Other authors
70
30
Manuscript drafting Candidate
Other authors
70
30
Manuscript review Candidate
Other authors
50
50
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Chapter 6 Apolipoprotein(a) kinetics in statin-treated patients with elevated plasma
lipoprotein(a) concentration
Published as Ma L, Chan DC, Ooi EM, Marcovina SM, Barrett PHR, Watts GF.
Apolipoprotein(a) kinetics in statin-treated patients with elevated plasma lipoprotein(a)
concentration. The Journal of Endocrinology & Metabolism 2019:104(12), 6247–6255.
Contribution percentage
Study concept and design Candidate
Other authors
50
50
Conduct of the study Candidate 100
Collection of blood samples Candidate 100
Laboratory analyses Candidate 100
Statistical analyses Candidate
Other authors
70
30
Kinetic analyses Candidate 100
Data interpretation Candidate
Other authors
60
40
Manuscript drafting Candidate
Other authors
70
30
Manuscript review Candidate
Other authors
50
50
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Chapter 7 Lipoprotein(a) and low-density lipoprotein apolipoprotein B metabolism
following apheresis
Klaus Parhofer and Elisa Waldmann were responsible for the clinical studies including
patient screening and recruitment. Elisa Waldmann was responsible for blood sample
collection and laboratory analyses.
Published as Ma L, Waldmann E, Ooi EMM, Chan DC, Barrett PHR, Watts GF, Parhofer
KG. Lipoprotein(a) and low-density lipoprotein apolipoprotein B metabolism following
apheresis in patients with elevated lipoprotein(a) and coronary artery disease. European
Journal of Clinical Investigation 2019:49(2), E13053.
Contribution percentage
Study concept and design Candidate
Other authors
20
80
Statistical analyses Candidate
Other authors
70
30
Kinetic analyses Candidate 100
Data interpretation Candidate
Other authors
60
40
Manuscript drafting Candidate
Other authors
70
30
Manuscript review Candidate
Other authors
50
50
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PUBLICATIONS, PRESENTATION & AWARDS
Publications
Ma L, Chan DC, Ooi EMM, Barrett PHR, Watts GF. Fractional turnover of apolipoprotein(a)
and apolipoprotein B-100 within plasma lipoprotein(a) particles in statin-treated
patients with elevated and normal Lp(a) concentration. Metabolism 2019:96, 443-
454 (Study 2)
Ma L, Chan DC, Ooi EM, Marcovina SM, Barrett PHR, Watts GF. Apolipoprotein(a)
kinetics in statin-treated patients with elevated plasma lipoprotein(a) concentration.
The Journal of Endocrinology & Metabolism 2019:104(12), 6247-6255 (Study 3)
Ma L, Waldmann E, Ooi EMM, Chan DC, Barrett PHR, Watts GF, Parhofer KG.
Lipoprotein(a) and Low-density lipoprotein apolipoprotein B metabolism following
apheresis in patients with elevated lipoprotein(a) and coronary artery disease.
European Journal of Clinical Investigation 2019:49(2), E13053. (Study 4)
Watts GF, Chan DC, Pang J, Ma L, Ying Q, Aggarwal S, Marcovina SM, Barrett PHR.
PCSK9 Inhibition with alirocumab increases the catabolism of lipoprotein(a)
particles in statin-treated patients with elevated lipoprotein(a). Metabolism 2020:
recently accepted. (Appendix I)
Presentations
Orals
Lipoprotein(a) Metabolism in Subjects at High Risk of Cardiovascular Disease – Australian
Atherosclerosis Society (AAS) 2016 Annual Scientific Meeting, Hobart, Australia,
2016.
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Within-patients Variation in Plasma Concentration of Lipoprotein(a): Implications of
screening programs and new therapies – Australian Atherosclerosis Society (AAS)
2017 Annual Scientific Meeting, Sydney, Australia, 2017.
Increased production of apolipoprotein(a) is the primary mechanism for high lipoprotein(a)
concentrations in statin-treated hypercholesterolaemic patients – Medical School
Research Day 2019, Perth, Australia, 2019.
Posters
Increased Lipoprotein(a) Concentration in Patients with Hypercholesterolaemia on Statin
Therapy – Cardiac Society of Australia and New Zealand (CSANZ) 2017 Annual
Scientific Meeting, Perth, Australia, 2017.
Lipoprotein(a) and LDL particle metabolism following apheresis – Australian
Atherosclerosis Society (AAS) 2017 Annual Scientific Meeting, Sydney, Australia,
2017.
Awards
UWA Graduate Research Student Travel Award (EAS 2016, Innsbruck)
Australian Atherosclerosis Society Student Travel Grant (AAS 2016. Hobart)
Australian Atherosclerosis Society Student Travel Grant (AAS 2017. Sydney)
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LIST OF TABLES
Chapter Table number Table Name Page
1 1.1 Physicochemical properties and composition
of Lp(a) and LDL
5
1.2 Radiolabelled isotope kinetic studies of Lp(a)
metabolism
14
1.3 Stable isotope kinetic studies of Lp(a)
metabolism in humans
16
1.4 Influence of standard lipid-lowering therapies
on Lp(a)
40
1.5 Principles of apheresis systems 43
1.6 Influence of newer lipid-lowering therapies on
Lp(a)
44
3 3.1
3.2
Lp(a)/apo(a) methods used for studies in this
thesis
SRM transitions used for the analyte and IS
peptide measuring apolipoprotein(a)
concentration
76
78
4 4.1 Clinical and biochemical characteristics in the
subjects studied
106
5 5.1 Clinical and biochemical characteristics in the
patients studied
112
6 6.1 Clinical and biochemical characteristics of the
patients
118
6.2 Plasma concentration, production rate and 120
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fractional catabolic rate of apolipoprotein(a) in
patients with elevated and normal
lipoprotein(a) concentration
7 7.1 Clinical characteristics of 13 patients 128
7.2 Plasma lipid, lipoprotein and apolipoprotein
concentrations of the patients immediately
before and after apheresis sessions
129
7.3 Kinetic indices of lipoprotein(a) and LDL-apoB
metabolism in patients undergoing apheresis
130
7.4 Published studies reporting LDL and
lipoprotein(a) fractional catabolic rates in
patients undergoing apheresis
131
7.5 Stable isotope studies reporting LDL and
lipoprotein(a) fractional catabolic rates in
subjects and undergoing apheresis
132
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LIST OF FIGURES
Chapter Figure number Figure Name Page
1 1.1 Structure of Lp(a) 3
1.2 Structural similarities of apo(a) to
plasminogen
6
1.3 Assembly of Lp(a) 11
1.4 Catabolism of Lp(a) 12
1.5 Frequency distributions of plasma Lp(a)
concentrations and of KIV-2 repeats, and the
association between Lp(a) mass and KIV-2
repeats, in the Danish general population
18
1.6 Frequency distribution of KIV-2 CNV alleles in
African, European and Asian populations
18
1.7 Pathobiology of Lp(a) 23
1.8 Observational studies of Lp(a) as a CVD risk
factor
26
1.9 Demonstration of residual risk of elevated
Lp(a)
28
1.10 Known and proposed mechanisms of
compounds that lower Lp(a)
37
1.11 The principle of the tracer method 49
1.12 Compartmental model to describe apo(a)
tracer kinetics
52
3 3.1
3.2
Quantification of apo(a) by LC-MS method
Immunoblot of the human plasma apo(a)
77
80
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isoforms
3.3 Experimental protocol for isolation and
measurement of isotopic enrichment of Lp(a)
83
3.4 Compartmental model to describe Lp(a)-
apo(a) and Lp(a)-apoB tracer kinetics
85
3.5 Compartmental model to describe Lp-apo(a)
tracer kinetics
86
4 4.1 Association between apo(a) LC-MS and
NLMDRL method in the subjects studied
107
4.2 Bland-Altman plot in log transformed value
with limits of agreement (LOA) for apo(a) LC-
MS and NLMDRL method.
108
4.3 Bland-Altman plot in absolute concentration
data with limits of agreement (LOA) for apo(a)
LC-MS and NLMDRL method.
109
5 5.1 Isotopic enrichment curves of Lp(a)-apo(a)
and Lp(a)-apoB
113
5.2 Association between the FCR of Lp(a)-apo(a)
and Lp(a)-apoB
113
5.3 Bland-Altman plot with limits of agreement
(LOA) for Lp(a)-apo(a) and Lp(a)-apoB FCR
113
6 6.1 Isotopic enrichment curve of Lp(a)- apo(a) 119
6.2 Association of apo(a) concentration and
metabolic parameters
121
6.3 Association of apo(a) fractional catabolic rate 121
xxxii
and production rate
7 7.1 Mean plasma concentration of Lp(a) and
LDL-apoB over 7 days following apheresis
130
7.2 Percentage change of Lp(a) and LDL-apoB
over 7 days following apheresis
130
xxxiii
LIST OF ABBREVIATIONS
The following abbreviations and terms are used in this thesis.
Abbreviations Full Name
ACN Acetonitrile
AIC
AIM-HIGH
Apo
Apo(a)
ApoB
ASCVD
ASO
Akaike information criterion
Atherothrombosis Intervention in Metabolic Syndrome with
Low HDL/High Triglyceride and Impact on Global Health
Outcomes
Apolipoprotein
Apolipoprotein(a)
Apolipoprotein B-100
Atherosclerotic cardiovascular disease
Antisense oligonucleotide
BMI Body mass index
CAD
CAVD
Coronary artery disease
Calcific aortic valve disease
CE
CES
Cholesterol ester
Cholesterol esterase
CETP Cholesterol ester transfer protein
CHD Coronary heart disease
CI
CKD
Confidence interval
Chronic kidney disease
CNV
CO
Copy number variation
Cholesterol oxidase
xxxiv
CSANZ
CV
Cardiac Society of Australia and New Zealand
Coefficient of variation
CVD
DALI
Cardiovascular disease
Direct adsorption of lipoproteins
DBP
DLCN
EC
Diastolic blood pressure
Dutch Lipid Clinic Network
Endothelial cell
ECG
ECM
EDTA
Electrocardiogram
Extracellular matrix
Ethylene diamine tetra acetic acid
ELISA
ERN
Enzyme linked immunosorbent assay
Extended release niacin
FCR Fractional catabolic rate
FDA
FSR
Food and Drug Administration
Fractional synthetic rate
GC-MS
GM
Gas chromatography-mass spectrometry
Geometric Mean
HDL
HDL-C
HeFH
HoFH
HELP
HMG-CoA
High density lipoprotein
High density lipoprotein cholesterol
Heterozygous familial hypercholesterolaemia
Homozygous familial hypercholesterolaemia
Heparin-induced extracorporeal LDL-precipitation
3-hydroxy-3-methyglutaryl-coenzyme A
HOMA
HR
Homeostasis model assessment
Hazard ratio
xxxv
IDL
IFCC
JUPITER
Intermediate density lipoprotein
International Federation of Clinical Chemistry
Justification for the Use of Statins in Prevention: An
Intervention Trial Evaluating Rosuvastatin
KIV-2
LA
LC-MS
Kringle IV type 2
Lipoprotein apheresis
Liquid chromatography-mass spectrometry
LDL
LDL-C
Low density lipoprotein
Low density lipoprotein cholesterol
LDL-R
LIPID
LOA
LRP
LRP1
LRP2
Low density lipoprotein receptor
Long-Term Intervention with Pravastatin in Ischaemic
Disease trial
Limits of agreement
Low density lipoprotein receptor-related protein
Low density lipoprotein receptor-related protein 1
Low density lipoprotein receptor-related protein 2
Lp(a)
Mab
MACE
MAP
MBGP
Lipoprotein(a)
Monoclonal antibody
Major adverse coronary event
Mean arterial pressure
Magnetic beads G-proteins
MTP
NHLBI
Microsomal triglyceride transfer protein
National Heart, Lung and Blood Institute
NHMRC
NLMDRL
National Health and Medical Research Council
Northwest Lipid Metabolism and Diabetes Research
xxxvi
OR
OxPL
PCSK9
Laboratories
Odds Ratio
Oxidised phospholipids
Proprotein convertase subtilisin/kexin type 9
PR Production rate
PS
PVDF
REVEAL
Pool size
Polyvinylidene fluoride
Randomized Evaluation of the Effects of Anacetrapib
through Lipid Modication
SAAM II Simulation, analysis and modelling software II
SAE Serious adverse event
SBP
SD
SDS
Systolic blood pressure
Standard deviation
Sodium dodecyl sulphate
SEM
SMC
SPSS
SR-B1
SRM
Standard error of the mean
Smooth muscle cell
Statistical Package for the Social Sciences
Scavenger receptor class B type 1
Selected reaction monitoring
T2D
TC
Type 2 diabetes
Total cholesterol
TFA Trifluoroacetic acid
TFAA Trifluoroacetic anhydride
TG
TRL
Triglyceride
Triglyceride-rich lipoproteins
xxxvii
TTR Tracer-tracee ratio
VLDL Very low density lipoprotein
VLDL-R
VTE
WHO
Very low density lipoprotein receptor
Venous thromboembolism
World Health Organization
2
1.1 General introduction to the role of lipoprotein(a) in
human health and diseases
Lipoprotein(a) [Lp(a)] is a plasma lipoprotein containing a low-density lipoprotein (LDL)-like
particle with one molecule of apolipoprotein B-100 (apoB-100) covalently bound through a
disulphide bond to a larger glycoprotein, apolipoprotein(a) [apo(a)]. An elevated plasma
concentration of Lp(a) is associated with cardiovascular disease (CVD). Due to the
association between elevated blood lipid concentrations and the risk of CVD, it is important
to understand the mechanisms, pathways and factors that regulate lipid and lipoprotein
metabolism. Understanding these pathways and associations will help to understand the
pathophysiology of dyslipidaemia and to design more effective interventions to lower CVD
risk.
One of the most common treatments for dyslipidaemia, a risk factor for CVD, is statins.
Statins have been shown to lower LDL-cholesterol (LDL-C) concentration and CVD
mortality by >20% in clinical trials (Baigent et al, 2005; Mills et al, 2008). However,
significant residual CVD risk remains even in patients on maximally tolerated statin therapy
(Mora, Glynn et al, 2012). Recent studies have reported that 30–50% of high-risk patients
have elevated plasma Lp(a) concentration (Mora, Wenger et al, 2012). Lp(a) has also
been shown to increase CVD risk independent of LDL-C and triglyceride concentrations
(Hoover-Plow & Huang, 2013; Nicholls et al, 2010; Parhofer, 2011). In the past few years,
major advances have been made in understanding the causal role of increased Lp(a)
concentration in CVD (Danesh et al, 2000; Kamstrup et al, 2013). Mendelian
randomisation studies have suggested that there is a causal role of Lp(a) in CVD
(Kamstrup et al, 2009). There is an inverse relationship between apo(a) isoform size and
Lp(a) plasma concentration. Typically, small apo(a) isoforms are associated with higher
3
CVD risk, and the type of apo(a) isoform may play a role in the metabolism of Lp(a). Given
that individuals with similar Lp(a) concentration may have different CVD risk (Koschinsky &
Marcovina, 2004), the relationship between Lp(a) concentration and metabolism should be
investigated further.
1.2 Molecular aspects of lipoprotein(a)
1.2.1 Structure and function
Structure
Lp(a) has a similar structure to LDL, with a core of a polar lipid surrounded predominantly
by a phospholipid monolayer with glycoproteins. LDL comprises a single glycoprotein,
apoB-100, whereas Lp(a) contains apo(a) bound to apoB-100 via a disulphide bond
(Figure 1.1). ApoB and apo(a) are present in Lp(a) in a 1:1 molar ratio (Nordestgaard et al,
2010).
Figure 1.1 Structure of Lp(a) (adapted from Nordestgaard et al, 2010).
4
Apo(a) is highly glycosylated and comprises O-linked oligosaccharides, which may affect
the apo(a) functionality by increasing resistance to proteolytic cleavage and thus affecting
the catabolism of Lp(a) and contributing to the atherogenic properties of this lipoprotein
particle (Garner et al, 2001).
Lp(a) typically has a higher density (1.006–1.125 g/mL) than LDL (1.019–1.063 g/mL), but
the range of particle size is dependent on the size of Lp(a), which implies that Lp(a) can be
found in the LDL and high-density lipoprotein (HDL) size range. However, a higher molar
ratio of phospholipids, free cholesterol, cholesterol esters and triglyceride was reported in
Lp(a) than in LDL (Table 1.1). This finding suggests that the lipoprotein component of
Lp(a) may be less dense than LDL (Fless et al, 1986).
Apo(a) contains 12–51 kringle IV (KIV)-like units as well as a KV-like unit and a protease
domain similar to that in plasminogen (Figure 1.2). The apo(a) protease domain contains
an amino acid substitution that prevents cleavage by plasminogen activators as well as
deletion (Gabel et al, 1995). There are 10 types of KIV in apo(a) based on amino acid
sequence, and all but KIV type 2 (KIV-2) are present in a single copy. KIV-2 has variable
numbers (1 to >40) depending on the allele size of LPA (Lackner et al, 1993), which
contributes to remarkable size polymorphism in Lp(a) across the population.
The structure of the Lp(a) particle, as well as its cellular distribution and metabolism, is due
to KIV units containing either strong or weak lysine-binding sites. Oxidative modification of
Lp(a) also induces biochemical and functional changes that promote atherogenesis
(Weisel et al, 2001). The assembly of Lp(a) particles occurs by non-covalent docking of
the KIV-5 to KIV-8 domains to the N-terminus of apoB-100, which leads to the covalent
binding of apo(a) to apoB-100 through a disulphide bond between the unpaired cysteine in
apo(a) in KIV-9 (Schmidt et al, 2016).
5
Table 1.1 Physicochemical properties and composition of Lp(a) and LDL (adapted
from Lippi & Guidi, 2000)
Lp(a) LDL
Physico-chemical properties
Molecular mass (kDa) 3800–4000 2900
Diameter (nm) 28.3 ± 0.5 25.9 ± 0.1
Density (g/mL) 1.006–1.125 1.019–1.063
Composition (%)
Protein 17–29 26–31
Carbohydrates 8 2
Free cholesterol 6–9 9
Esterified cholesterol 35–46 40–43
Triglycerides 4–8 4–6
Total phospholipids 17–24 22
6
Figure 1.2 Structural similarities of apo(a) to plasminogen (adapted from Boffa &
Koschinsky, 2016).
Different KIV domains in apo(a) are responsible for its specific functions. KIV-9 comprises
a single unpaired cysteine that is involved in the disulphide linkage to apoB-100 in Lp(a)
(Koschinsky et al, 1993). KIV-10 contains a high-affinity lysine-binding site, whereas KIV-
5–8 each contains a low-affinity lysine-binding site (Ernst et al, 1995; Rahman et al, 2002).
The low-affinity binding sites in KIV-7 and KIV-8 have been shown to facilitate the initial
non-covalent interactions with lysine residues in apoB that lead to the formation of
disulphide bonds during Lp(a) assembly (Becker et al, 2001; Gabel & Koschinsky, 1998).
The KIV-10 lysine-binding site mediates interaction of Lp(a) with lysine-containing
substrates because the low-affinity binding sites are masked in the context of the Lp(a)
particle (Ernst et al, 1995). The KIV-10 domain has also been shown to be a key potential
mediator of Lp(a) pathogenicity (Hughes et al, 1997).
Function
The physiological function of Lp(a) remains unclear, and several functions have been
proposed. Components of the Lp(a) particle may assist in linking the cholesterol transport
7
and the fibrinolytic system (Miles & Plow, 1990). Some studies have shown that Lp(a)
modulates coagulation and fibrinolysis under in vitro conditions (Boffa & Koschinsky, 2015;
Koschinsky, 2006). Lp(a) may also be involved in tissue repair. Apo(a) is recognised by
different molecules and receptors on the surface of macrophages, endothelial cells and
fibroblasts (Hughes et al, 1997; Pillarisetti et al, 1997). Studies have shown interactions
between Lp(a) and components of the vascular wall and extracellular matrix, including
fibrin (Kostner & Bihari-Varga, 1990). It has been suggested that transport of cholesterol to
the sites of injury and wound healing may occur through the binding of Lp(a) to fibrin
(Brown & Goldstein, 1987). Recent studies have suggested that Lp(a) functions as a
preferential carrier of oxidised phospholipids OxPLs. The atherogenicity of Lp(a) may be
partly due to the correlation of Lp(a) concentration with OxPL (Tsimikas et al, 2005). The
KIV-10 lysine-binding site has been suggested to mediate the interaction between OxPLs
and Lp(a) (Leibundgut et al, 2013). Pathobiology of Lp(a) and its association with OxPLs
are later discussed in section 1.4.3.
1.2.2 Genetics
The concentration of Lp(a) can vary up to a 1000-fold and is partly determined by
polymorphisms in the gene, LPA, which codes for apo(a). LPA is located on the long arm
of chromosome 6 at 6q2.6–2.7, adjacent to the human plasminogen gene. Despite sharing
a high degree of homology, plasminogen is almost universally expressed whereas LPA
expression is limited to just a few species such as old-world monkeys, apes and humans
(Lawn, 1996; Lawn et al, 1995). The human LPA likely appeared through duplication of the
plasminogen gene during primate evolution about 40 million years ago. LPA is also
present in the European hedgehog, where it is unrelated to the human apo(a) gene, which
suggests that these genes evolved independently in the human and hedgehog (Lawn et al,
8
1997).
The most common LPA polymorphism is the KIV-2 size polymorphism, a copy number
variant that is defined by a variable number of repeats, which results in differently sized
isoforms of apo(a). The number of KIV-2 repeats correlates inversely with Lp(a)
concentration (Kamstrup et al, 2009). There are >20 different apo(a) isoforms with varying
molecular weight (300–800 kDa); the smaller isoforms are associated with higher plasma
Lp(a) concentration (Parhofer et al, 1999).
The copy number variation (CNV) of Lp(a) is an independent risk factor for coronary artery
disease (CAD) (Wu et al, 2014). CNV is defined by the changes in DNA segments that
involve deletions, insertions and duplications. KIV-2 CNV is a multi-allelic CNV harboured
by the LPA locus. The number of KIV-2 copies varies from 1 to >40, and this explains
>95% of the size heterozygosity in most ethnic groups (Enkhmaa et al, 2016).
The heterogeneous distributions of the different sized LPA alleles vary according to ethnic
background. Asians have distribution of KIV-2 CNV shifted towards the longer alleles in
comparison to Africans and Europeans, who show similar size distributions. The function
of KIV-2 CNV has not been established. However, the kringle domains are known to be
independent structural and functional domains, and their main functions seem to involve
the interactions of domains and other proteins (Schmidt et al, 2016).
The plasma Lp(a) concentration and LPA genotype have a clear association with CVD risk.
A recent study established that elevated plasma Lp(a) concentration and its corresponding
LPA risk genotype are associated with an increased risk of heart failure. This association
also appears to be partly facilitated by myocardial infarction and aortic valve stenosis (See
section 1.4).
9
1.2.3 Metabolism
Despite evidence supporting the concept that Lp(a) is a causal risk factor for CVD, the
metabolism of Lp(a) in humans is poorly understood. The primary site of Lp(a) synthesis is
thought to be the liver (Hoover-Plow & Huang, 2013). However, the site of Lp(a) assembly
remains controversial, and studies have reported both the intracellular and extracellular
assembly of Lp(a) (Kostner & Kostner, 2017). The synthesis and secretion of apo(a) of
different sizes is also not well understood.
Plasma Lp(a) concentration and isoform size are determined mainly by LPA. The inverse
association between Lp(a) concentration and isoform size is mainly dependent on the
hepatic production of apo(a) as opposed to the rate of catabolism (Jenner et al, 2005).
Apo(a) enters the endoplasmic reticulum lumen where it is folded and transported to the
Golgi apparatus or may also be released into the proteasome pathway (Kronenberg, 2016).
Most large apo(a) isoforms are degraded, whereas most small isoforms are secreted
(Edelstein et al, 1994; Wang et al, 1999).
An early study on Lp(a) compare the specific binding of Lp(a) and LDL to cultured human
fibroblasts (Krempler et al, 1980). Lp(a) was shown to exhibit some specific binding and to
suppress the activity of 3-hydroxy-3-methyglutaryl-coenzyme A reductase but to a lesser
extent as LDL.
A study injected iodine-labelled Lp(a) and LDL into rabbits and measured the decay in
plasma radioactivity and the uptake of radiolabelled isotopes into different organs (Scanu,
2012). The liver accumulated the most radiolabelled Lp(a) and LDL, followed by the kidney,
spleen, brain and gut. These studies had limitations, such as the use of a heterologous
10
system in which LDL and Lp(a) were degradable. Nevertheless, this study provided
information about the primary site for Lp(a) metabolism (Scanu, 2012).
Lp(a) assembly
The site of Lp(a) assembly remains controversial. Studies have demonstrated that Lp(a)
assembly may be extracellular, intracellular or on the hepatocyte surface (Figure 1.3). The
assembly of Lp(a) has been reported as extracellular in baboon hepatocytes (White et al,
1994). However, the assembly of Lp(a) was found to be intracellular in a study of primary
human hepatocytes (Bonen et al, 1997). An in vivo study established that Lp(a) is broken
down and reassembled in the plasma and that apo(a) does not remain covalently linked to
apoB (Demant et al, 2001). Another study suggested that Lp(a) is assembled intracellularly
from apo(a) and newly synthesised LDL (Frischmann et al, 2012). The formation of Lp(a)
occurs through the extracellular coupling of apo(a) to apoB-100 on the surface of
hepatocytes (Kronenberg, 2016). There are uncertainties as to whether very low-density
lipoprotein (VLDL) binds to apo(a) initially. However, one study suggested that Lp(a) in
triglyceride-rich lipoprotein (TRL) is unlikely to be the precursor for Lp(a) in the LDL and
HDL size range (Reyes-Soffer et al, 2017). Additionally, elevated Lp(a) levels in FH may
potentially be associated with VLDL-independent, direct secretion of LDL that occurs in FH
patients (Soutar et al, 1977; Teng et al, 1986). After hepatic secretion of Lp(a), apo(a) may
dissociate from the parent lipoprotein and reassemble with apoB-100-containing particles
in the circulation (Jenner et al, 2005), but not all studies are consistent with this notion
(Krempler et al, 1980).
Free apo(a) is not detected at significant concentrations in plasma, which contradicts the
notion that apo(a) recycles in the circulation (Reyes-Soffer et al, 2017). Plasma Lp(a)
11
concentration is inversely associated with triglyceride concentration, which suggests a
possible metabolic relationship between Lp(a) coupled to TRLs and triglycerides. Lowering
plasma triglyceride concentration in hypertriglyceridaemic subjects has also been shown to
elevate plasma Lp(a) concentration (Hiraga et al, 1993). Hepatic apoB-100 synthesis may
be increased through an intracellular PCSK9–apoB-100 protein interaction and inhibition of
apoB-100 degradation (Sun et al, 2012), which may increase the coupling of apoB-100
with apo(a) and, thus, Lp(a) assembly.
Figure 1.3 Assembly of Lp(a). Three possible sites have been proposed: (A)
intracellular; (B) cell surface; and (C) extracellular (adapted from Hoover-Plow &
Huang 2013).
12
Lp(a) catabolism
Although the liver is the primary site of Lp(a) catabolism, the kidney and artery wall may
also clear Lp(a) from plasma. Many receptors have been proposed to mediate the
clearance of Lp(a) from the liver, including the LDL receptor (LDL-R) (Floren et al, 1981;
Tam et al, 1996) and other members of the LDL-R family such as the VLDL receptor
(VLDL-R) (Argraves et al, 1997) and the LDR-R-related protein 1 (LRP1) (Reblin et al,
1997) and LRP2 (Niemeier et al, 1999). Catabolism by scavenger receptor class B type 1
(SR-B1) (Yang et al, 2013) and plasminogen receptor (Tam et al, 1996) may also play a
role (Figure 1.4).
Figure 1.4 Catabolism of Lp(a). Apo(a) may be degraded to fragments in the plasma,
liver or other tissues, and the fragments excreted into the urine (A). Lp(a) and apo(a)
and its fragments bind to extracellular matrix proteins in the vascular wall (B). Lp(a)
may bind to VLDL-R or megalin and be taken up by hepatocytes, fibroblasts or
macrophages (C) (adapted from Hoover-Plow & Huang 2013).
13
Lp(a) catabolism may be related to LDL catabolism because of similarity in structures.
However, the role of the LDL-R in Lp(a) clearance remains controversial. Several studies
have demonstrated that LDL-R plays a role in Lp(a) binding and uptake (Havekes et al,
1981; Krempler et al, 1983). In mice, the clearance of Lp(a) is significantly increased when
LDL-R is overexpressed (Hofmann et al, 1990). The FCR of Lp(a) is not altered in
homozygous FH (HoFH) compared with healthy controls (Rader et al, 1995), and Lp(a)
catabolism is similar between LDL-R-knockout and wild-type mice (Cain et al, 2005). The
LDL from Lp(a) can only be catabolised by the LDL-R after apo(a) has been released,
which implies that apo(a) may mask the apoB-100-binding domain of the receptor (Knight,
1994). Statins lower LDL-C concentration by upregulating LDL-R activity but do not affect
plasma Lp(a) concentration (Slunga et al, 1992). The VLDL-R is expressed in the heart,
skeletal muscle and adipose tissue, but has a low expression level in the liver (Gåfvels et
al, 1993; Webb et al, 1994). A study of transgenic mice showed that Lp(a) can be
catabolised by the human VLDL-R (Argraves et al, 1997). In cells expressing LRP2, the
uptake and degradation of Lp(a) is about 2-fold greater than that of controls lacking this
receptor. LRP1 plays a role in the clearance of protease-inhibitor complexes and
chylomicron remnants, but the role of Lp(a) remains controversial (März et al, 1993). Cell
culture studies suggest that SR-B1 may also serve as a receptor for Lp(a). A study of SR-
B1-overexpressing mice reported increased plasma clearance of exogenously added Lp(a)
compared with SR-B1-deficient mice (Yang et al, 2013). Another study has shown that
genetic variation within the gene encoding SR-B1 is associated with high concentrations of
both HDL-C and Lp(a) (Yang et al, 2016). Additionally, Lp(a) internalisation and recycling
were reduced in human hepatoma cells in which the plasminogen receptor KT was
knocked out (Sharma et al, 2017).
14
Table 1.2 Radiolabelled isotope kinetic studies of Lp(a) metabolism
Patients Type of
Detection
FCR (pools/day) PR
(mg/kg/day)
Literature Ref
Apo(a)
heterozygotes
(n=3)
EI Lp(a) – 0.25 Lp(a) – 7.9 Rader et al, 1994
Normal (n=11) EI Lp(a) – 0.32 Lp(a) – 2.1 Rader et al, 1993
Normal (n=9) EI Lp(a) – 0.31 Lp(a) – 5.0 Krempler et al,
1980
Normal (n=13) GC-MS Lp(a) – 0.26 Lp(a) – 4.6 Krempler et al,
1983
GC-MS, gas chromatography–mass spectrometry; EI, electroimmunoassay. Apo(a)
heterozygotes refers to isoform alleles.
Additionally, plasma Lp(a) concentration is significantly higher in familial
hypercholesterolaemia (FH) patients with an LDL-R null allele than in controls (Alonso et al,
2014). However, it appears that under physiological conditions, the LDL-R does not play a
major role in the catabolism of Lp(a) (Kostner & Kostner, 2017). Many studies have utilised
a radiolabelled (Table 1.2) or stable isotope (Table 1.3) to determine the metabolic
parameters including fractional catabolic rate (FCR) and production rate (PR). A study
concluded that LDL and Lp(a) are cleared via the same mechanism (Krempler et al, 1980).
By contrast, A study involving patients with HoFH demonstrated that LDL-R is not
important for the catabolism of Lp(a) (Rader et al, 1995). In another turnover study,
elevated Lp(a) concentration in FH patients was shown to be caused by defective LDL-R
and PCSK9 gain-of-function mutations. It has been suggested that the effects of Lp(a)
15
concentration depend on the type of mutation in the LDL-R or other mechanisms that
affect the Lp(a) concentration. In addition, PCSK9 inhibitors, which act through the LDL-R
pathway, lower the Lp(a) concentration by about 30%, whereas statins do not.
Although the liver is the main site of Lp(a) catabolism, there is significant evidence
supporting the role of the kidney in Lp(a) clearance (Figure 1.4). Plasma Lp(a)
concentration is increased in patients with chronic kidney disease (CKD), and their Lp(a)
concentration generally decreases after successful kidney transplantation (Kronenberg et
al, 1996; Kronenberg et al, 1994). In people with normal renal function, Lp(a) concentration
is significantly lower in the renal vein than in the ascending aorta, which implies that Lp(a)
is removed by the renal circulation (Kronenberg et al, 1997).
16
Table 1.3 Stable isotope kinetic studies of Lp(a) metabolism in humans
Patients Type of
detection
FCR
(pools/day)
PR
(nmol/kg/day)
Reference
Healthy (n=9) GC-MS Apo(a) – 0.25
ApoB – 0.30
Apo(a) – 1.16
ApoB – 1.28
Frischmann et al, 2012
Haemodialysis
(n=7) GC-MS Apo(a) – 0.16
ApoB – 0.13
Apo(a) – 1,10
ApoB – 0.83
Frischmann et al, 2007
Healthy (n=3) LC-MS Apo(a) – 0.22 Apo(a) – 1.28 Zhou et al, 2013
Lp(a)
concentrations
Low (n=6)
Intermediate (n=8)
High (n=9)
GC-MS Low
Apo(a) – 0.43
ApoB – 0.76
Intermediate
Apo(a) – 0.29
ApoB – 0.48
High
Apo(a) – 0.14
ApoB – 0.31
Low*
Apo(a) – 0.14
ApoB – 0.42
Intermediate*
Apo(a) – 0.37
ApoB – 0.93
High*
Apo(a) – 0.65
ApoB – 2.19
Jenner et al, 2005
Healthy (n=7) GC-MS Apo(a) – 0.27
ApoB – 0.26
Apo(a) – 0.15*
ApoB – 0.15*
Demant et al, 2001
Normal (n=5) GC-MS Apo(a) – 0.16
ApoB – 0.14
Not reported Gaubatz et al, 1993
GC-MS, gas chromatography–mass spectrometry; LC-MS, liquid chromatography–mass
spectrometry. *reported in mg/kg/per day
17
1.3 Epidemiology of lipoprotein(a)
Epidemiological studies using the Mendelian randomisation approach have shown that
elevated Lp(a) concentration is a risk factor for CVDs including, atherosclerosis,
myocardial infarction and aortic valve stenosis (Nordestgaard & Langsted, 2016).
1.3.1 Lipoprotein(a) distribution and reference values
Plasma Lp(a) concentration has a positively skewed distribution (Figure 1.5) and can vary
by more than a 1000-fold between people within the same population (Crawford et al,
2008). The correlation between the apo(a) size polymorphism and Lp(a) plasma
concentrations was found to vary greatly among individuals of different ethnicity, as do
plasma Lp(a) concentrations (Marcovina et al, 1996; Sandholzer et al, 1991). One study
found that the apo(a) allele frequencies were different among different populations
(Sandholzer et al, 1991). In that study, the size variation of apo(a) explained from 19% in
Sudanese to 77% in Malays of the variability in plasma Lp(a) concentrations.
The frequency distributions of different sized LPA alleles are heterogeneous across ethnic
groups. As shown in Figure 1.6, Asian people have a frequency distribution of KIV-2 CNV
sizes shifted toward the longer alleles compared with African and European people with
similar size distributions (Schmidt et al, 2016).
18
Figure 1.5 Frequency distributions of (A)
plasma Lp(a) concentrations and of (B)
KIV-2 repeats, and (C) the association
between Lp(a) mass and KIV-2, in the
Danish general population (Figure from
Nordestgaard & Langsted, 2016).
Figure 1.6 Frequency distribution of KIV-2 CNV alleles in African, European and
Asian populations. Short repeats are more frequent in Africans and long repeats in
Asians (Figure from Schmidt et al, 2016).
19
1.3.2 Geographic variation in lipoprotein(a) concentration
In the general population, plasma Lp(a) concentrations vary greatly between individuals
and between different ethnicities. People of African descent have higher Lp(a)
concentrations compared with those from European or Asian backgrounds. The mean
Lp(a) concentration in people of African descent is about twice that in Caucasians. A
heterogeneous distribution of Lp(a) concentration has been reported in Asian populations;
for example, Lp(a) concentration is higher in Indian than in Chinese people. The Lp(a)
distribution pattern among Chinese is closer to that of Caucasian populations, whereas the
pattern in Indians is intermediate between those of African and Caucasian populations
(Knapp et al, 1993; Srinivasan et al, 1991).
1.3.3 Genetic epidemiology
Studies involving Mendelian randomisation design have shown that elevated Lp(a)
concentration predict a 3-fold increased risk in the general population. In particular,
rs10455872 and rs3798220 minor alleles and low KIV-2 repeats carriers are at increased
risk of CVD. The rs10455872 variant is significantly associated with aortic valve stenosis.
This association has been found in many ethnic groups, particularly in European, African
and Hispanic cohorts (Kamstrup et al, 2014; Thanassoulis et al, 2013).
1.3.4 Physiological determinants
Lp(a) concentration is primarily genetically determined in more than 90%, and factors such
as physical activity, sex and age appear to make very small contributions. However, some
20
studies have shown that these factors may affect the Lp(a) concentration. Some of these
factors are discussed below.
Pregnancy
Studies have shown that findings in association of Lp(a) and pregnancy have been
variable. Studies have shown that Lp(a) concentrations increase 2–3-fold during
pregnancy (Zechner et al, 1986; Manten et al, 2003; Lippi et al, 2007). However, other
studies have not shown pregnancy-induced changes in Lp(a) (Rymer et al, 2002; Silliman
et al, 1994).
Menopause
Elevated Lp(a) concentration has been reported in postmenopausal women compared with
pre-menopausal women in a Japanese study, as well as in the Framingham Offspring, in
which there was no significant difference after adjustment for age (Jenner et al, 1993;
Ushioda et al, 2006). Another study showed no effect of menopause on Lp(a)
concentration in black and white women (Selby et al, 1994). By contrast, postmenopausal
women taking hormone-replacement therapy show a more consistent reduction in plasma
Lp(a) concentration compared with those not on hormone-replacement therapy (Danik et
al, 2008; Salpeter et al, 2006).
21
Sex
Studies have shown that findings in association of Lp(a) and sex have been variable.
Studies across population groups have shown no significant difference in Lp(a)
concentration between males and females. In another study, a small but significant
increase in Lp(a) concentration was observed in females compared with males for both
Africans and Caucasians (Srinivasan et al, 1991). A recent study also observed a
significantly higher Lp(a) concentration in females than in males among healthy Arabian
adolescents (Akanji et al, 2011). In a Japanese study, Lp(a) concentration was significantly
elevated in females compared with males (Nago et al, 1995). Although some studies have
indicated that elevated Lp(a) is more prominent in females than in males, there are many
potential confounders, such as ethnicity, apo(a) size distribution, menopausal status and
differences in assay methodology.
Age
Studies in newborns have shown that Lp(a) concentration increases from birth to postnatal
day 7 and an additional increase up to 8 months (Labeur et al, 1989). Studies in
adolescents have reported an increase in Lp(a) concentrations aged 11–17 years
(Srinivasan et al, 1991). Studies of adults have shown variable results. Lp(a) was
associated with age in women but not in men in one study (Jenner et al, 1993). However,
another study reported that age was significantly associated with increasing Lp(a)
concentration in both men and women (Slunga et al, 1993). Another study reported no
association between age and Lp(a) concentration (Gaw et al, 1999). Therefore, the
influence of age on Lp(a) concentration remains unclear.
22
Exercise
Many studies have been unable to demonstrate an association between Lp(a)
concentration and physical activity (MacAuley et al, 1996; Selby et al, 1994). A large study
involving Finnish children and young adults reported an inverse association between Lp(a)
concentration and physical activity (Taimela et al, 1994). Another study also reported that
physical fitness was inversely associated with Lp(a) concentration in young children and
adolescents with diabetes mellitus (Austin et al, 1993). However, other studies have
shown that exercise has no effect on Lp(a) concentration (Hellsten et al, 1989; Hubinger et
al, 1995). Other studies have not found any association between BMI and Lp(a)
concentration between men and women (Selby et al, 1994; Slunga et al, 1993).
1.4 Pathobiology of lipoprotein(a)
Lp(a) has both pro-atherosclerotic (LDL-like) and pro-thrombotic (plasminogen-like) effects.
The pro-atherosclerotic effects of Lp(a) include promoting cell adhesion, formation of foam
cells through the internalisation of oxidised Lp(a) by macrophages and direct stimulation of
vascular smooth muscle formation (Spence & Koschinsky, 2012; Figure 1.7). The pro-
thrombotic effects of Lp(a) are partly related to its protein component, apo(a), which
exhibits structural homology with plasminogen and plasmin. Therefore, Lp(a) may bind to
the extracellular matrix (ECM) and leads to altered activation and migration of cells, and
inhibition of plasminogen activation and plasmin activity (Bowden et al, 1994; Hoover-Plow
& Huang, 2013; Spence & Koschinsky, 2012).
23
Figure 1.7 Pathobiology of
Lp(a). Potential
mechanisms are
categorised into pro-
thrombotic, pro-
inflammatory, and pro-
atherosclerotic. SMC:
smooth muscle cell; EC:
endothelial cell; Plg:
plasminogen; OxPL:
oxidised phospholipids
(adapted from Ellis et al,
2017).
1.4.1 Thrombosis
Recent studies have suggested that Lp(a) is a risk factor for venous thromboembolism
(VTE). Lp(a) concentration is largely determined by the genetic variation in apo(a), one of
the protein components of Lp(a), which has structural similarities to plasminogen. Lp(a)
concentration >300 mg/L is considered to be a risk factor for VTE (Nowak-Göttl et al,
1997). Another study has shown that Lp(a) is an independent risk factor for VTE in
humans (Marcucci, 2003). However, other studies have not found a strong association
between Lp(a) concentration and VTE (Rodger et al, 2010 & Vormittag et al, 2007).
24
1.4.2 Fibrinolysis
Lp(a) may behave as an inhibitor of fibrinolysis through apo(a), which may compete with
plasminogen for fibrin interaction because of their structural similarities. The mechanism
by which Lp(a) exerts its atherogenic and/or thrombogenic effects is not clear. However,
Lp(a) is associated with fibrin deposits in atherosclerotic plaques. A study has shown that
the structural heterogeneity of apo(a) acts as a competitive inhibitor of plasminogen for
fibrin binding that inhibits the formation of plasmin and decreases fibrinolysis (Hervio et al,
1995).
1.4.3 Oxidative stress
Lp(a) can carry OxPLs and increase the expression of adhesion molecules and
chemokines, which can promote inflammation and instability in atherosclerotic plaque. A
study investigated the relationship between lipoproteins and oxidative stress in psoriasis, a
systemic disease associated with abnormalities in multiple organs and an increased risk of
CVD (Ferretti et al, 2012). Modifications of plasma lipid and lipoproteins, such as
increased concentrations of triglycerides, cholesterol, and Lp(a), were more prominent in
people with psoriasis than in controls. Oxidised LDL has been observed in lesional skin
and serum in subjects with psoriasis (Tekin et al, 2007). The associations between Lp(a)
concentration and markers of lipid peroxidation suggest that people with higher
concentration of Lp(a) are more exposed to oxidative damage (Ferretti et al, 2012).
Several mechanisms have been proposed to explain how Lp(a) influences atherosclerosis.
One of the mechanisms involves direct deposition of Lp(a) on the arterial wall, similar to
that of LDL and oxidised LDL. Lp(a) is more likely to undergo oxidation than LDL, which
25
might facilitate uptake by macrophages and lead to the formation of foam cells, precursors
of atherosclerosis (Argraves et al, 1997). Another mechanism relates to the inverse
correlation between Lp(a) concentration and vascular reactivity; that is, an increase in
Lp(a) plasma concentration may induce endothelial dysfunction (Wu et al, 2004).
1.4.4 Inflammation
The relationship between inflammation and plasma Lp(a) concentration is not clearly
understood. Some studies have suggested that inflammation elevates, reduces or does
not change Lp(a) expression and concentration (Pirro et al, 2017). Lp(a) may induce the
expression of pro-inflammatory cytokines and increase the activated state in the
endothelium, which can lead to an invasion of inflammatory cells into the arterial wall (Pirro
et al, 2017).
1.5 Significance of lipoprotein(a) in clinical medicine
1.5.1 Atherosclerotic cardiovascular disease
Studies have confirmed the association between Lp(a) concentration and the risk of
atherosclerotic cardiovascular disease (ASCVD). People with Lp(a) concentration >50
mg/dL are at 2.3-fold higher risk of ASCVD than are those with a normal Lp(a)
concentration (Kostner et al, 1981). A meta-analysis involving 27 prospective studies,
which included 5500 people, found a clear independent association between Lp(a) and
atherosclerosis (Danesh et al, 2000).
26
Elevated plasma Lp(a) concentration is associated with coronary and cerebral artery
atherosclerosis (Kolovou, 2017). Figure 1.9 represents the data from different studies
showing an increased risk of CVD with elevated Lp(a) concentration. Epidemiological
studies and meta-analyses have demonstrated that the risk of CVD is flat up to an Lp(a)
concentration of about 24 mg/dL and then increases with elevated Lp(a) concentration up
to a risk ratio of about 1.5 at an Lp(a) concentration of 192 mg/dL (Figure 1.8, left panel). A
Mendelian randomisation study demonstrated that patients with genetically elevated Lp(a)
concentration had an increased hazard ratio (HR) (Figure 1.8, middle panel). Other studies
have reported on the genome-wide association between LPA single nucleotide
polymorphisms (SNPs) and the risk of CVD. A meta-analysis using a gene score involving
LPA SNPs (rs10455872 and rs3798220) showed odds ratios (ORs) for CAD of 1.51 for
one variant and 2.57 for two or more variants (Figure 1.8, right panel).
Figure 1.8 Observational studies of Lp(a) as a CVD risk factor: (A) Classical
epidemiology, (B) Mendelian randomisation, (C) Genome-wide association (Figure
from Tsimikas, 2016).
27
Three large randomised trials, including the AIM-HIGH (Atherothrombosis Intervention in
Metabolic Syndrome with Low HDL/High Triglyceride and Impact on Global Health
Outcomes), JUPITER (Justification for the Use of Statins in Prevention: An Intervention
Trial Evaluating Rosuvastatin), and LIPID (Long-Term Intervention with Pravastatin in
Ischaemic Disease trial), found that Lp(a) concentration remained a predictor for CVD
events (Albers et al, 2013; Khera et al, 2014; Nestel et al, 2013) even as patients under
aggressive statin therapy reached LDL-C concentrations as low as 113, 53, and 62 mg/dL,
respectively (Figure 1.9).
In the AIM-HIGH study, patients were randomised to simvastatin plus placebo or
simvastatin plus extended-release niacin (ERN). ERN reduced Lp(a) concentration by 21%
but did not reduce CVD events in any quartile of Lp(a) (Figure 1.9). However, these
patients were not selected for elevated Lp(a) concentration, and it is unclear whether
different results would have been obtained in patients with elevated Lp(a).
In the JUPITER study, patients took rosuvastatin or placebo. Baseline Lp(a) concentration
was independently associated with CVD in patients on placebo (HR 1.18) and patients on
statin (HR 1.27) (Figure 1.9).
In the LIPID study, patients were randomised to pravastatin or placebo. Baseline Lp(a)
concentration was associated with total CVD, CHD, and coronary events. For events after
year 1, an increase in Lp(a) concentration was associated with adverse outcomes for total
CVD and CHD events (Figure 1.9).
28
Figure 1.9 Demonstration of residual risk of elevated Lp(a) (Figure from Tsimikas,
2016).
In a recent meta-analysis, 29069 patients from seven randomised, placebo-controlled
statin outcome trials were collated to calculate the HRs for CVD events (Willeit et al, 2018).
In this meta-analysis, elevated baseline and on-statin Lp(a) concentration were associated
with CVD risk (Willeit et al, 2018).
1.5.2 Aortic valve stenosis
Aortic valve stenosis is the most common type of valve disease, although the mechanism
is not well understood and there are no effective medical therapies. LPA seems to be the
most powerful genetic risk factor for aortic valve stenosis (Thanassoulis et al, 2013).
Elevated plasma Lp(a) concentration was found to be associated with aortic valve stenosis
(Yu et al, 2018). Lp(a) is an independent predictor of CHD based on epidemiological and
genetic studies (Garcio et al, 2017; Katsiki et al, 2017). Recent epidemiological and
Mendelian randomisation studies have shown that Lp(a) concentration was associated
with the pathogenesis of aortic valve disease and CHD. The largest epidemiological study
to investigate the association of elevated Lp(a) concentration with CHD risk included 36
29
studies with more than 126,000 participants (Ergou et al, 2009). The risk ratio for CHD was
1.13 per 3.5-fold for elevated Lp(a) concentration. The risk of CHD was constant until an
Lp(a) concentration of about 24 mg/dL, after which it increased continuously with higher
Lp(a) up to a risk ratio of 1.5 at 192 mg/dL.
1.5.3 Other medical conditions
Circulating Lp(a) concentration may be elevated in some medical conditions such as FH,
hypertension, obesity, rheumatic disease, metabolic syndrome, nephrotic syndrome, end-
stage renal insufficiency, heart failure, microvascular angina, or infection by human
immunodeficiency virus. By contrast, medical conditions such as liver failure lowers
plasma Lp(a) concentration. A study reported increased serum Lp(a) concentration in
people with type 1 diabetes mellitus (Kolovou, 2017).
Familial hypercholesterolaemia
Patients with heterozygous familial hypercholesterolaemia (HeFH) have elevated Lp(a)
concentration independent of apo(a) polymorphism (Utermann et al, 1989). A study has
demonstrated that Lp(a) was an independent risk factor for CVD in patients with FH
patients than those without FH (Santos, 2014). In another study, patients with HeFH had
approximately double the concentration of Lp(a) compared with those in the control group
(Mbewu et al, 1991). These studies suggest that people with elevated Lp(a) concentration
in the presence of high LDL-C concentration have a higher probability of having HeFH.
30
Hypertension
Studies have found no differences in Lp(a) concentration between hypertensive and
normotensive people (Flesch et al, 1994; Steinmetz et al, 1993). However, Lp(a) appears
to be a major contributor to target organ damage in hypertensive patients (Catena et al,
2005). In a multivariate analysis, Lp(a) was the best predictor of the presence of organ
damage, followed by systolic blood pressure (SBP) (Sechi et al, 1997). Organ damage in
hypertensive patients was also associated with small apo(a) isoforms, implying a genetic
predisposition for the development of organ damage (Sechi et al, 1997).
Chronic kidney disease
Patients with CKD have an increased risk of CVD events and have elevated Lp(a)
concentration. A study demonstrated a positive effect of dietary changes that involved a
supplemented vegetarian diet, which reduced Lp(a) concentration in patients with CKD
(Monzani et al, 1996).
Diabetes
Despite evidence supporting the link between Lp(a) and CVD events, the association with
diabetes is unclear. A prospective cohort study of 2308 patients with type 2 diabetes (T2D)
reported that the effect of Lp(a) on CVD events may differ between patients with T2D and
the general population (Qi et al, 2011). A recent case control study of 143,087 Icelanders
reported that low Lp(a) concentration increases T2D risk. Reduction of Lp(a) concentration
in patients with elevated to population median concentration is predicted to decrease CAD
risk without increasing T2D risk (Gudbjartsson et al, 2019).
31
1.6 Analytical biochemistry
Lp(a) has a complex structure, which poses a considerable challenge for quantifying Lp(a)
plasma concentration accurately and reliably. Most methods to measure Lp(a)
concentration have used immunochemical methods, such as the enzyme-linked
immunosorbent assay (ELISA), nephelometry, immunoturbidimetry, and dissociation-
enhanced lanthanide fluorescent immunoassay. However, many available assays use
polyclonal antibodies that have been raised against epitopes within the KIV-2 domain. As a
consequence, Lp(a) concentration is overestimated in people with a large apo(a) isoform
and underestimated in those with a small apo(a) isoform (Marcovina et al, 2018). This can
be resolved if all laboratories used molar concentrations traceable to the same reference
material with a known molar concentration and an antibody that recognises an epitope
(Brown et al, 2010; Marcovina et al, 2018).
There is currently no reference method for measuring Lp(a) concentration recommended
by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). As a
consequence, interpretation and comparison between studies may be difficult if Lp(a)
concentration is measured differently. To address this issue, the IFCC developed an Lp(a)
reference material, which was then approved by the US National Heart, Lung and Blood
Institute (NHLBI) and the World Health Organization (WHO). To evaluate this reference
material, Lp(a) was measured using 22 different commercially available assays in 30
samples with a wide range of Lp(a) concentrations and apo(a) isoform sizes. The findings
showed that there was a significant variation in Lp(a) concentration between the different
methods tested despite the use of a reference material. The inter-assay variation may
reflect differences in apo(a) isoform bias, the antibodies used, and assay precision,
robustness, and sensitivity (Marcovina et al, 2000). Currently, there are no commercial
assays that are isoform independent (Cegla et al, 2019). Assays using Denka Seiken
32
reagents are currently the most reliable commercial assay, partly due to the effect of
isoform size being reduced, by use of several calibrators covering a range of Lp(a)
concentrations (Cegla et al, 2019).
Estimation of Lp(a) concentration has involved mainly the mass concentration for the past
decades. However, methods to measure the mass concentration result in poor estimations
because of variability in apo(a) and particle lipid content. Recent studies have reported
Lp(a) in molar concentration based on 1:1 relationship of apo(a) to Lp(a) particle number.
Simple conversion between mass and molar units is unreliable because of the issues of
variability in particle mass, assay specificity, calibration, and traceability. As stated above,
the major complications in measuring Lp(a) concentration relate to the different methods
used. Therefore, to minimise measurement variability and inconsistency, a common
guideline that uses one method to measure Lp(a) concentration specifically is required.
1.6.1 Principle of the assays
An accurate and precise measurement of Lp(a) requires the following.
• The antibody should be specific to the Lp(a) being measured.
• The Lp(a) being measured should have the same structural characteristics as the
analyte in the assay calibrator.
• To have consistent and comparable results, an accuracy-based target value should
be assigned to the assay calibrator.
33
1.6.2 Description of the assays
A variety of immunochemical methods, such as ELISA, immunoturbidimetry, nephelometry,
and dissociation-enhanced lanthanide fluorescent immunoassay, are used for the
measurement of Lp(a) concentration. Each of these is discussed below.
ELISA
Lp(a) concentration can be measured using ELISA. The Northwest Lipid Metabolism and
Diabetes Research Laboratories (NLMDRL) method uses an ELISA method, which is
considered a well established assay for apo(a) measurement in human plasma or serum
(Tsimikas et al, 2018). The assay involves the use of a monoclonal antibody (mAb) that
specifically targets the epitope present in the apo(a) KIV-9 domain and is independent of
apo(a) isoform size (Marcovina & Albers, 2016).
Immunoturbidimetric method
The immunoturbidimetric method is a well-established method for measuring Lp(a)
concentration. This method uses an antibody specific to Lp(a), which causes agglutination
when dispensed to a sample containing Lp(a), and measures turbidimetry.
Nephelometric assay
The nephelometric assay to measure serum Lp(a) concentration involves the use of a
specific antibody to Lp(a), which causes agglutination and generate a high rate of light-
scatter formation (Gillery et al, 1993).
34
Dissociation-enhanced lanthanide fluorescent immunoassay
Dissociation-enhanced lanthanide fluorescent immunoassay involves the use of a
polyclonal antibody against apoB and a polyclonal antibody against Lp(a). The antibodies
are both labelled with lanthanides to improve the specificity and sensitivity of the assay
(Hemmilä, 1988).
Liquid chromatography–mass spectrometry method
Apo(a) concentration can be measured using a liquid chromatography–mass spectrometry
(LC-MS) system. It involves the use of a signature peptide LFLEPTQADIALLK, which was
selected using a protein sequence database. The peptide was selected to maximise
sensitivity, specificity, and stability. This specific peptide mimics the proteolytic domain
located in the peptidase S1 region of apo(a) (Croyal et al, 2015).
1.6.3 Evaluation and comparison of assays
The nephelometry assay of Lp(a) concentration generates weak signals depending on the
antibodies used, which can lead to misinterpretation of data because of background noise
and or nonspecific reactions (Gillery et al, 1993). The dissociation-enhanced lanthanide
fluorescent immunoassay has minimal sensitivity, and this limits its use in analyses of
Lp(a) present at elevated concentrations (Hemmilä, 1988).
The LC-MS method to measure apo(a) concentration is very sensitive and precise. A study
has shown that this method is reproducible and has a high degree of robustness (Zhou et
al, 2013).
35
The different methods for measuring Lp(a) and apo(a) are compared in Chapter 4.
1.6.4 Standardisation and precision
A recent study evaluated the Lp(a) measurements using the following MAbs:
• MAb a-6 targets the epitope located in apo(a) KIV-2
• MAb a-5 targets the epitope located in both apo(a) KIV-1 and KIV-22
• MAb a-40 targets the unique apo(a) epitope in KIV-9
The analysis included 723 samples for the measurements of Lp(a) with various MAbs. The
average bias between MAb a-5 and MAb a-40 was highly correlated with the number of
apo(a) isoform sizes in the sample. Lp(a) concentrations were higher using a-5 than using
a-40 in samples with >21 apo(a) isoform sizes and were lower in samples containing <21
isoform sizes. The measured Lp(a) concentrations in samples containing 18 apo(a)
isoform sizes were underestimated by 10%, whereas in samples with 25 isoform sizes
were overestimated by 20%. The results of this study clearly show that apo(a) size
heterogeneity has a significant effect on the accuracy of Lp(a) measurements (Marcovina
& Albers, 2016). The standard used was a purified Lp(a) that had been isolated from
human plasma containing an apo(a) with 21 KIV motifs. The standard was used to
calibrate the assay, and multiples analyses were performed over a period of days. The
means of the values were the assigned values of the assay calibrator (Marcovina & Albers,
2016).
36
1.6.5 Analytical issues and limitations
The variability in the size of apo(a) poses difficulty in the development of immunoassays to
measure Lp(a) concentration in plasma and serum samples. The variability of the size in
apo(a) reflects the expression of two different genetically determined apo(a) isoforms
(Marcovina & Albers, 2016). At a recent meeting, Dr. Marcovina stated that Lp(a)
concentration is underestimated in samples with small apo(a) isoform size, smaller than
the isoforms in the calibrator, and that values are overestimated in samples with isoforms
larger than those in the calibrator (Marcovina et al, 2018).
To develop appropriate immunoassays to measure Lp(a) concentration, a suitable
antibody is required. Using a reducing agent to dissociate apo(a) from Lp(a) and using an
antibody against apo(a) may be a way to measure the concentration of Lp(a) accurately.
However, many of the reducing agents may not work well in specifically dissociating the
disulphide bond between apo(a) and apoB because it may also cleave the three intra-
chain disulphide bonds in each of the kringle domains. The cleavage of these bonds
contributes to the altered apo(a) conformation and may therefore make the method
inaccurate. Given the size variation in apo(a) between and within individuals, the ability to
use a single assay calibrator to measure Lp(a) accurately is questionable. To do so will
require specific antibodies directed at the KIV-2 repeats appearing in each person.
1.7 Treatments for elevated lipoprotein(a) concentration
Elevated Lp(a) concentration has been shown to play a causal role in premature CVD in
many studies. Although a beneficial outcome of lowering Lp(a) concentration has not been
37
observed, many clinical and experimental studies, including Mendelian randomisation
studies, have demonstrated a causal relationship between Lp(a) concentration and CVD.
Figure 1.10 Known and proposed mechanisms by which compounds may lower
Lp(a) concentration (adapted by Bos et al, 2014).
1.7.1 Standard/older lipid-lowering pharmacotherapies
Statins
The effect of statins on Lp(a) concentration is controversial. Statins lower LDL-C
concentration and may either not affect or increase Lp(a) concentration (Table 1.4); the
effects may be dose dependent (Ky et al, 2008). For example, a study has shown an
38
approximate 30% increase in Lp(a) concentration in a patient group treated with statins
compared with a placebo group (Kostner et al, 1989). In another study, 3896 people on
statins had an overall 11% increase in Lp(a) concentration and a 24% increase in OxPLs
contained in apoB-100 (Yeang et al, 2016). In a recent study, statins significantly
increased the plasma concentration of Lp(a). The mean percentage change from baseline
ranged from 8.5% to 19.6% in the statins group and –0.4% to –2.3% in the placebo group
(Tsimikas et al, 2019).
Niacin
Nicotinic acid (niacin) targets apoB-containing lipoproteins and ultimately reduces Lp(a)
concentration. In the AIM-HIGH trial, niacin and background statin therapy was shown to
reduce Lp(a) concentration by 19% compared with the placebo. However, there were no
beneficial effects on CVD outcomes (Albers et al, 2013). Studies have shown that the
reduction in Lp(a) concentration by niacin results primarily from the reduction in apo(a) and
apoB-100 production (Croyal et al, 2015; Ooi et al, 2015; Figure 1.10).
Fibrates
Fibrates, also known as fibric acid derivatives, are agonists of the peroxisome proliferator-
activated receptor. Fibrates effectively raise HDL-C concentration and lower triglyceride
concentration (Birjmohun et al, 2005). The proposed mechanism occurs through activation
of transcription factors that mediate the synthesis of apolipoproteins, apoA, and other lipids,
increase the expression of lipoprotein lipase, and decrease the synthesis of hepatic apoC-
III (Staels et al, 1998; Auwerx et al, 1996). In terms of Lp(a) lowering, gemfibrozil reduced
39
the concentration of Lp(a) and significantly decreased plasma triglyceride concentration
(Fereshetian et al, 1998; Table 1.4).
Ezetimibe
In a study, ezetimibe reduced the concentration of Lp(a) by about 30%. However,
ezetimibe is often used with simvastatin, which has no additional effect on Lp(a)
concentration (Nozue et al, 2010; Table 1.4).
Fish oil
The effect of fish oil on Lp(a) concentration is contradictory. One study has shown that
omega-3 fatty acid supplements reduced serum Lp(a) concentration (Herrmann et al,
1995). However, other studies have shown no effect of omega-3 fatty acid on Lp(a)
concentration (Schmitz et al, 2002; Svensson et al, 2008).
40
Table 1.4 Influence of standard lipid-lowering therapies on Lp(a) concentration
Author (year) Therapy Principal result
Kostner (1989)
Yeang (2016)
Statins
Statins
éLp(a) concentration (30%)
éLp(a) concentration (11%)
Tsimikas (2019) Statins éLp(a) concentration (19%)
Croyal (2015) Niacin ê Lp(a) concentration
Ooi (2015) Niacin ê Lp(a) concentration
Fereshetian (1998) Fibrates êLp(a) concentration (33%)
Nozue (2010) Ezetimibe êLp(a) concentration (30%)
Hermann (1995) Fish Oils êLp(a) concentration (14%)
Svensson (2008) Fish Oils No effect
Schmitz (2002) Fish Oils No effect
1.7.2 Lipoprotein apheresis
Lipoprotein apheresis (LA) has been in clinical use for over 30 years. LA removes serum
lipids such as LDL-C, Lp(a), and triglycerides from the blood circulation. Various types of
LA are available, such as heparin-induced LDL-precipitation apheresis, polyacrylate
adsorption from whole blood, simple double-filtration plasmapheresis, dextran-sulfate
adsorption from plasma and blood, and apoB100 immunoadsorption. LA has traditionally
41
been used in high-risk patients with FH as well as those with elevated LDL-C
concentration with CVD treated with statins. Studies have shown that LA reduces the
concentrations of Lp(a) by about 60-70% (Waldmann & Parhofer, 2016). However, the
increase is rapid and LA must be repeated regularly, every week or two in some countries.
Guidelines for several countries recommend the use of LA as a secondary therapy to
lower LDL-C in high-risk patients in addition to maximally tolerated lipid-lowering
medication. A few countries also recommend the use of LA in patients with high Lp(a)
concentrationl and a high risk of CVD (Derfler et al, 2015; Stefanutti, 2010).
Studies have also evaluated the effect of LA on CVD incidence (Koziolek et al, 2010;
Stefanutti, 2009; Tatamia et al, 1992). A large longitudinal cohort study of 120 patients
investigated the relationship between LA and Lp(a) concentration as well as major adverse
coronary event (MACE). LA reduced Lp(a) concentration by 73.3% and the MACE rate per
patient by 86.4% (Kassner et al, 2015). LA also reduces the concentrations of oxidised
phospholipids and lipoprotein-associated phospholipase A2, both of which are associated
with high CVD risk (Kiechl et al, 2007). The German Lipoprotein Apheresis Registry has
shown that cardiovascular events were significantly reduced after the regular use of LA
(Schettler et al, 2015). Another study has shown that LA reduced CVD events in patients
with elevated Lp(a) concentration irrespective of the LDL-C concentration compared with
patients with low Lp(a) concentration but elevated LDL-C concentration (Von Dryander et
al, 2013). The Pro(a)-LiFe study investigated the impact of regular LA in patients with
elevated Lp(a) concentration. The study included 170 patients with elevated Lp(a)
concentration and progressive CVD disease. After 5 years of LA therapy, there were
reductions in major adverse cardiac events and other major non-cardiac events (Roeseler
et al, 2016).
42
Types of apheresis
Different types of apheresis systems can be used to lower LDL and Lp(a) concentrations,
and each is based on different principles, as shown in Table 1.5.
Heparin-induced extracorporeal LDL precipitation (HELP) was introduced in 1983
(Armstrong et al, 1983). ApoB-containing lipoproteins such as LDL and Lp(a) form a
complex with several other proteins in an acidic setting (pH 5.12). In this LA procedure, the
complexes are precipitated and eliminated, which simultaneously eliminates fibrinogen.
The elimination of fibrinogen may be seen as an advantage but also reduces the amount
of plasma that can be treated in one session.
Direct adsorption of lipoproteins (DALI) was the first whole-blood system and was first
used in 1996 (Bosch et al, 1997). Positively charged apoB binds to negatively charged
polyacrylate ions bound to a column. All apoB-containing lipoproteins are eliminated.
Apheresis with dextran sulphate similarly uses a negative charged cellulose-bound dextran
sulphate to eliminate apoB-containing lipoproteins such as Lp(a), LDL, IDL, and VLDL
(Yokoyama et al, 1984).
Lipid filtration/membrane differential filtration is a technique based on the elimination of
particles by size (Matsuda et al, 1995). The filter holds back LDL, Lp(a), and fibrinogen,
and the remaining plasma components pass through the filter without being eliminated.
43
Table 1.5 Principles of apheresis systems
Type of apheresis Principle
HELP Precipitation of a complex consisting of
heparin, LDL, Lp(a), fibrinogen, and CRP
DALI Negatively charged polyacrylate anions with
positively charged apoB
Dextran sulphate Negatively charged dextran sulphate with
positively charged apoB
Filtration LDL and Lp(a) eliminated from plasma by
filters based on size
1.7.3 Newer therapies
With the advances in genetics, epidemiology and understanding the metabolism of Lp(a),
new therapies to reduce the concentration of Lp(a) have emerged. These include
proprotein convertase subtilisin/kexin type 9 monoclonal antibody (PCSK9-mAb), apoB
production inhibitors, microsomal triglyceride transfer protein inhibitors, apo(a) antisense
therapies, and many more. Apo(a) antisense oligonucleotide (ASO) is the only therapy that
specifically targets Lp(a).
44
Table 1.6 Influence of newer lipid-lowering therapies on Lp(a)
Author (year) Therapies Principal result
Reyes-Soffer
(2017)
PCSK9 mAb éLp(a) FCR, –Lp(a) PR, ê Lp(a) concentration
Watts (2018) PCSK9 mAb
PCSK9 mAb + statin
–Lp(a) FCR, êLp(a) PR, ê Lp(a) concentration
éLp(a) FCR, –Lp(a) PR, ê Lp(a) concentration
Nandakumar
(2018)
Mipomersen éLp(a) FCR, –Lp(a) PR, ê Lp(a) concentration
Stefanutti (2015) MTP inhibitor ?Lp(a) FCR, ê(?)Lp(a) PR, ê Lp(a)
concentration
Thomas (2017) CETP inhibitor –Lp(a) FCR, êLp(a) PR, ê Lp(a) concentration
Viney (2016) Apo(a) ASO ?Lp(a) FCR, êLp(a) PR, êê Lp(a)
concentration
Graham (2016) Apo(a) ASO ?Lp(a) FCR, êLp(a) PR, êê Lp(a)
concentration
Apo(a) ASO ?Lp(a) FCR, êLp(a) PR, êê Lp(a)
concentration
45
Proprotein convertase subtilisin/kexin type 9 inhibitor
Proprotein convertase subtilisin/kexin type 9 mAb (PCSK9-mAb) intervenes in the binding
process of PCSK9 to the hepatic LDL-R, which promotes lysosomal degradation leading to
an increase in LDL-C concentration (Bergeron et al, 2015). Recent studies have
developed therapeutic approaches that prevent the binding of PCSK9 with hepatic LDL-R
(mAbs) and inhibit the synthesis of PCSK9 in the endoplasmic reticulum (Tsimikas et al,
2015; Viney et al, 2016; Watts et al, 2018). Recent clinical trials have demonstrated that
PCSK9-mAb induces a dose-dependent decrease in the concentrations of LDL-C (40–
70%), apoB (45–60%) and Lp(a) (25–50%) (Reyes-Soffer et al, 2017; Watts et al, 2018;
Table 1.6). Lp(a) and LDL-C concentrations and their reductions by PCSK9-mAb also
predicted the risk of MACE after recent acute coronary syndrome (Bittner et al, 2020).
Mipomersen
ApoB production inhibitors, such as ASOs to apoB, inhibit mRNA translation and splicing,
which leads to mRNA degradation by RNase (Rosie et al, 2007; Figure 1.10). Mipomersen
is an ASO that targets apoB and leads to a dose-dependent reduction in apoB, non-HDL-C,
and Lp(a) concentrations. The efficacy of mipomersen in combination with maximal statin
therapy in FH patients was confirmed in phase II and phase III studies, which reported a
significant reduction in Lp(a) concentration. A recent study has shown that mipomersen
increased the FCR of Lp(a) by 27% and reduced Lp(a) concentration by 21%
(Nandakumar et al, 2018; Table 1.5). Mipomersen has been approved by the US FDA only
for the use of HoFH patients (Norata et al, 2012).
46
Apolipoprotein(a) antisense oligonucleotide therapy
The apolipoprotein(a) ASO molecule IONIS-APO(a)Rx specifically targets the mRNA of
apo(a) (Figure 1.10). After a positive outcome in the trial involving transgenic mice, a
phase I trial involving healthy volunteers with Lp(a) concentration ³25 nmol/L was
conducted (Merki et al, 2011). Lp(a) concentration was significantly reduced by varying
doses of apo(a) ASO. The highest dose of 300 mg reduced the concentration of Lp(a) by
nearly 80%. The effect was constant, and Lp(a) concentration remained reduced 84 days
after the last dose (Tsimikas et al, 2015).
Microsomal triglyceride transfer protein inhibitor
Microsomal triglyceride transfer protein (MTP) mediates the formation of lipoproteins
containing apoB in the liver and the intestine (Figure 1.10). Lomitapide is an MTP inhibitor
that has been shown to reduce LDL-C, apoB, and Lp(a) concentrations in patients with
HoFH or hypercholesterolaemia (Cuchel et al, 2007; Samaha et al, 2008). However,
lomitapide has been observed to have adverse effects such as an increase in liver enzyme
and hepatic fat accumulation, and the drug is restricted for use only in patients with HoFH.
Lomitapide has been approved by the FDA for the treatment of patients with HoFH.
Cholesteryl ester transfer protein inhibitors
Inhibitors of cholesteryl ester transfer protein (CETP) increases HDL-C and reduces LDL-
C concentrations. In a phase II trial, anacetrapib lowered Lp(a) concentration by 50%
(Teramoto et al, 2013). Evacetrapib, as a monotherapy or with statins, significantly
reduces the concentrations of atherogenic apoB-containing lipoproteins such as Lp(a) and
47
LDL (Nicholls et al, 2016). Obicetrapib, a newer agent, lowered Lp(a) by 37% at week 12
in another phase II trial (Hovingh et al, 2015). The Randomized Evaluation of the Effects of
Anacetrapib through Lipid Modification (REVEAL) trial of anacetrapib has recruited about
30,000 participants and provides a robust assessment of the efficacy and safety of
anacetrapib in people with high-risk CVD who are receiving effective LDL-lowering
treatment (Landray et al, 2017). All clinical use of CETP inhibitors have been discontinued.
Thyromimetics
Thyroid hormone receptor agonists promote lipolysis in adipose tissue and hepatic
expression of the LDL-R and HDL-R. Patients with hypothyroidism have elevated Lp(a)
concentration, which is reduced with treatment. A double-blinded, multicentre, placebo-
controlled study reported that eprotirome (hormone receptor isoform b-selective
analogues) reduced Lp(a) concentration by up to 40% (Ladenson et al, 2010). All clinical
use of thyromimetics have been discontinued.
1.8 Tracer kinetic methods in human lipoprotein
research
Plasma lipid and lipoprotein concentrations are measured to identify lipid disorders caused
primarily by defects in catabolism and/or production of lipoproteins. Simply measuring lipid
and lipoprotein concentrations will not provide information about the mechanisms
contributing to lipid disorders (Burnett & Barrett, 2002). Therefore, kinetic studies using
stable isotopes are crucial to understanding the regulation of lipoproteins (Barrett et al,
2006).
48
Stable isotope methods were first introduced in 1935 for studying cholesterol
(Schoenheimer & Rittenberg, 1935). However, because of the high cost and lack of
commercial availability, the method was replaced by radioactive labelling methods. Kinetic
studies of lipoproteins were developed in animals and humans using exogenous labelling
with radioactive iodine. Given the hazards of radiation exposure, stable isotopes were
preferred later on. Stable isotope techniques involve the use of endogenous labelling and
allows one to directly estimate the PR of all lipoproteins of interest (Lichtenstein et al,
1990; Packard, 1995). A study that compared the use of exogenous and endogenous
labelling found that these produce similar results (Ikewaki et al, 1993). Stable isotope
studies became more widely used as mass spectrometers became more efficient and
affordable. The use of gas chromatography–mass spectrometry (GC-MS) provides a
sensitive measurement of the isotopic enrichment of lipoproteins.
1.8.1 Principles
The aim of substrate kinetics studies is to determine the rate of production and clearance
of a substrate from a system (Wolfe, 1999). A system refers to a biological construct in
which the substance under investigation, for this instance apolipoprotein, may exist in
pools within the whole body, organ, or cellular or subcellular environment (Figure 1.11).
The system should be in a steady state, where the rates of input and output for the
unlabelled tracee are equal. A tracee is defined as the substance of interest, for example
apo(a), to be traced kinetically. A tracer is a labelled substance, in this instance labelled
leucine. A tracer should be physically, chemically and biologically identical with the tracee
and should be quantitatively detectable.
49
Production and catabolic rates can be determined using mathematical modelling involving
functions and differential equations that can be generated using the program SAAM II (The
Epsilon Group, VA, USA).
Figure 1.11 The principle of the tracer method. The unlabelled tracee (white circles)
and labelled tracer (black circles) are mixed in the biological system.
1.8.2 Definition of terms
The kinetic parameters relevant to this research thesis are defined below:
Pool is an amount of a homogeneous substance that is distinct from other substances in
the system, which can flow into or out from a compartment.
Pool size (PS) is the amount of tracee in a specific pool in which the tracer is introduced
and from which samples measuring its mass will be taken.
50
FCR is the fraction of tracee lost irreversibly from a pool per unit time.
PR is the rate at which the tracee moves through a pool.
1.8.3 Isotope labelling methods
Stable isotope tracer methods require an accurate and precise instrument that can detect
and quantify the abundance of a labelled isotope. Plasma samples are collected, and a
labelled isotope, such as D3 leucine, is used as a tracer to measure the kinetics of Lp(a).
Other stable isotopes such as 13C, 15N and 18O are commonly used as stable isotopes for
metabolic tracer studies. This is known as isotopic enrichment and is commonly measured
using GC-MS (Frischmann et al, 2012).
Radiolabelled isotope tracer methods involve an injection of radioiodinated Lp(a) into an
individual. This allows for quantification of the radiolabelled isotope in plasma using a
gamma counter and the generation of curves. For example, the FCR is generated from the
plasma radioactivity curves using a computer-assisted curve-fitting technique (Rader et al,
1994).
1.8.4 Clinical protocols
In this thesis, a single bolus of D3 leucine was used to study apo(a) and apoB-100 of Lp(a)
kinetics. D3 leucine was used because it has several advantages over other isotopes:
leucine is an abundant essential amino acid that is metabolised mainly in the periphery
and is labelled on three atoms, which allows a more sensitive measurement (Wolfe, 1992).
51
1.8.5 Analytical techniques
Samples must be derivatised to convert the analyte into a derivative more suitable for
chromatographic analysis, which is a compound of a similar chemical structure. In this
thesis, free leucine was mixed with a derivatising agent, trifluoroacetic acid/trifluoroacetic
anhydride (TFA/TFAA), which converts leucine and other amino acids to volatile
oxazolinones.
1.8.6 Analytical aspects of kinetic studies
Linear regression
Early studies used linear regression models to determine kinetic parameters (Cohn et al
1990; Cryer et al, 1986). This approach assumes that the system of interest is in a steady
state and that output is irreversibly removed from the same pool as the input. This
approach can be used to determine the initial rate of enrichment and to estimate the
fractional synthetic rate (FSR), equivalent to the FCR in a steady-state system, by dividing
the initial rate by the precursor plateau isotopic enrichment (Cohn et al, 1990; Cryer et al,
1986). However, this approach does not account for the heterogeneity of lipoprotein
metabolism, which is inherently nonlinear by nature. Therefore, the linear regression
method represents a non-physiological method to determine the kinetic parameters and is
not recommended (Parhofer et al, 1991).
52
Mono-exponential function
The mono-exponential function has been used to estimate the FSR. This approach
assumes that the pool is a homogeneous population of particles. However, numerous
studies have shown that the lipoprotein pool is heterogeneous (Fisher et al, 1980; Packard
et al, 1984).
Multi-compartmental model
In this thesis, a compartmental model was constructed using SAMM II modelling software
(The Epsilon Group, VA, USA) to fit it to the tracer enrichment data for plasma apo(a) and
apoB. To account for the leucine tracer data, two linked models were used: plasma leucine
and apo(a) leucine enrichment (Figure 1.12). The leucine compartment consists of a four-
compartment subsystem (compartments q1–q4) that describes the plasma leucine kinetics.
The apo(a) compartment consists of a two-compartment subsystem (compartments q10–
q11) that describes the plasma apo(a) kinetics.
Figure 1.12 Compartmental model to describe apo(a) tracer kinetics
53
Steady-state kinetics
The estimation of lipoprotein kinetics of a compartment is governed by the principle of
mass balance. At any timepoint, the rate at which the material mass is produced is
equivalent and constant to the rate of clearance. The kinetics of a system is determined by
the difference between the rates of tracer input and tracer clearance. Given that the kinetic
performance of the tracer is the same as that of the tracee, the determination of tracer
kinetics provides a measure of the metabolic parameters of the tracee. A mathematical
model was developed to study the metabolism of Lp(a) in Studies 2 and 3 of this thesis.
Non-steady-state kinetics
Non-steady state kinetics of cholesterol were first calculated in 1978 (Apstein et al, 1978).
Under the non-steady state, assumptions about catabolism and/or PR are required to
develop a mathematical model that best fits the system. Stable isotopes and mathematical
models have been previously used to study the metabolism of Lp(a) and apoB in the non-
steady state (Parhofer et al, 1999). A mathematical model was developed to study the
metabolism of Lp(a) and LDL–apoB in Study 4 of this thesis. The model used a single
compartment to determine the kinetics of Lp(a) and LDL–apoB and was based on the rate
of increase in concentration after apheresis. A single compartment implies that the pools of
Lp(a) and LDL–apoB are kinetically homogeneous, and the model also assumes that
apheresis does not alter the FCR or PR. Although LA does not alter apoB metabolism in
the long term, it has been shown that the LDL-R activity is increased immediately after
apheresis (Parhofer et al, 2000).
55
Lp(a) is an LDL-like protein comprising two protein subunits: apo(a) and apoB-100. Lp(a)
acts as a transport protein for cholesterol, fats and proteins in the blood. At high
concentrations (>50 mg/dL), Lp(a) is reported to be an independent risk factor for CVD;
however, the metabolism of Lp(a) remains unclear.
The work reported in this thesis aimed to understand better the metabolism of Lp(a)
through the following studies.
1) Comparison of plasma apo(a) concentrations as measured by a novel LC-MS
method and an apo(a) ELISA, a well established assay.
2) Comparative analysis of apo(a) and apoB-100 kinetics in the Lp(a) particle.
3) Determination and comparison of apo(a) kinetics in statin-treated patients with or
without elevated plasma Lp(a) concentration.
4) Investigation of Lp(a) and LDL metabolism following apheresis.
The philosophical method used in this thesis was to acquire new knowledge through the
empirical process. The empirical process requires a general hypothesis based on
observations statements, hypothesis testing, and making inferences from the observations.
The current chapter provides an overview of the studies reported in Chapters 4–7 with
respect to their individual study rationale, hypotheses, aims, designs and statistical
analyses. The specific study designs and analyses are described in detail in the following
chapters. The aim of this chapter is to provide an overview of how the results of these
independent investigations can be combined to increase understanding of the role of Lp(a)
in CVD.
By better understanding Lp(a) metabolism and the methods used to measure Lp(a), the
work outlined in this thesis will provide insights into the role that Lp(a) plays in CVD and
whether targeting elevated Lp(a) concentration is an effective therapeutic strategy.
56
2.1 Study 1: Comparison of methods for measuring
plasma lipoprotein(a) particle concentration
2.1.1 Rationale
Elevated plasma Lp(a) concentration is directly associated with an increased risk for CVD.
However, there is currently no standard method for measuring plasma Lp(a) concentration.
Measurement of Lp(a) is complex, and this study attempted to correlate the LC-MS
method with a well-established ELISA method measured at the Northwest Lipid
Metabolism and Diabetes Research Laboratories (NLMDRL).
2.1.2 Hypothesis
There is good agreement between plasma apo(a) concentrations as measured by LC-MS
and NLMDRL methods.
2.1.3 Aim
To validate the novel LC-MS method for measuring plasma apo(a) concentration through
comparative analysis with the well established NLMDRL method.
57
2.1.4 Study design
This study was undertaken using a cross-sectional design. Plasma samples were collected
from 91 subjects and analysed concurrently using both LC-MS and ELISA. Comparative
analyses of the resulting apo(a) concentrations were used to validate the new LC-MS
method.
2.1.5 Statistical analyses
Data were assessed for normality using the Shapiro–Wilk test. Skewed variables were
logarithmically transformed to normalise their distribution. Associations between the
plasma concentrations of Lp(a) and apo(a) were examined using simple linear regression,
and the agreement was measured using Lin’s concordance correlation. Statistical
significance was defined at the 5% level using a two-tailed test. All data were analysed
using IBM SPSS Statistics (version 21.0; IBM Corp., Armonk, NY, USA).
2.2 Study 2: Fractional turnover of apolipoprotein(a) and
apolipoprotein B-100 within plasma lipoprotein(a)
particles in statin-treated patients with elevated and
normal Lp(a) concentration.
58
2.2.1 Rationale
Lp(a) consists of two proteins: apo(a) and apoB. However, it is not known whether these
two protein constituents are metabolised at the same rate. This study compared protein
turnover to provide insight into how Lp(a) and its constituent parts respond to current
therapies. This work will be useful for determining whether the catabolic rate of these
proteins is coupled or uncoupled.
2.2.2 Hypothesis
The FCRs of apo(a) and apoB within plasma Lp(a) particles are coupled.
2.2.3 Aim
To measure the FCR of the Lp(a) constituent proteins, apo(a) and apoB-100.
2.2.4 Study design
This study was undertaken using a cross-sectional study design. Plasma samples were
obtained from 10 patients with elevated plasma Lp(a) concentration and 10 clinically
matched patients with normal plasma Lp(a) concentration. The kinetics of Lp(a)–apo(a)
were assessed using stable isotopes, GC-MS and multi-compartmental modelling.
59
2.2.5 Statistical analyses
Data were assessed for normality using the Shapiro–Wilk test. Skewed variables were
logarithmically transformed to normalise the distribution. Associations between the
catabolic rates of Lp(a)–apoB and Lp(a)–apo(a) were examined using simple linear
regression. Statistical significance was defined at the 5% level using a two-tailed test. All
data were analysed using IBM SPSS Statistics (version 21; IBM Corp., Armonk, NY, USA).
2.3 Study 3: Apolipoprotein(a) kinetics in statin-treated
patients with elevated plasma lipoprotein(a)
concentration
2.3.1 Rationale
Lp(a) exhibits both pro-atherogenic and pro-thrombotic effects. These effects are thought
to be driven more strongly by the apo(a) component of Lp(a) than by apoB-100. Statins
potentially decrease the hepatic synthesis and availability of apoB and consequently may
affect the production of Lp(a). There is evidence that apo(a) recycles in the circulation
following catabolism of the Lp(a) particle. As a consequence upregulation of the LDL-R,
statins may lead to greater relative clearance of apoB from plasma and could accelerate
the recycling of apo(a). Statins could therefore theoretically bear on the coupling of apo(a)
and apoB. Examining the kinetics of apo(a) in patients on statins may provide insights into
60
how Lp(a) assembly, secretion and catabolism can affect its pro-atherogenic and pro-
thrombotic effects.
2.3.2 Hypothesis
The hepatic production of apo(a) is increased in statin-treated patients with elevated
plasma Lp(a) concentration.
2.3.3 Aim
To determine the kinetics of apo(a) in statin-treated patients with or without elevated
plasma Lp(a) concentration.
2.3.4 Study design
This study used an unmatched case–control design to compare the kinetics of apo(a) in
patients taking statins. Plasma samples were collected from 14 patients with elevated
plasma Lp(a) concentration and 15 patients with similar clinical and biochemical
characteristics with normal plasma Lp(a) concentration. The kinetics of apo(a) were
measured using stable isotopes, GC-MS and multi-compartmental modelling.
2.3.5 Statistical analyses
61
Data were assessed for normality using the Shapiro–Wilk test. Skewed variables were
logarithmically transformed to normalise the distribution. Comparative analyses were
performed using paired t tests. Associations between the kinetic determinants of apo(a)
were examined using simple linear regression. Statistical significance was defined at the
5% level using a two-tailed test. All data were analysed using IBM SPSS Statistics (version
21; IBM Corp., Armonk, NY, USA).
2.4 Study 4: Lipoprotein(a) and low-density lipoprotein–
apolipoprotein-B metabolism following apheresis in
patients with elevated lipoprotein(a) concentration and
coronary artery disease
2.4.1 Rationale
LA is a therapeutic technique that lowers LDL concentration in patients with CAD by
separating and removing lipoproteins from their blood. This process also enables the
determination of the catabolism and PR of lipoproteins (e.g. Lp(a) and LDL) in the non-
steady-state condition. Comparisons of the metabolic rates of Lp(a) and LDL would greatly
increase the understanding of the relationship between elevated concentrations these
lipoproteins and the risk profiles of patients with CAD.
2.4.2 Hypothesis
62
The FCR of Lp(a) is slower than that of LDL–apoB in patients with elevated Lp(a)
concentration and CAD undergoing LA.
2.4.3 Aim
To investigate the kinetics of Lp(a) and LDL–apoB particles in patients with elevated Lp(a)
concentration and CAD undergoing LA.
2.4.4 Study design
This study used a cross-sectional study design and was performed using samples
collected from 13 patients with elevated Lp(a) concentration undergoing LA.
2.4.5 Statistical analyses
Data were assessed for normality using the Shapiro–Wilk test. Skewed variables were
logarithmically transformed to normalise the distribution. Comparative analyses between
LDL and Lp(a) metabolism were performed using paired t tests. Statistical significance was
defined at the 5% level using a two-tailed test. All data were analysed using IBM SPSS
Statistics (version 21; IBM Corp., Armonk, NY, USA).
2.5. Conclusion, overall discussion and future
directions
63
The final chapter of this thesis combines the findings from the research chapters and
discusses the broader implications of these investigations. This chapter also discusses the
limitations of this work and how future investigations will help to continue to elucidate the
role of Lp(a) in CVD.
The research findings from each chapter are discussed as outlined below.
1) An overview of the investigative design
2) Results of the current investigations and their relevance to current understanding in
the field
3) Practical implications arising from this work, including acknowledgment of the
limitations arising from the current work
4) Avenues for future investigations
5) Overall conclusions.
65
3.1 Subjects
In Study 1, 92 subjects with a wide range of Lp(a) concentration (1.16–476 nmol/L) were
recruited from the Lipid Disorder Clinics at Royal Perth Hospital (n=29), and the general
community through an advertisement placed on newspapers and the internet (n=63).
In Study 2, 10 patients aged 18–70 years were recruited from the Lipid Disorder Clinics at
Royal Perth Hospital. All patients had an elevated plasma Lp(a) concentration of ³0.8 g/L
(or ³170 nmol/L, i.e., 95th population percentile), were on statins and aspirin, and had a
moderate-to-high ASCVD risk or history of ASCVD events. Ten patients of similar clinical
and biochemical characteristics, also on statins, with a normal Lp(a) concentration of £0.3
g/L (or £70 nmol/L) were also recruited from the lipid clinic.
In Study 3, 14 patients aged 18–70 years were recruited from the Lipid Disorder Clinics at
Royal Perth Hospital. All patients had an elevated plasma Lp(a) concentration of ³0.8 g/L
(or ³170 nmol/L, i.e., 95th population percentile), were on statins and aspirin, and had a
moderate-to-high ASCVD risk or history of ASCVD events. Fifteen patients of similar
clinical and biochemical characteristics, also on statins, with a normal Lp(a) concentration
of £0.3 g/L (or £70 nmol/L) were also recruited from the lipid clinic.
In Study 4, 13 patients aged 18–70 years attending the Munich University Hospital
(Department of Internal Medicine IV, Germany) to be treated with regular apheresis (³3
months, weekly or biweekly) were recruited. All patients had an elevated Lp(a)
concentration (>0.5 g/L), hypercholesterolaemia (total cholesterol >5.2 mmol/L) and history
of CAD. The study was performed as part of routine care and was not specific to the
project.
66
3.2 Inclusion criteria
3.2.1 Study 1
The inclusion criteria were Caucasian, aged 18–70 years, BMI <35 kg/m2, fasting plasma
triglyceride concentration <1.7 mmol/L and LDL-C concentration <4.9 mmol/L.
3.2.2 Studies 2 and 3
The inclusion criteria were Caucasian, aged 18–70 years, taking statin and aspirin,
moderate-to-high CVD risk based on an Australian tool for assessing absolute CVD risk,
elevated Lp(a) concentration (³0.8 g/L, n=14) or normal Lp(a) concentration (£0.3 g/L,
n=15).
3.2.3 Study 4
The inclusion criteria were Caucasian, aged 18–70 years, elevated Lp(a) concentration
(>0.5g/L), hypercholesterolaemia (pre-treatment LDL-C concentration >3.2 mmol/L) and
CAD.
67
3.3 Exclusion criteria
3.3.1 Studies 1–3
The exclusion criteria were a cardiovascular event in the past 6-months, history of venous
thromboembolism and/or pulmonary embolism, diabetes, secondary hyperlipidaemia, renal
disease (creatinine >130 µmol/L, including proteinuria, or nephrotic syndrome),
hypertriglyceridaemia (fasting triglyceride concentration >3.5 mmol/L), hepatic dysfunction
(alanine aminotransferase >3´ the upper limit of normal), anaemia (haemoglobin <125 g/L),
haematological disorder, current smoker or taking lipid-lowering therapy known to have a
major effect on plasma Lp(a) concentration (e.g., hormonal replacement therapy, niacin or
PCSK9 monoclonal antibodies).
3.3.2 Study 4
The exclusion criteria were severe liver disease, CKD, proteinuria, uncontrolled thyroid
disease or other endocrinologic disease, pregnancy, malignant disease, high alcohol
consumption (women >70 g/week, men >140 g/week), drug abuse or taking niacin.
3.4 Protocol for screening
Following an expression of interest, potential participants were interviewed over the
telephone to ascertain their initial suitability according to the entry criteria. A brief
explanation of the study and the time commitment required was provided. About 200
people were screened over the telephone during a 2-year period (Studies 1–3). Suitable
68
participants were invited to attend a screening visit. Participants were screened at the
Linear Clinical Research Centre (Sir Charles Gairdner Hospital) and the Research Studies
Unit (Royal Perth Hospital). The following measurements were taken at screening: height,
weight, blood pressure, electrocardiogram, medical examination, full blood count, plasma
renal biochemistry (urea, electrolytes, creatinine, urinary protein excretion), liver and
muscle enzymes tests, fasting insulin and glucose, and fasting lipid, lipoprotein and
apolipoprotein profiles.
Subjects who fulfilled the entry criteria were notified via telephone and letter to participate.
Each subject was given a detailed explanation of the study objectives, procedures and
time commitment involved. The studies were approved by the relevant ethics committees,
and all subjects provided informed written consent.
3.5 Anthropometric measurements
Height and weight were measured with the subject wearing light clothing without shoes.
Height was measured using a fixed stadiometer, and weight was measured using a DI-160
electronic scale (Digi, Ontario, CA) to the nearest 0.1 kg. BMI was calculated as the weight
in kg divided by the square of the height in metres.
3.6 Blood pressure
A Dinamap 1846SX instrument was used to measure resting blood pressure in all subjects
in this thesis (Critikon, Florida, USA). The unit is a semi-automatic oscillometric recorder
designed to measure SBP, diastolic blood pressure (DBP), mean arterial pressure and
heart rate. The equipment can determine measure SBP in the range 30–245 mmHg and
69
DBP 10–210 mmHg. Repeated measurements were programmed to be taken at 2-minute
intervals. Four measures were recorded over 8 minutes and were printed on the automatic
paper print out. The first measurement was discarded, and the mean of the remaining
three readings was calculated and used in the analyses.
3.7 Protocol for blood measurements
Blood was collected in the morning after a 12-hour overnight fast, unless otherwise
indicated. Subjects were rested supine for 20 minutes before a 21-gauge scalp vein
needle was inserted intravenously to an antecubital view of the forearm by a trained nurse.
Blood was collected into the appropriate Vacutainer blood collection tube for the sample
required (Becton Dickinson, Rutherford, NJ, USA). Blood was either sent immediately for
routine analysis or centrifuged, aliquoted and stored at –80 °C for later analysis.
3.8 Protocol for stable isotope infusion
3.8.1. Preparation of leucine infusion
The dose of D3 leucine powder (5 mg/kg of body weight) was calculated and weighed. The
powder was then dissolved in sterile 0.9% sodium chloride for injection at a final
concentration of 15 mg/mL. Under the laminar flow cabinet, the solution was drawn into
sterile syringes, filtered aseptically through a 0.2-micron hydrophilic membrane and stored
in a sterile vial. The vial was capped with a silver metal cap and kept at 4 °C. The D3
leucine was purchased from Cambridge Isotope (MA, USA) and prepared within 3 days of
injection by the Department of Pharmacy, Royal Perth Hospital.
70
3.8.2 Stable isotope infusion
Patients were admitted to the Research Studies Unit, Medical Research Foundation
Building at Royal Perth Hospital after a 12-hour fast. They were studied in the semi-
recumbent position and were allowed water alone. An intravenous bolus of D3 leucine (5
mg/kg body weight) was administered via a superficial vein in the antecubital fossa. A
cannula was placed in a superficial vein in the antecubital fossa of the other arm for blood
collection at 5, 10, 20, 30, 40 and 60 minutes and 1.5, 2, 2.5, 3, 4, 5, 6, 8 and 10 hours
from the time the stable isotope was administered. After the end of the blood collection,
subjects were given a snack and allowed to go home. Additional fasting blood samples
were collected in the morning on the following 4 days of the same week (24, 48, 72 and 96
hours). Serial blood samples were taken for GC-MS analysis for the isotope leucine
enrichment of plasma Lp(a)–apo(a) and apoB-100.
3.9 Protocol for apheresis
The study was performed as part of routine care, which included the use of the following
four different apheresis systems for removing LDL and Lp(a).
• Octonova (DIAMED Medizintechnik, Cologne, Germany): series membrane filtration
to eliminate LDL and Lp(a) from plasma based on size properties (Waldmann et al,
2017; Matsuda et al, 1995.
• Kaneka (Kaneka, Osaka, Japan): electrostatic interaction of negatively charged
dextran sulphate with positively charged apoB (Waldmann et al, 2017; Yokoyama et
al, 1984).
71
• Plasmat Futura (B. Braun Medical Inc., Bethlehem, PA): heparin-induced
extracorporeal precipitation of a complex comprising heparin, LDL, Lp(a) and
fibrinogen at pH 5.12 (Armstrong et al, 1983).
• DALI (Fresenius HemoCare Adsorber Technology, St. Wendel, Germany):
electrostatic interaction of negatively charged polyacrylate anions with positively
charged apoB (Waldmann et al, 2017; Bosch et al, 1997).
Patients were treated with regular apheresis (³3 months, weekly or biweekly). Patients
were not fasted at the time of blood withdrawal because the apheresis procedure is
tolerated better in a non-fasting state. Plasma samples were collected immediately before
and after apheresis, and 1, 2 and 3 hours, and 1, 2, 3, 5 and 7 days after apheresis.
3.10 Laboratory methods
All whole blood samples were processed into serum or plasma within 1 hour of collection.
To obtain serum, the blood was allowed to stand for 30 minutes and then centrifuged at
1000 g for 15 minutes at 20 °C. To obtain plasma, blood was centrifuged immediately at
1000 g for 15 minutes at 4 °C. The biochemical parameters were measured using an
Architect c16000 analyser and corresponding reagents mentioned in this thesis (Abbott
Diagnostics, Abbott Laboratories, Abbott Park, IL, USA).
3.10.1 Total cholesterol
Principle: This assay involves the following biochemical reactions:
1. Cholesterol ester + H2O !"#$%&'%(#$%&'%(*&%+⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯- cholesterol + R.COOH
72
2. Free cholesterol + O2 !"#$%&'%(#$#./0*&%+⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯- cholest-4-ene-3-one + H2O2
3. 2H2O2 + 4 aminophenazone + phenol 1%(#./0*&%+⎯⎯⎯⎯⎯⎯⎯- 4 quinoneimine + H2O
The amount of red quinoneimine produced is stoichiometrically related to the concentration
of cholesterol and is detected spectrophotometrically at 500 nm. The inter-assay
coefficient of variations (CVs) at 2.71 mmol/L and 6.39 mmol/L were 1.40% and 1.06%,
respectively.
3.10.2 Triglycerides
Principle: This assay involves the following biochemical reactions:
1. Triglyceride + 3H2O $/1#1(#'%/2$/1*&%+⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯- glycerol + 3R.COOCH
2. Glycerol + ATP 3$4!%(#$5/2*&%+⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯- glycerol-3-phosphate + ADP
3. Glycerol-3-phosphate + O2 3$4!%(#$676
1"#&1"*'%#./0*&%+⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯- dihydroxyacetone + H2O2
4. 2H2O2 + 4-aminoantipyrin + 4-chlorophenol 1%(#./0*&%+⎯⎯⎯⎯⎯⎯⎯- quinoneimine + 2H2O2 + HCl
The amount of red quinoneimine produced is stoichiometrically related to the concentration
of cholesterol and is detected spectrophotometrically at 500 nm. The inter-assay CVs at
1.01 mmol/L and 2.12 mmol/L were 4.06% and 2.24%, respectively.
3.10.3 HDL-cholesterol
Principle: This assay involves the following biochemical reactions:
73
1. Non-HDL unesterified cholesterol +accelerator + DSBmT89:1%(#.0/*&%+⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯- colourless
end product
2. HDL-C + HDL-specific detergent +⎯⎯⎯⎯⎯⎯⎯⎯- HDL disrupted
3. HDL-C + H2O + O2 HD- specific detergent 8;<:89+⎯⎯⎯⎯⎯- cholest-4-en-3-one + H2O2
4. H2O2+ DSBmT + 4-aminoantipyrine 1%(#./0*&%+⎯⎯⎯⎯⎯⎯⎯⎯- blue colour complex
The amount of blue complex produced is stoichiometrically related to the concentration of
HDL-C and is detected spectrophotometrically at 500 nm. The inter-assay CVs at 0.84
mmol/L and 1.83 mmol/L were 2.76% and 2.68%, respectively.
3.10.4 LDL-cholesterol
The concentration of LDL-C in plasma was estimated using the Friedewald calculation
(mmol/L):
LDL-cholesterol = total cholesterol – HDL-cholesterol – (0.46 ´ triglyceride)
3.10.5 Total apoB
Principle: This assay involves the formation of insoluble antigen–antibody complexes in
solution and in the presence of antibody excess. The produce is detected by the change in
absorbance of transmitted light at 340 nm as a function of turbidity. The increase in
absorbance of the test samples was compared to that of standard concentrations of apoB.
The inter-assay CVs at 0.41 g/L, 0.93 g/L and 1.39 g/L were 2.79%, 1.03% and 1.46%,
respectively.
74
3.10.6 LDL-apoB
Principle: This method is based on a sequential ultracentrifugation method and Lowry
protein estimation. In brief, an aliquot of 1 mL of plasma was added to an ultracentrifuge
tube. Sodium chloride solution was then added to adjust the density to 1.019 kg/L, and the
tube was centrifuged at 5000 rpm for 16 hours at 4 °C (Optima XL-100K, Beckman Coulter,
Australia). Using a syringe and blunted needle, 1 mL of the VLDL–IDL supernatant from
the very top of the solution was aspired slowly without disturbing the remaining solution.
The LDL fraction (density 1019–1.063 g/L) was sequentially isolated by the same
ultracentrifuge procedure with 1 mL of NaCl solution (21.56% w/w). ApoB in the LDL
fraction was precipitated with 50% isopropanol. The extraction of the precipitate with 100%
isopropanol removes neutral lipid. The delipidated apoB was then made soluble in alkaline
deoxycholate solution and the amount estimated using the Lowry method with a bovine
serum albumin standard.
3.10.7 Glucose
Principle: This assay involves the following biochemical reactions:
1. Glucose + ATP "%.#5/2*&%+⎯⎯⎯⎯⎯⎯⎯- glucose-6-phosphate + ADP
2. Glucose-6-phosphate + NAD =$>!#&%6?61"#&1"*'%
0%"40(#3%2*&%+⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯- 6-phosphogluconate + NADH
The amount of NADH produced is stoichiometrically related to the concentration of glucose
and is detected spectrophotometrically at 340 nm. The inter-assay CVs at 4.75 mmol/L
and 16.45 mmol/L were 2.09% and 1.99%, respectively.
75
3.10.8 Insulin
Principle: This assay involves the sample binding to the anti-insulin-coated microparticles
and anti-insulin acridinium-labelled conjugate. After washing, pre-trigger and trigger
solutions were then added to the reaction mixture, and the resulting chemiluminescent
reaction was measured as relative light units. The inter-assay CVs at 37.32, 87.01 and
193.47 µU/mL were 1.78%, 2.59% and 2.08%, respectively.
3.10.9 Insulin resistance
The Homeostasis Model Assessment (HOMA) score was calculated as follows:
@ABC =EFGHIJK(MMIF/O)´QRJGFQR(MS/O)
22.5
3.10.10 Dutch Lipid Clinic Network Score
The Dutch Lipid Clinic Network (DLCN) score was used to define FH phenotypically and
was categorised as definite, probable, possible and unlikely (Chan et al, 2018).
3.11 Measurements of lipoprotein(a) concentration
In this thesis, plasma Lp(a) concentration was measured using four different assays: the
well established ELISA used in the NLMDRL, a novel, simple and sensitive LC-MS method
for quantification of Lp(a) particle number in nmol/L, and two commercially available
76
assays (Abbott Quantia and DiaSys 21FS) that measure Lp(a) mass in g/L. Table 3.1
summarises the methods used in the four studies in this thesis.
Table 3.1 Lp(a)/apo(a) methods used for studies in this thesis
Study 1 Study 2 Study 3 Study 4
NLMDRL ü ü ü
LC-MS ü ü ü
Abbott Quantia ü ü
DiaSys 21FS ü
3.11.1 NLMDRL method
Principle: The measurement uses an ELISA in which the wells are coated with the murine
mAb a-6 directed to apo(a) KIV-2 repeats, which captures the apo(a) particles, and the
detection involves the murine mAb a-40, which is directed to a single epitope present in
apo(a) KIV-9 and is conjugated with horseradish peroxidase. Because KIV-9 is present in
only one copy per apo(a) molecule, this immunoassay is not sensitive to apo(a) size
heterogeneity. The method is approved by the IFCC (Marcovina et al, 2000) because it is
isoform independent and traceable to IFCC reference material. Moreover, the NLMDRL
method uses five independent assay calibrators with values ranging from low to high (or
apo(a) isoform sizes from large to small) to minimise the impact of apo(a) size on the
accuracy of Lp(a) measurement. The inter-assay CVs were at 11–22% (Marcovina et al,
2000).
77
3.11.2 LC-MS method
Principle: This measurement uses a synthetic peptide (LFLEPTQADIALLK) that targets the
proteolytic domain of apo(a) for quantification following a standardised sample trypsin
digestion procedure and LC-MS (Proteomics International – commercial laboratory, Queen
Elizabeth II Medical Centre, Perth, WA). This LC-MS method is assumed to not be
sensitive to apo(a) isoform size heterogeneity for detection and standardisation. In brief,
proteins were precipitated by adding 75 µL of acetonitrile (ACN) to human plasma sample
(50 µL), followed by centrifugation at 1000 g for 5 minutes at 4 °C. Protein pellets were
resuspended in 100 µL of 50 mM ammonium bicarbonate buffer and 50 µL of IS spike-in
solution (125 nM D3-labeled peptide LFLEPTQADIA[D3-L]LK in 50 mM ammonium
bicarbonate buffer). Pellets were digested using trypsinisation with 1% formic acid to
quench digestion. The samples were then subjected to solid-phase extraction using an
Oasis HLB µElution plate (Waters Corporation) with 60% ACN/0.1% formic acid (elution
buffer). Ten microlitres of each sample was injected into an Ekspet microLC 200 system
(Eksigent) coupled to a QTrap 6500 mass spectrometer (Sciex) for analysis (Figure 3.1).
The selected reaction monitoring (SRM) transitions used for the analyte and the IS peptide
are shown in Table 3.2. The synthetic LFLEPTQADIALLK and D3-labeled peptide
(i.e. LFLEPTQADIALLK and LFLEPTQADIA[D3-L]LK, respectively) were used as the
calibrator and internal standard for the calculation of apo(a) concentration by LC-MS.
78
Table 3.2 Selected reaction monitoring transitions used for the analyte and IS
peptide
Peptide Q1
(mz)
Q2 (m/z) Time
(ms)
DP (V) CE (V) CXP (V)
LFLEPTQADIALLK 786.6 1069.6 25 140 34 9
LFLEPTQADIA[D3-L]LK 788.1 1072.6 25 140 34 9
Figure 3.1 Quantification of apo(a) by the LC-MS method.
79
3.11.3 Abbott Quantia method
Principle: This method is an immunoturbidimetric method in which the Quantia reagent
comprises a suspension of polystyrene latex coated with IgG anti-human Lp(a). When a
sample containing Lp(a) is mixed with the reagent, agglutination occurs, which can be
measured by turbidimetry (Abbott Diagnostics, Abbott Laboratories, Abbott Park, IL, USA).
The inter-assays CV at 0.068 g/L, 0.146 g/L and 0.213 g/L were 6.37%, 2.28% and 1.71%,
respectively. This method was used for the screening and selection of patients for Studies
2 and 3.
3.11.4 DiaSys 21FS method
Principle: This method is an immunoturbidimetric method with latex particles coated with
specific anti-Lp(a) antibodies. Agglutination occurs when a sample containing Lp(a) is
mixed with the reagent. The amount of agglutination is measured optically via sample
turbidity using a Response 910 analyser (DiaSys Diagnostic Systems) and is directly
proportional to the amount of Lp(a) in the sample. This Lp(a) method correlates well with
the NLMDRL method (y = 0.03x – 0.13, R = 0.89). The inter-assay CVs at 0.21 g/L, 0.52
g/L and 0.83 g/L were 1.63%, 1.53% and 2.40%, respectively.
3.12 Determination of apolipoprotein(a) isoform size
Principle: Apo(a) isoform size is measured using a highly sensitive sodium dodecyl
sulphate (SDS)-agarose gel electrophoresis procedure followed by immunoblotting
(Northwest Lipid Metabolism and Diabetes Research Laboratories, University of
80
Washington, Seattle, WA). This procedure has a sensitivity of 5 fmol of apo(a). In this
system, a constant relationship exists between the relative migration of apo(a) in the
agarose gel and the number of KIV-2 repeats determined by pulsed-field electrophoresis.
In brief, apo(a) size isoform can therefore be identified from its migration distance on the
gel (Figure 3.2). To enhance the isoform detection, a variable volume of plasma calculated
based on Lp(a) concentration is applied to the gel. In a heterozygous person, when the
immunoblotting signal of one of the two isoforms predominates, the predominant form is
used to describe the apo(a) size of that person. When both isoforms appeared to be
equally expressed, the mean KIV number is used. The major apo(a) isoform is expressed
in terms of the number of KIV repeats.
Figure 3.2 Immunoblot of the human plasma apo(a) isoforms. Lane 1, apo(a) kringle
4 standards ranging from 15 to 38; lanes 2–6 plasma samples (Marcovina et al, 2012).
3.13 Analytical methods for isotopic enrichment
81
3.13.1 Extraction of free leucine from plasma
Free leucine was extracted from plasma using ion-exchange chromatography with cation
resin (AG 50 W-X8, 200-40 mesh, BioRad, Hercules, CA, USA). The resin was prepared
by washing with deionised water and was allowed to settle until the resin bed and
supernatant were of equal volume before the supernatant was discarded. To prepare an
ion-exchange column, for each time sample point, a short Pasteur pipette was plugged at
the tip with cotton wool and 200 µL of the mixed resin solution was added. The resin was
acidified with 1 mL of 6 M HCl and rinsed twice with deionised water.
Whole blood was collected at specific time points into EDTA spray-coated tubes over 96
hours. Blood samples were immediately centrifuged at 3000 rpm for 10 minutes at 4 °C,
and the plasma was removed and stored at –80 °C until further analysis. After thawing at
room temperature, the plasma was vortexed and centrifuged at 30000 rpm for 5 minutes.
An 20 µL aliquot of plasma was mixed with 160 µL of water and, while the mixture was
vortexed, 20 µL of 60% perchloric acid was added to precipitate the plasma proteins. After
precipitation, the mixture was centrifuged at 3000 rpm for 15 minutes at 4 °C, and the
supernatant was diluted with water without disturbing the precipitate. The whole
supernatant was then transferred to the ion-exchange column. Each column was washed
twice with 1 mL of water. The leucine was eluted twice with 0.5 mL of 3 M ammonia and
collected into glass vials. The elute was then dried on a heating block at 70 °C overnight
and at 110 °C for 2 hours before derivatisation. This method was used in Studies 2 and 3.
3.13.2 Derivatisation of leucine
82
The dried samples were derivatised by adding 100 µL of derivatising agent, trifluoroacetic
acid/trifluoroacetic anhydride (TFA/TFAA, 1:1) to form oxazolinone derivatives. The
samples were capped and heated at 110 °C for 5 minutes on a heating block. After cooling
the samples, 400 µL of cyclohexane and 1 mL of deionised water were added. The
samples were mixed for vigorously 30 seconds and centrifuged at 3000 rpm for 5 minutes
at 4 °C. The top layer of cyclohexane contained the extracted derivatives, and the water
layer contained trifluoracetic acid. The oxazolinone derivatives were measured by GC-MS
with negative-ion chemical ionisation. The ions measured were at mass-to-charge ratios
(m/z) of 209 and 212.
Leucine isotopic tracer/tracee ratio at each time point (t) was calculated as:
%Tracer/tracee (at time=i)
= 100 ´ [(area 212)/(area 209)]t=I – 100 ´ 100 ´ [(area 212)/(area 209)]t=0
3.13.3 Isolation and measurement of the isotopic enrichment of
lipoprotein(a) in whole plasma
Lp(a) from plasma was isolated using an immunoprecipitation method, as previously
described. Briefly, 250 µL of a 1:10 dilution of human Lp(a) antibody (Advy Chemical Ltd,
Mumbai, India) was immobilised onto 80 µL of magnetic Dynabead G-proteins (MBGP) by
incubation for 1.5 hours at room temperature. The magnetic beads coated with Lp(a)
antibody-MBGP were then incubated overnight with 80 µL of plasma at 4 °C, which
allowed the isolation of Lp(a) from plasma as an Lp(a)–Lp(a) antibody-MBGP complex.
Lp(a) was eluted from the beads and then reduced by dithiothreitol in sample buffer. The
83
apo(a) protein was separated by SDS-PAGE and transferred onto polyvinylidene fluoride
(PVDF) membrane by Western blotting. The membrane was then stained with amido black
staining solution and destained using methanol. The apo(a) band was excised and
hydrolysed with 200 µL of 6 M HCl at 110 °C overnight. After evaporation of the HCl, the
amino acids were derivatised using an oxazolinone method, and isotopic leucine
enrichment (tracer/tracee ratio) was determined as described earlier. This method was
used in Studies 2 and 3.
Figure 3.3 Experimental protocol for isolation and measurement of isotopic
enrichment of Lp(a).
84
3.13.4 Isolation and measurement of isotopic enrichment of
lipoprotein(a) in LDL/HDL fractions
A sequential ultracentrifugation method was used to isolate the LDL–HDL fraction using a
Beckman titanium 50.4i fixed-angle rotor (Optim XL-100K, Beckman Coulter, Fullerton,
Australia). In brief, 3 mL of EDTA plasma sample obtained at each time point was
collected in an ultracentrifuge tube (Beckman, CA, USA) and centrifuged at 50000 rpm for
16 hours at 20 °C. To collect the VLDL fraction (density <1.0063 kg/L), 1 mL of the
supernatant from the very top of the solution was aspirated without disturbing the rest of
the solution. The solution was then adjusted to 1.019 g/L by adding 1 mL NaCl (6.5% w/w)
and centrifuged again at 50000 rpm for 16 hours at 20 °C to isolate the IDL fraction
(density 1.006 to 1.019 kg/L). The LDL fraction (density 1.019–1.063 kg/L) was
sequentially isolated using the same ultracentrifuge procedures with 1 ml NaCl solution
(21.56% w/w). The HDL fraction (density 1.063–1.21 kg/L) was then isolated using the
same ultracentrifuge procedures with 1.3 ml NaBr solution (45% w/w). Equal volumes of
the LDL and HDL fractions (800 µL each) were then pooled for subsequent isolation and
measurement of the isotopic enrichment of Lp(a) as described earlier. This method was
used in Study 2.
3.14 Kinetic analyses and mathematical modelling
3.14.1 Study 2
A compartmental model was developed using SAAM II software (The Epsilon Group, VA,
USA) to fit it to the tracer enrichment data for Lp(a)–apo(a) and Lp(a)–apoB. Three linked
85
models were used to account for the leucine tracer data: plasma leucine, Lp(a)–apo(a) and
Lp(a)–apoB leucine enrichment (Figure 3.4). Part of the model comprises a four-
compartment subsystem (compartments 1–4) that describe the plasma leucine kinetics.
This subsystem is connected to an intrahepatic delay compartment (compartment 5 and 6)
that accounts for the time required for the synthesis and secretion of Lp(a)–apo(a) and
Lp(a)–apoB into plasma compartment 7 and 8, respectively. The FCR of apo(a) and apoB
were estimated after fitting the model to the apo(a) tracer/tracee ratio.
Figure 3.4 Compartmental model to describe Lp(a)–apo(a) and Lp(a)–apoB tracer
kinetics
3.14.2 Study 3
A similar compartmental model was constructed using SAAM II software (The Epsilon
Group, VA, USA) to fit it to the tracer enrichment data for Lp(a)–apo(a). Two linked models
were used to account for the leucine tracer data: plasma leucine and apo(a) leucine
86
enrichment (Figure 3.5). A four-compartment subsystem (compartments 1–4) was used to
describe the plasma leucine kinetics, with an intrahepatic delay compartment
(compartment 5) that accounts for the time required for the synthesis and secretion of
Lp(a)–apo(a) into plasma compartment 7. The FCR of Lp(a)–apo(a) was estimated after
fitting the model to the apo(a) tracer/tracee ratio. The PR of Lp(a)–apo(a) was calculated
as the product of FCR (pool/day) and apo(a) pool size (nmol) divided by individual body
weight, and is expressed as nmol/kg/day. Pool size was derived by multiplying plasma
volume [body weight (kg) ´ 0.045] and the plasma apo(a) concentration (nmol/L). A
correction factor was applied to adjust for the decrease in relative plasma volume in
subjects who were overweight/obese (Riches et al, 1998).
Figure 3.5 Compartmental model to describe Lp–apo(a) tracer kinetics
3.14.3 Study 4
A compartmental model was developed using SAAM II software (The Epsilon Group, VA,
USA) to fit it to Lp(a) concentration data and estimate the following parameters:
87
concentration immediately before apheresis, concentration immediately after apheresis
and FCR. The study used a single-compartment model with constant PR and reset the
Lp(a) or LDL–apoB concentration to simulate the effect of apheresis treatment. The
catabolism was estimated by the rebound of Lp(a), which was assumed to be in the steady
state. FCR was determined by fitting a mono-exponential model to the concentration data.
The use of SAAM II is more advantageous than using a first order disappearance constant,
as SAAM II modelling uses all the concentration data simultaneously while the first order
disappearance constant takes concentration data as absolute concentration and ignores
any errors associated with them.
The PR was determined using the following equation:
PR (mg/kg/day) = FCR (pools/day) ´ (estimated immediately before apheresis
concentration (mg/L) ´ estimated plasma volume (L) [0.045 ´ body weight (kg)])
The model uses a single-compartment model to determine the kinetics of Lp(a) and LDL–
apoB, which implies that plasma Lp(a) is kinetically homogeneous. This model also
assumes that apheresis does not alter the FCR or PR.
3.15 Statistical analyses
Data analysis in this thesis was performed using IBM SPSS Statistics (version 21; IBM
Corp., Armonk, NY, USA). For all analyses, a P-value of £0.05 was considered to be
significant.
88
3.15.1 Descriptive statistics
Mean (W̅) is the sum of the values divided by the number of samples.
W̅ = WY +W[ + ⋯+W2
R
Geometric mean (GM) measures an average for sets of positive numbers that are
interpreted according to their product and not their sum.
]B = (WYW[ …W2)Y2
3.15.2 Dispersion statistics
Standard deviation (SD) measures the spread of the dataset from the mean. The SD was
calculated by taking the square root of the variance:
_` = a∑(WY − d)[
R − 1
Standard error of mean (SEM) is the SD of the sample means of a population size and
depends on both the population variance and the sample size (n):
_fB =ag[
R
Coefficient of variation (CV) is used to describe the variability of a set of observations
around their mean. It is calculated as follows:
hi =_`dW100%
89
Confidence interval (CI) is a range of intervals that the true value lies between.
W̅ ± mJ
√R
where m is determined by the confidence interval table and s is the SD.
3.15.3 Distribution of data
The normality of the data was assessed using the Shapiro–Wilk test. The null hypothesis
of a test assumes that the sample came from a normally distributed population and test is
more appropriate for small sample sizes (n<50). All skewed variables were log-
transformed before statistical analysis.
3.15.4 Independent-samples t test
The t test was used to evaluate the differences in means between groups (for example,
comparing baseline characteristics between two groups). The t test can be used in studies
with small sample sizes (e.g. n=10) provided that the variables are normally distributed
and the variance does not differ between the two groups. The independent-samples t test
uses the following equation:
o =dY −d[
_f(dY −d[)
where dY and d[ are the mean of two groups and _f(dY −d[) is the standard error of the
mean difference between the groups. SE can be calculated by:
90
_f(dY −d[) = _pY
2q+
Y
2r
where S is the pooled SD of both populations.
The corresponding P-value was then derived from the t distribution with (RY +R[ − 2)
degrees of freedom.
3.15.5 Paired-samples t test
The paired t test is used to evaluate whether the mean difference is significantly different
from zero for a single group or two groups that have been matched. The paired-samples t
test uses the following equation:
o =st̅ − 0u
_f(t̅)
where t̅ is the observed mean difference.
The corresponding P value was then derived from the t distribution with (RY +R[ − 2)
degrees of freedom.
3.15.6 Correlational analysis
Correlational analysis is used to statistically evaluate the strength and direction of the
relationship between two variables. A correlation coefficient ranges from –1 to +1. The
direction of the relationship is reflected in the sign of the coefficient.
91
In this thesis, Pearson’s correlation coefficient ® was used. The correlation coefficient,
given two sets of variables, X and Y, is calculated as:
v =∑(.w6.̅)(4w64x)
y∑(.w6.̅)r ∑(4w64x)r
where W2 and z2 are the values of X and Y for the nth individual.
3.15.7 Linear regression
Y = a + bX
where a = intercept when x = 0 and b = slope
The slope of the regression line (regression coefficient) is calculated as:
{ =∑(.w6.̅)(4w64x)
∑(.w6.̅)r .
3.15.8 Bland–Altman analysis
Bland–Altman analysis was used to evaluate the agreement between paired readings in
Studies 1 and 2. The mean of the two readings is plotted against the difference between
the two readings with bias and 95% limit of agreement (LOA) as the window of agreement
(Altman & Bland, 1983).
92
3.15.9 Lin’s concordance correlation
The concordance correlation coefficient was also used to evaluate the agreement between
paired readings in Studies 1 and 2 (Lin, 2000). The concordance correlation coefficient (rc)
ranges from 0 to 1, and the strength of agreement is classified into four categories: almost
perfect (rc>0.99), substantial (rc>0.95–0.99), moderate (rc=0.90–0.95) and poor (rc<0.90).
The concordance correlation coefficient, given two sets of variables, X1 and X2, is
calculated as:
v! = f[(}Y −}[)[]
gY[ +g[[ + (dY −d[)[
3.16 Sample size and power calculations
The sample size and power calculations are integral to good study design. For a given
study, a sufficient number of subjects is required to increase the possibility of detecting the
true effect of the hypotheses tested. In this thesis, the sample size calculations were
based on a method of Campbell et al (1995). To compute sample size using this method,
one needs to specify three components: (1) power, (ii) significance level and (iii) difference
parameter (d). In this thesis, power was set at least >80% and significance level (two
sided) at 5%. The difference parameter d is a ratio of the expected differences to the
measurement variability and is calculated as follows:
t = KW�KHoKtMKÄRtQÅÅKvKRHK
JoÄRtÄvttKÇQÄoQIR
93
Study 1
The sample size calculation was based on the hypothesis that the LC-MS and NLMDRL
methods to measure apo(a) concentration have a close agreement. Given a correlation of
>0.99, a sample size of 91 patients was estimated to provide >95% power to detect an
association between the LC-MS and NLMDRL methods at a two-sided a = 0.05 (Watts et
al, 2018).
Study 2
The sample size calculation was based on the hypothesis that the FCRs of apo(a) and
apoB within plasma Lp(a) particles are coupled. Given a correlation of >0.99 (Watts et al,
2018), a sample size of 10 patients per group was estimated to provide >85% power to
detect an association between the apo(a) and apoB FCRs at a two-sided a = 0.05.
Study 3
The sample size calculation was based on the hypothesis that patients with elevated Lp(a)
concentration would have a higher PR than those with a normal Lp(a) concentration.
Assuming PRs for apo(a) of 0.83 nmol/kg/day in patients with elevated Lp(a) concentration
and 0.25 nmol/kg/day in those with normal Lp(a) concentration (Chan et al, 2019), and a
within-group SD of 0.20 nmol/kg/day, a sample size of 14 patients per group was
estimated to provide >90% power to detect a difference in apo(a) PR with an a = 5%.
94
Study 4
The sample size calculation was based on the hypothesis that Lp(a) is catabolised more
slowly than LDL–apoB. Given FCRs of 0.15 pools/day for Lp(a) and 0.24 pools/day for
LDL–apoB determined from previously published studies (Armstrong et al, 1989; Thiery et
al, 1996) and a within-group SD of 0.09 pools/day, a sample size of 13 participants was
estimated to provide >80% power to detect a difference between Lp(a) and LDL–apoB
FCRs for 0.10 pools/day for a two-sided a = 0.05.
96
Abstract
Background: Lp(a) contains a complex structure, and the accurate and reliable
quantification of plasma Lp(a) concentration is challenging. The NLMDRL method is a well
established assay and has been used in many studies. However, the method is available
mainly for academic research and is not available in clinical practice. The LC-MS method
has recently been proposed as an alternative to immunoassays for measuring apo(a)
concentration because it is not sensitive to apo(a) isoform size heterogeneity as required
for detection and standardisation. The aim of this study was to compare the validity of a
new LC-MS method with the well established NLMDRL method for measuring apo(a)
concentration in human plasma.
Methods: Plasma Lp(a) concentration was measured using LC-MS and NLMDRL
methods in samples from 91 people who had a wide range of Lp(a) concentration (1.16–
476 nmol/L).
Results: The apo(a) concentration measured by the LC-MS method was highly correlated
with that measured using the NLMDRL method (r=0.977, y=1.10x – 0.11, P<0.001). The
mean difference between the two methods in log-transformed values was –0.03, with 95%
LOAs of –0.45 to 0.45. This was equivalent to –-15.5 nmol/L (95% LOA –79.5 to 48.5
nmol/L) in absolute concentration values. This finding is consistent with the substantial
overall agreement for apo(a) concentration measured using the LC-MS and NLMDRL
methods (rc=0.967, 95% CI 0.954–0.977).
Conclusion: Apo(a) concentration measured by the LC-MS method correlates strongly
with that measured using the well establised isoform-independent NLMDRL immunoassay.
97
4.1 Introduction
Lp(a) is a complex lipoprotein comprising one molecule of the glycoprotein apo(a)
covalently bound to apoB-100-containing LDL-like particles (Nordestgaard et al, 2010).
Lp(a) is highly heterogeneous in structure because of the variable number of kringle
repeats in the apo(a) portion of the molecule. Lp(a) is known to have potent pro-
atherogenic, anti-thrombolytic and inflammatory properties, although not all
epidemiological data support a causal relationship between elevated Lp(a) concentration
and increased risk of ASCVD (Danesh et al, 2000; Kamstrup et al, 2009; Saleheen et al,
2017). The reason for this is unclear but may relate to the lack of standardisation of the
immunoassays used to measure Lp(a) concentration.
Several immunochemical methods, such as ELISA, nephelometry and immunoturbidimetry,
are currently used to measure Lp(a) concentration in human plasma. However, the high
degree of size heterogeneity of apo(a) hinders the development of suitable routine
immunoassays for the accurate and precise measurement of Lp(a) concentration
(Marcovina & Albers, 2016). This is because of different antibodies directed to the Lp(a)
particle and the size of the apo(a) isoform(s) in the calibrator. From a laboratory
perspective, two issues should be considered to improve the standardisation of routine
assay for measurement of Lp(a)concentration. Firstly, the antibodies selected to capture
specific apo(a) epitope should not be affected by apo(a) heterogeneity. Secondly, the
calibrators used in the assay should be traceable to the WHO/IFCC secondary reference
material. However, not all available immunoassays meet these criteria (Cegla et al, 2019).
Moreover, Lp(a) is routinely reported as the apo(a) particle molar concentration (in nmol/L)
or mass (as g/L). Measuring the apo(a) molar concentration reflects the number of
circulating Lp(a) particles, whereas measuring Lp(a) mass reflects the mass of the entire
98
Lp(a) particle including the total mass of apoB, apo(a) and various amounts of cholesteryl
esters, free cholesterol, triglycerides and phospholipids (Tsimikas et al, 2018). Hence, it is
not feasible to apply a single conversion factor to reliably convert the values from g/L to
nmol/L or vice versa, which can compound the use of clinical values for risk stratification
and therapeutic interpretation (Tsimikas et al, 2018). Recent expert guidelines recommend
that Lp(a) concentration should be expressed in nmol/L (Tsimikas et al, 2018).
An isoform-independent method to measure Lp(a) concentration and reported in nmol/L of
apo(a), has been developed by the NLMDRL to minimise the impact of apo(a)
heterogeneity. Briefly, the NLMDRL method uses a mAb (MAb a-40) that specifically
captures the apo(a) particles in a unique apo(a) epitope located in KIV-9 (Marcovina et al,
2000). The method uses calibrators that are traceable to the WHO/IFCC reference
material. Hence, the NLMDRL assay is considered to be a well established assay for
measurement of apo(a) concentration (expressed as nmol/L) in human plasma. However,
the method is available mainly for academic research and is not available in clinical
practice (Cegla et al, 2019).
With the increasing use of LC-MS in clinical laboratories, standardised quantification using
LC-MS has been applied to apo(a) in human plasma. The LC-MS method has recently
been proposed as an alternative to immunoassays for measuring apo(a) concentration
because it is not sensitive to apo(a) isoform size heterogeneity for detection and
standardisation (Ellis et al, 2017). However, the relationship between apo(a) molar
concentration measured by LC-MS compared with that measured using the well
established immunoassay remains to be demonstrated formally.
The aim of this study was to investigate the agreement between a new LC-MS method and
the well established NLMDRL method for measuring apo(a) molar concentration in human
plasma.
99
4.2 Methods
Subjects
Ninety-one Caucasian subjects aged 18–70 years were recruited from the Lipid Disorder
Clinics at Royal Perth Hospital and the general community. The subjects were selected for
a wide range of Lp(a) concentration (1.16–476 nmol/L). None had diabetes, renal disease
(plasma creatinine >130 µmol/L), hepatic dysfunction (alanine aminotransferase >3 times
the upper limit of normal), anaemia (haemoglobin <125 g/L), haematological disorder or
CVD events in the past 6 months. Subjects provided informed written consent, and the
study was approved by the Royal Perth Hospital (Royal Perth Hospital, Perth, Western
Australia) and Bellberry Human Research Ethics Committees (Bellberry Ltd, Eastwood,
South Australia).
Clinical protocol and biochemical assays
Eligible volunteers were admitted to the site metabolic ward after a 14-hour fast. Height
and weight were measured. BMI was calculated in kg/m2. Arterial blood pressure was
recorded after 3 minutes in the supine position using a Dinamap 1846 SX/P monitor
(Critikon, Tampa, FL, USA). Venous blood was collected for biochemical analyses. Plasma
lipid, lipoprotein, apolipoprotein and glucose concentrations were determined by standard
enzymatic methods.
Quantification of apo(a) concentration
100
LC-MS method
This method was performed at Proteomics International (Queen Elizabeth II Medical
Centre, Perth, WA), and the values are reported in nmol/L. Plasma apo(a) concentration
was measured following a standardised sample trypsin digestion procedure and LC-MS. A
specific peptide LFLEPTQADIALLK, located in the proteolytic domain of apo(a), was
selected for apo(a) quantification. The synthetic LFLEPTQADIALLK and D3-labeled
peptide (i.e. LFLEPTQADIALLK and LFLEPTQADIA[D3-L]LK, respectively) were used as
the calibrator and internal standard for the calculation of apo(a) concentration by LC-MS.
The inter-assay CVs were < 5%. Full details of laboratory protocols have been previously
described (Zhou et al, 2013).
ELISA
Apo(a) concentration was measured at the NLMDRL of the University of Washington
(Seattle, WA, USA), and the values are reported in nmol/L. The murine mAb a-6 is
directed to an epitope present in apo(a) KIV-2 for capturing all free and bound apo(a)
particles, and the detection murine mAb a-40 is directed to an epitope present in apo(a)
KIV-9. This ELISA has been demonstrated to be insensitive to apo(a) isoform size
heterogeneity and uses five independent assay calibrators that are traceable to the
WHO/IFCC secondary reference material. Full details of the laboratory protocols have
been previously described (Marcovina et al, 2000).
Determination of apo(a) isoform size
101
Apo(a) isoform size was determined using highly sensitive SDS-agarose gel
electrophoresis followed by immunoblotting (Northwest Lipid Metabolism and Diabetes
Research Laboratories, University of Washington, Seattle, WA) and the apo(a) isoforms
are expressed as the respective number of KIV repeats. Most people have two circulating
apo(a) types of different size and, therefore, the predominantly expressed apo(a) isoform
was used for statistical analysis. Full details of the laboratory protocols have been
previously described (Marcovina et al, 1996).
Statistical analyses
All analyses were performed using IBM SPSS Statistics (version 21; IBM Corp., Armonk,
NY, USA). Data are presented as mean ± SD unless otherwise indicated. The Shapiro–
Wilk test was used to determine whether the variables were normally distributed. Skewed
variables were log-transformed to normalise their distribution. Associations between the
results of the LC-MS and NLMDRL methods were examined using simple and partial
correlational analyses. Agreement between the LC-MS and NLMDRL methods was also
evaluated using Bland–Altman analysis, and the LOA and Lin’s concordance correlation
coefficients (rc) reported. Statistical significance was defined at the 5% level using a two-
tailed test.
4.3 Results
Clinical and biochemical characteristics
Table 4.1 shows the clinical and biochemical characteristics of the 91 participants. On
average, they were middle-aged, non-obese, normotensive and non-diabetic, and had
normal plasma lipid and lipoprotein profiles. Apo(a) concentrations measured by the LC-
102
MS and NLMDRL methods were similar to other published data (Watts et al, 2018). Fifty-
seven participants (63%) had a small apo(a) isoform at a cut-off of £22 KIV repeats.
Comparison of LC-MS and NLMDRL methods for measurement of apo(a)
concentration
Figure 4.1 shows the association of plasma apo(a) concentration measured using the LC-
MS and NLMDRL methods in the 91 participants. The apo(a) molar concentration
measured using the two methods was strongly correlated (r=0.977, y=1.10x – 0.11,
P<0.001). However, the correlation was less between the two methods for apo(a)
concentrations below 30 nmol/L (n=56, r=0.804, y=1,01x – 0.04). Apo(a) isoform size was
significantly and inversely correlated with plasma apo(a) concentration measured by the
LC-MS and ELISA methods (r=–0.594 and –0.633, respectively, P<0.001 for both). The
correlation for plasma apo(a) concentration between the two assays remained significant
after adjustment for apo(a) isoform size (r=0.950, P<0.001).
The Bland–Altman plot of log-transformed data for the NLMDRL and LC-MS methods for
apo(a) measurement is shown in Figure 4.2. The mean difference between the two
methods in log-transformed value was –0.03, with 95% LOAs of –0.45 to 0.45. This was
equivalent to –15.5 nmol/L (95% CI LOA –79.5 to 48.5 nmol/L) in absolute concentration
values (Figure 4.3). These values are consistent with the substantial overall agreement for
apo(a) concentration measured using the LC-MS and NLMDRL methods (rc=0.967, 95%
CI 0.954–0.977). When considering the cut-off of 75 nmol/L as an indicator of increased
risk of ASCVD, this bias resulted in reclassification of only two patients (2.2%) from the
lower to higher risk category.
103
4.4 Discussion
The major finding of this study was that apo(a) concentrations measured using the LC-MS
method showed significant correlation and substantial agreement with the values
measured using the NLMDRL method in Caucasians. The significant correlation of plasma
apo(a) concentration measurement between the two assays was independent of apo(a)
isoform size.
Lp(a) has become a focus of research interest because of the link between elevated
plasma Lp(a) concentration and an increased risk of ASCVD reported in epidemiological
studies (Cegla et al, 2019). A variety of methods, including the Lp(a)–cholesterol assay
and quantitative Lp(a) immunofixation electrophoresis, have been proposed for separating
and measuring Lp(a) (Guadagno et al, 2015; Tsimikas, 2016). However, the reliability and
precision of these methods are suboptimal. Current available immunoassays for routine
measurement of Lp(a) concentration are complicated by the high degree of structural
heterogeneity of apo(a) isoforms, which complicates the choice of assay antibodies and
standards for apo(a) measurement (Ellis et al, 2017).
In this study, a novel LC-MS method was used to measure the molar concentration of
apo(a). This LC-MS method offers several advantages over traditional routinely available
immunoassays for apo(a) measurement in terms of the issues relating to assay antibodies
and reference materials. More specifically, this assay does not require specific antibodies
because it directly targets and detects the signal of a unique peptide for apo(a) (Zhou et al,
2013). The use of synthetic peptide calibrators also minimises the influence of different
apo(a) isoform sizes in the calibrator materials. Consistent with an earlier technical report,
there was excellent correlation and agreement between the LC-MS method and the
NLMDRL method (Chan et al, 2019).
104
We further demonstrated that the correlation of this LC-MS method with the NLMDRL
method was not influenced by apo(a) isoform size. However, we found a weaker
correlation between the two methods at low apo(a) concentration. This suggests that the
precision of this LC-MS method is not optimal for measurement of apo(a) concentration in
people with a low concentration, and this warrants further improvement. Nevertheless, the
potential bias of the LC-MS method only resulted in a reclassification of two patients
(2.2%) from a lower to higher risk category. Hence, we consider that the apo(a)
concentration measured by the LC-MS could be reliably used for diagnostic interpretation
and clinical decision making in the context of Lp(a)-mediated ASCVD risk, particularly in
those with elevated Lp(a) concentration. The LC-MS method could potentially replace
immunoassays such as the Quantia method for routine use.
This study has some limitations. The sample size was small but sufficiently statistically
powered for simple correlational and agreement analyses. However, a larger sample size
is required in future studies to define the Lp(a) cut-off value that reflects an increased risk
of ASCVD. A recent evidence-based guideline suggests that an Lp(a) concentration of
>100 nmol/L is a risk threshold for ASCVD (Tsimikas et al, 2018). The LC-MS method may
have a lower throughput and higher cost relative to traditional immunoassays. However,
the method allows the measurement of multiple proteins from a single sample (2–5 µL),
which should reduce the overall cost of sample analysis. It is noteworthy that the LC-MS
method can measure isotope enrichment for apo(a) and other lipoproteins (apoC and
apoE), which is particularly useful and cost-effective for studying the metabolism of Lp(a)
and related lipoproteins.
In conclusion, the data demonstrate that there is a close agreement between apo(a) molar
concentrations measured by the LC-MS method and those measured using the well
established isoform-independent immunoassay. Further studies are required to assess the
105
standardisation employed in both methods and the reproducibility of the LC-MS method
across the range of apo(a) molar concentration. Also, additional studies on the precision of
the assay would be required for its clinical application.
106
Table 4.1 Clinical and biochemical characteristics in the subjects studied.
Data are presented as mean ± SD or geometric mean (95% confidence interval).
Characteristics n=91
Age (years) 40.3 ± 14.7
Male (%) 87.0
Body mass index (kg/m2) 26.5 ± 4.37
Systolic blood pressure (mmHg) 126 ± 11.1
Diastolic blood pressure (mmHg) 76.4 ± 9.54
Glucose (mmol/L) 5.38 ± 0.42
Total cholesterol (mmol/L) 4.60 ± 0.81
Triglyceride (mmol/L) 1.04 ± 0.49
HDL-cholesterol (mmol/L) 1.23 ± 0.29
LDL-cholesterol (mmol/L) 2.89 ± 0.64
Total apolipoprotein B (g/L) 0.85 ± 0.17
LC-MS apo(a) (nmol/L] 29.0 (22.5, 37.6)
NLMDRL apo(a) (nmol/L) 32.0 (24.0, 42.8)
Predominant apo(a) isoform (KIV repeats) 21.0 (20.0, 22.1)
107
Figure 4.1 Association between apo(a) concentration measured using the LC-MS
and NLMDRL methods in the subjects studied.
108
Figure 4.2 Bland–Altman plot of log transformed values with limits of agreement
(LOA) for the LC-MS and NLMDRL methods to measure apo(a) concentration. Lin’s
concordance correlation coefficient (rc) with 95% CI.
109
Figure 4.3 Bland–Altman plot of absolute concentrations with limits of agreement
(LOA) for the LC-MS and NLMDRL methods to measure apo(a) concentration. Lin’s
concordance correlation coefficient (rc) with 95% CI.
110
Chapter 5
Study 2: Fractional turnover of
apolipoprotein(a) and apolipoprotein B-
100 within plasma lipoprotein(a) particles
in statin-treated patients with elevated
and normal Lp(a) concentration.
Ma L, Chan DC, Ooi EMM, Barrett PHR, Watts GF. Metabolism 2019:96, 443-
454
115
Chapter 6
Study 3: Apolipoprotein(a) kinetics in
statin-treated patients with elevated
plasma lipoprotein(a) concentration.
Ma L, Chan DC, Ooi EM, Marcovina SM, Barrett PHR, Watts GF.
The Journal of Endocrinology & Metabolism 2019:104(12), 6247-6255
125
Chapter 7
Study 4: Lipoprotein(a) and Low-density
lipoprotein apolipoprotein B metabolism
following apheresis in patients with
elevated lipoprotein(a) and coronary
artery disease.
Ma L, Waldmann E, Ooi EMM, Chan DC, Barrett PHR, Watts GF, Parhofer KG. European
Journal of Clinical Investigation 2019:49(2), E13053.
136
8.1 Overview and principal findings
This thesis is presented as a review of the background, philosophical perspectives, study
designs and the methods used, which are followed by a series of studies designed to test
the hypotheses. The studies presented in this thesis have investigated the metabolism of
Lp(a) particles, as reflected by Lp(a)–apo(a) and Lp(a)–apoB concentrations, in patients at
high risk of ASCVD. This chapter provides an overview of the chapters presented,
limitations, implications, suggestions for future studies and conclusion.
Chapter 1 reviewed the current literature on Lp(a) metabolism, genetics, pathophysiology,
measurements and treatments for elevated Lp(a) concentration.
Chapter 2 presented the anatomy of the thesis, which comprises the rationale,
hypotheses, aims, designs and statistical analyses. The principal focus of this chapter was
to identify the hypotheses and aims of the overall project. This thesis included four
separate studies focusing on Lp(a), and this chapter brought together the rationales and
hypotheses of these studies.
Chapter 3 provided details of the clinical methods, biochemical assays, kinetic analyses,
mathematical modelling and statistical analyses. Study 1 compared the Lp(a)
concentrations measured using the NLMDRL and LC-MS methods. The NLMDRL method
measured Lp(a) concentration using a mAb that specifically targets the epitope present in
the apo(a) KIV- 9 domain and is, thus, independent of apo(a) isoform size. The LC-MS
method was performed using an LC-MS/MS equipped with electrospray ionisation and
ultra-performance liquid chromatography. This method measured apo(a) concentration by
using a synthetic peptide (LFLEPTQADIALLK) that targets the proteolytic domain of Lp(a).
Study 2 examined the catabolic rates of Lp(a)–apo(a) and Lp(a)–apoB. Study 3
investigated the metabolic parameters in patients with elevated and normal Lp(a)
137
concentrations. Both of these studies used the same kinetic analyses and mathematical
modelling. Isotopic enrichment using GC-MS was determined using ion monitoring of the
derivatised samples at m/z values of 212 and 209. SAAM II software (The Epsilon Group,
VA, USA) was used to fit the model to the Lp(a) concentration data. Lp(a)–apo(a) and
Lp(a)–apoB metabolic parameters, including the FCR and PR, were derived following a fit
of the compartment model to the Lp(a) tracer/tracee ratio data. Study 4 investigated the
catabolism of Lp(a) and LDL–apoB in patients undergoing apheresis. Lp(a) concentration
was measured using the Response 910 Analyser and Lp(a) 21FS kits (DiaSys Diagnostic
Systems), and LDL–apoB concentration was measured using the Lowry method. All
statistical analyses were conducted using IBM SPSS Statistics (version 21; IBM Corp.,
Armonk, NY, USA), and a P value of £0.05 was considered to be significant.
Chapter 4 (Study 1) presented the findings of a method comparisons between apo(a)
concentration measurement using LC-MS and the reference NLMDRL method in 92
patients with a wide range of Lp(a) concentration (0.03–2.41 g/L, 1.16–476 nmol/L). This
study’s findings showed a substantial correlation and agreement between the LC-MS
method and the NLMDRL method. The results support the use of the LC-MS method for
measurement of apo(a) concentration in clinical practice.
Chapter 5 (Study 2) investigated the catabolism of Lp(a)–apo(a) and Lp(a)–apoB in 20
patients with elevated Lp(a) concentration (³0.8g/L; n=10) or normal Lp(a) concentration
(£0.3 g/L; n=10) using stable isotope techniques and compartmental modelling. Plasma
apo(a) concentration was measured using LC-MS. All patients were on statin therapy and
were studied in the fasting state. The FCRs did not differ significantly between Lp(a)–
apo(a) and Lp(a)–apoB in statin-treated patients with elevated or normal Lp(a)
concentration. The FCR of Lp(a)–apo(a) correlated significantly with that for Lp(a)–apoB in
patients with elevated or normal Lp(a) concentration. Lin’s concordance test showed
138
substantial agreement between the FCRs for Lp(a)–apo(a) and Lp(a)–apoB in patients
with elevated or normal Lp(a) concentration. The study findings show that the apo(a) and
apoB proteins within Lp(a) particles have similar FCRs and are therefore tightly coupled as
an Lp(a) holoparticle in statin-treated patients with elevated or normal Lp(a) concentration.
Chapter 6 (Study 3) investigated the association between metabolic parameters of Lp(a)
and Lp(a) concentration in patients with elevated Lp(a) concentration (n=14) or normal
Lp(a) concentration (n=15) using stable isotope techniques and compartmental modelling.
Plasma apo(a) concentration was measured using LC-MS. All patients were on statin
therapy and were studied in the fasting state. The PR for Lp(a)-apo(a) was higher in
patients with an elevated Lp(a) concentration than in those with a normal Lp(a)
concentration. The FCR of Lp(a)–apo(a) did not differ significantly between the groups. In
univariate analysis, plasma Lp(a)–apo(a) concentration was significantly and positively
associated with the PR for Lp(a)–apo(a) in both patient groups. There was no significant
association between plasma Lp(a)–apo(a) concentration and the FCR in either of the
groups. The study findings show that elevated plasma Lp(a) concentration is a
consequence of increased hepatic production of Lp(a) particles in statin-treated patients.
Chapter 7 (Study 4) investigated the catabolic rates of Lp(a) and LDL–apoB in samples
from 13 patients undergoing apheresis because of elevated Lp(a) concentration (>500
mg/L) and CAD. The FCR was significantly lower for Lp(a) than for LDL–apoB, although
the corresponding PRs did not differ significantly. No significant associations were
observed between the FCRs and PRs for Lp(a) and LDL–apoB. The results suggest that,
in patients with elevated Lp(a) concentration, different metabolic pathways are involved in
the catabolism of Lp(a) and LDL particles.
139
8.2 Study limitations
There are several limitations and assumptions related to the studies in this thesis.
First, the sample sizes for Studies 2 and 3 were too small to demonstrate a significant
association between apo(a) concentration and FCR or a true difference in the Lp(a) FCR
between patients with elevated and normal Lp(a) concentrations. Nevertheless, this
sample size was similar to that used in previous studies.
Second, only Caucasian individuals with hypercholesterolaemia were studied in this thesis.
It is possible that the metabolism of Lp(a) might have been different in patients with more
specific abnormalities (FH, hypertriglyceridaemia, and or diabetes) or ethnic groups.
Future studies should include subjects with different ethnic background and other
characteristics.
Third, Study 1 did not extensively compare the measurement of apo(a) by the LC-MS and
ELISA methods in subjects with intermediate apo(a) concentration (i.e. 70–170 nmol/L).
Future studies should validate the LC-MS method over a wide range of Lp(a) plasma
concentration.
Fourth, The apo(a) concentration determined by the LC-MS method was used for
analyses in Studies 1-3. Although the LC-MS method didn’t have perfect agreement with
the NLMDRL method, study findings were not altered when using the data of apo(a)
concentration determined by the NLMDRL method (See appendix I).
Fifth, Study 2 did not measure the turnover of Lp(a)–apo(a) and Lp(a)–apoB isolated from
the LDL–HDL fractions. This may have affected the Lp(a)–apo(a) and Lp(a)–apoB kinetic
data because of VLDL contamination. However, a previous study found no significant
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differences between Lp(a)–apo(a) kinetic data derived from whole plasma and LDL–HDL
fractions (Watts et al, 2018).
Sixth, Studies 2 and 3 excluded those with extremely low plasma Lp(a) concentration and
focused only on the kinetics of the predominant apo(a) isoform because the gel separation
method is not sufficiently sensitive for isolating minor apo(a) isoforms and for precisely
measuring isotopic enrichment, especially in patients with low apo(a) concentration. Future
studies should analyse the kinetics of the minor apo(a) isoforms because their kinetics
may differ from the predominant apo(a) isoform, which may affect the overall findings.
Seventh, Study 3 examined only subjects receiving statins. Recent experimental data
suggest that statin treatment increases LPA expression and apo(a) production in HepG2
cells (Romagnuolo et al, 2017). However, a study has demonstrated that high-dose
atorvastatin (80 mg/day) did not significantly alter Lp(a) kinetics in subjects with average
Lp(a) concentration within the normal range (Watts et al, 2018). Whether these findings
apply to subjects with elevated Lp(a) concentration needs further investigation.
Eighth, in Studies 2 and 3, the kinetics of LDL–apoB was not examined because of
possible interference (or contamination) of Lp(a)–apoB within LDL fractions, especially in
patients with high apo(a) concentration. Future studies should remove Lp(a) from the LDL
fraction to investigate the kinetics of LDL–apoB.
Finally, in Study 4, the kinetic model was based on certain assumptions. It was assumed
that the PRs and FCRs of Lp(a) and LDL–apoB remain constant during lipoprotein
apheresis. However, LDL-R activity may increase following apheresis. The study also
investigated the metabolic parameters under non-steady state. The kinetic results for Lp(a)
and LDL might have been different if these parameters were measured under the steady
state in the absence of apheresis.
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8.3 Implications of the research findings
Elevated Lp(a) concentration is an important risk factor for CVD, calcific aortic valve
disease, peripheral vascular disease and other conditions. However, reliable measurement
of Lp(a) concentration is challenging because of problems with the standardisation of Lp(a)
assays. The mechanisms responsible for elevated Lp(a) concentration in high-risk patients
are also not fully understood. The present studies attempted to address these research
gaps firstly by validating an LC-MS method for apo(a) measurement (Study 1) and
secondly by using stable isotope techniques (Studies 2 and 3) and lipoprotein apheresis
(Study 4) for the dynamic assessment of Lp(a) metabolism.
The findings of Study 1 indicate that the LC-MS method is a reliable and accurate method
to measure Lp(a) molar concentration when compared with a reference ELISA. This LC-
MS assay is not affected by apo(a) size heterogeneity, which overcomes the inherent
analytical problems associated with the routine use of antibodies and calibrators. However,
specific Lp(a) cut-points for the LC-MS method are needed to identify people at risk of
CVD who may require intervention. Although the LC-MS assay is not readily available for
high-throughput screening, a potentially wider application may be to combine
measurements of apo(a) concentration and isotopic enrichment as part of tracer-based
kinetic studies.
The findings of Study 2 demonstrated that the FCRs of Lp(a)–apo(a) and Lp(a)–apoB are
similar in statin-treated patients with elevated Lp(a) concentration. We also confirm this
observation in statin-treated people with a normal Lp(a) concentration. This observation
provides new evidence that apo(a) and apoB within the Lp(a) particle are tightly coupled
during its residence in the circulation and are cleared together as a holoparticle, which
refutes the notion of apo(a) recycling. Future kinetic studies of specific inhibitors of apoB or
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apo(a) synthesis on the turnover of Lp(a)–apo(a) and Lp(a)–apoB may help define further
the coupling of apo(a) and apoB within Lp(a) particles.
The principal aim of Study 3 was to investigate the kinetic mechanism for elevated Lp(a)
concentration in patients on optimal cholesterol-lowering therapy. The novel findings were
that patients with elevated Lp(a) concentration have higher hepatic production of apo(a)
than do patients with normal Lp(a) concentration. The present results also suggest that the
association between plasma Lp (a) concentration and PR is independent of apo(a) isoform
size. This supports the clinical use of agents that lower the production of Lp(a) with the aim
of targeting elevated plasma Lp(a) concentration and attenuating the risk of ASCVD
against background statin therapy.
An important controversial issue of Lp(a) research is whether the LDL-R is responsible for
the removal of Lp(a) particles. In Study 4, the rebound of Lp(a) and LDL–apoB
concentrations in patients undergoing apheresis was investigated to estimate the
corresponding FCRs during non-steady state. There was a difference in the FCRs of Lp(a)
and LDL–apoB in patients with elevated Lp(a) concentration. The slower removal of Lp(a)
than of LDL implies that more frequent apheresis would be required to treat individuals
with elevated Lp(a) concentration compared with elevated LDL alone. The results also
provide new evidence that the LDL-R may not play a major role in Lp(a) catabolism under
normal physiological conditions. However, recent evidence showing increased clearance
of Lp(a) with statin and PCSK9 mAb combination treatment may relate to
supraphysiological upregulation of the hepatic LDL-R and/or decreased competition of
Lp(a) with LDL particles for LDL-R uptake (Watts et al, 2018).
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8.4 Suggestions for future studies
The work contained in the thesis is based on the acquisition of new knowledge by
empirical observation and experimentation, hypothesis testing and deductive and inductive
reasoning. The findings and study limitations discussed above have led to the generation
of future research questions that may be tested in future studies. These are discussed
below.
8.4.1 Study population
The sample population in this thesis was middle-aged Caucasians with a normal or
elevated Lp(a) concentration. Genetic factors are known to alter Lp(a) metabolism.
Therefore, future studies should include patients with FH, hypertriglyceridaemia or
combined hyperlipidaemia, as well as subjects of other ethnic groups.
8.4.2 Integration of other lipoproteins and kinetic parameters
The liver appears to have the ability to secrete and remove lipoproteins of a wide variety of
sizes and compositions, such as VLDL, IDL and LDL. The investigation of TRL kinetics
may be important to understanding the overall integration of these lipoproteins with Lp(a)
particles in subjects with normal or elevated Lp(a) concentration. Further development of
the kinetic model involving Lp(a) should integrate and study these kinetic parameters
under the postprandial state.
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8.4.3 Measurement of lipoprotein(a) concentration
Lp(a) concentration can be quantified using different kinds of immunochemical methods,
and the ELISA is used most often in the clinical laboratory. However, there is a lack of
global standardisation of the different methods for measuring Lp(a) concentration because
of the use of different calibrators and antibodies (Marcovina & Albers, 2016). Apo(a)
concentration measured by the LC-MS method is robust, but future studies should
investigate the inter- and intra-variability in fasting and non-fasting samples, the effects of
freeze–thaw cycles, evaluation of analytical aspects, and cut-off values for CAD risk
assessment in different racial/ethnic groups including Africans and Asians.
8.4.4 Molecular, biological and genetic studies of lipoprotein(a)
metabolism
As outlined in the literature review, the synthesis, assembly and clearance of Lp(a) remain
largely undefined. Many gaps exist in the knowledge about Lp(a) biosynthesis and
catabolism. Future studies should investigate whether the binding of apo(a) to apoB-100
occurs outside or within the circulation and whether the bond between apo(a) and apoB-
100 is irreversible. The contribution of receptor-mediated catabolism to the clearance of
plasma Lp(a) is also debated, and there is evidence both for and against a role for LDL-Rs
(Cain et al, 2005; Floren et al, 1981; Rader et al,1995; Tam et al, 1996). The involvement
of other cellular receptors, including the VLDL-R, LRP1, SR-BI and plasminogen receptor
in Lp(a) catabolism also merits further investigation.
Lp(a) is a quantitative heritable trait regulated by the LPA locus. LPA directly determines
apo(a) size polymorphism and is responsible for >90% of the variation in Lp(a)
145
concentration (Enas et al, 2019). Further studies are needed to explore the impact of
apo(a) size polymorphism, CNV and SNPs on Lp(a) metabolism across different ethnic
populations.
8.4.5 Treatment modalities
The findings in this thesis suggest that Lp(a) has a wide range of plasma concentrations
that are determined kinetically chiefly by the PR and, to a lesser extent, the catabolism of
Lp(a). Further kinetic studies should examine the effect on Lp(a) metabolism of statin in
monotherapy or in combination with other treatment modalities, as discussed below.
Statins
The effect of statins on Lp(a) is controversial. Statins lower LDL-C concentration and may
either not affect or cause an increase in Lp(a) concentration (Ky et al, 2008; Tsimikas et al,
2019; Sahebkar et al, 2017). Future studies should investigate the mechanisms by which
statins can increase Lp(a) concentration. Understanding why statins can increase Lp(a)
concentration in some patients will help in determining whether this is part of the residual
risk.
Fibrates
A recent meta-analysis has shown that fibrates have a significantly greater effect in
reducing plasma Lp(a) concentration than statins (Sahebkar et al, 2017). The addition of
fibrates to statins can increase further the reduction in Lp(a) concentration by statins.
146
Reducing apo(a) expression and increasing Lp(a) clearance by fibrates and statins may
have a beneficial effect by decreasing the risk of CVD. Future studies should investigate
the mechanisms underlying the effects of these two therapies and compare the effects
with those of other conventional Lp(a)-lowering agents as well as the newer therapies.
PCSK9 monoclonal antibodies
PCSK9-mAbs, such as evolocumab and alirocumab, have been shown to reduce plasma
Lp(a) concentration by 20–50% in clinical trials (Reyes-Soffer et al, 2017; Watts et al,
2018). PCSK9 inhibits Lp(a) internalisation in hepatocytes through the LDL-R under in vitro
conditions, which suggests that inhibition of PCSK9 and concomitant statin therapy could
result in supraphysiological concentrations of the LDL-R and very low concentrations of
LDL (Romagnuolo et al, 2017). This could lead to significant clearance of Lp(a). Despite
the significant reduction in Lp(a) by PCSK9-mAb, the mechanism of action is not clearly
understood. A recent study investigated the effect of PCSK9-mAb on Lp(a) metabolism in
statin-treated patients with elevated Lp(a) concentration (Appendix I). In this study,
alirocumab lowers elevated plasma Lp(a) concentrations by accelerating the catabolism of
Lp(a) particles. The study suggested that while inhibition of PCSK9 with alirocumab lowers
Lp(a) via upregulation of the LDL pathway, further reduction in Lp(a) concentration to
prevent ASCVD requires targeting the production of Lp(a) particles in order to fully address
the residual risk attributed to lowering LDL-C alone. Future studies could examine the
apo(a) ASO or small interfering RNAs on lowering Lp(a) in targeting apo(a) production
against background of statin and PCSK9 mAb therapy.
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RNA-based therapies
Apo(a) antisense oligonucleotide. The IONIS-APO(a)RX specifically targets bound LPA
transcripts for degradation by RNase H1, which leads to a reduction in apo(a) translation
(Merki et al, 2011). This has been shown to reduce Lp(a) concentration by about 90% and
has a favourable safety and tolerability profile (Graham et al, 2016). This significant
reduction in Lp(a) concentration by apo(a) ASO was expected, as production is known to
be the major driver of Lp(a) concentration. Future kinetics studies should investigate the
effects of apo(a) ASO on Lp(a) metabolic parameters and CVD risk.
Small interfering RNA. Inclisiran is a small interfering RNA therapy that targets PCSK9
mRNA within hepatocytes and reduces the production of LDL-C significantly (Ray et al,
2018). It has been shown to reduce Lp(a) concentration by about 26%. The mechanism is
unclear, but it has been proposed that Lp(a) concentration is reduced by inclisiran via
intracellular inhibition of PCSK9 (Ray et al, 2018). PCSK9 inhibition reduces Lp(a)
concentration via extracellular inhibition of PCSK9. Future studies should investigate the
metabolic pathways and the difference in metabolic parameters between inclisiran and
PCSK9 mAb.
ApoB antisense oligonucleotide. Mipomersen is an apoB ASO that targets apoB mRNA
by inducing the degradation of bound transcripts by RNase h1 (Rosie et al, 2007).
Mipomersen reduces Lp(a) concentration by 20–50%. However, mipomersen is not
available because of its side-effect profile (Norata et al, 2012). Mipomersen has been
shown to increase the mean FCR of Lp(a) by 27% but has no effect on the PR
(Nandakumar et al, 2018). These findings were unexpected because the reduction in Lp(a)
concentration was affected by the FCR and not the PR, despite the significant reduction in
the apoB PR. Future studies could investigate the effects of mipomersen on Lp(a)
metabolism in patients with elevated Lp(a) concentration.
148
Microsomal triglyceride transfer protein inhibitor. Lomitapide is an MTP inhibitor. MTP
is required for the assembly of apoB-100-containing lipoproteins. Inhibition of MTP results
in the reductions in chylomicron and VLDL synthesis and secretion, which leads to lower
LDL concentration (Cuchel et al, 2007; Samaha et al, 2008). One study found that
lomitapide reduced Lp(a) in a dose-dependent manner, which resulted in a 17% decrease
in plasma Lp(a) concentration (Stefanutti et al, 2015). The mechanism of action has not
been studied, and it may be worthwhile to investigate the kinetics of changes in Lp(a)
concentration caused by MTP inhibitors.
Cholesteryl ester transfer protein inhibitor. Inhibitors of CETP, such as evacetrapib and
obicetrapib, have been shown to reduce Lp(a) concentration by up to 40% (Nicholls et al,
2016; Hovingh et al, 2015). Similar to apoB ASO, these are not used therapeutically
because of the side-effect profile. However, the mechanism responsible for CETP
inhibition may assist in future studies investigating Lp(a) metabolism.
Inhibitory effects on the expression of Lp(a)
Future studies could alter expression of LPA by using therapies such as anti-TNF-α, TGF-
b and PPAR modulators, and further investigate how these therapies could impact on
Lp(a) mediated risk of CVD.
8.4.6 Guidelines
The findings from this thesis may inform three topics addressed by the guidelines and
recommendations on Lp(a). These include assays for Lp(a) (Cegla et al, 2019; Mach et al,
149
2019; Wilson et al, 2018), use of apheresis in high risk patients (Cegla et al, 2019) and
knowledge of Lp(a) kinetics (Tsimikas et al, 2018; Wilson et al, 2018).
These guidelines have suggested that there is a critical need for better standardisation of
Lp(a) measurement in routine clinical practice (Cegla et al, 2019; Mach et al, 2019; Wilson
et al, 2018). Commercial assays for Lp(a) do not generally use antibodies that are isoform
independent (Scharnagl et al, 2019). The LC-MS method could be used as a reference
method for newly developed assays. However, LC-MS method would require further
standardisation.
The HEART UK guideline recommend that regular apheresis should be considered for
patients with progressive CAD and Lp(a) greater than 150 nmol/L despite maximal lipid
lowering therapy (Cegla et al, 2019). The findings from Study 4 agrees with the guideline
and suggest that frequent apheresis would be required to treat individuals with elevated
Lp(a) compared with LDL alone, as Lp(a) particles appear to be catabolised slower than
LDL.
The recent introduction of apo(a) antisense therapy has demonstrated an ability to
significantly lower plasma Lp(a) levels and it is known that the mechanism involves
reduction of apo(a) mRNA levels. However, it is important to fully explore the kinetic
mechanisms by which these reductions occur (Tsimikas et al, 2018). Study 3 findings
provide a kinetic rationale for the use of therapies that target synthesis of apo(a) and
production of Lp(a) particles in patients with elevated Lp(a).
8.5 Conclusion
Elevated plasma Lp(a) concentration is an important risk factor for atherosclerosis, and
this may account for the increased residual risk of CVD in patients with elevated Lp(a)
150
concentration. This thesis aimed to increase the understanding of Lp(a) metabolism in
patients with elevated Lp(a). The findings of the studies presented in this thesis support
the notion that Lp(a) metabolism is driven primarily by production and that catabolism may
play a role to a lesser degree (Study 3). Another novel finding was that the FCRs of the
apo(a) and apoB proteins within Lp(a) particles are tightly coupled (Study 2). The slower
FCR of Lp(a) FCR than LDL–apoB following apheresis suggests that the LDL-R may not
be responsible for Lp(a) catabolism in non-steady conditions (Study 4). The results of this
work suggest several areas for future research in patients with elevated Lp(a), including
studies aimed at understanding the kinetic bases of other atherogenic lipoproteins (VLDL
and LDL) and the mechanisms of actions of statins together with PCSK9-mAb and/or RNA
therapies on Lp(a) metabolism.
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APPENDIX I
PCSK9 Inhibition with alirocumab
increases the catabolism of
lipoprotein(a) particles in statin-treated
patients with elevated lipoprotein(a)
Watts GF, Chan DC, Pang J, Ma L, Ying Q, Aggarwal S, Marcovina SM, Barrett PHR.
Metabolism 2020, recently accepted.
191
PCSK9 Inhibition with alirocumab increases the catabolism of lipoprotein(a)
particles in statin-treated patients with elevated lipoprotein(a)
Gerald F. Watts,a, b, Dick C. Chan,b Jing Pang,b Louis Ma,b Qidi Ying,b Shashi Aggarwal,c
Santica M. Marcovina,d P. Hugh R. Barrett e
aLipid Disorders Clinic, Department of Cardiology, Royal Perth Hospital, Perth, Australia;
bSchool of Medicine, University of Western Australia, Perth, Australia; cLinear Clinical
Research Ltd, Perth, Australia; dNorthwest Lipid Metabolism and Diabetes Research
Laboratories, Division of Metabolism, Endocrinology, and Nutrition, Department of
Medicine, University of Washington , Seattle, USA. eFaculty of Medicine and Health,
University of New England, Armidale, Australia
Clinical Registry: ACTRN12617000401358 (https://www.anzctr.org.au)
Address for correspondence Winthrop Professor Gerald F Watts, School of Medicine,
University of Western Australia, Royal Perth Hospital, GPO Box X2213, Perth, WA 6847,
Australia.
Tel: +61-8-9224-0245; E-mail: [email protected]
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ABSTRACT
Background: Lipoprotein(a) (Lp(a)) is a low-density lipoprotein (LDL) particle containing
apolipoprotein(a) (apo(a)) covalently linked to apolipoprotein B-100 (apoB). Statin-treated
patients with elevated Lp(a) have an increased risk of atherosclerotic cardiovascular
disease (ASCVD). Recent trials show that proprotein convertase subtilisin/kexin type 9
(PCSK9) inhibition decreases Lp(a) and cardiovascular events, particularly in high risk
patients with elevated Lp(a). We investigated the kinetic mechanism whereby alirocumab,
a PCSK9 inhibitor, lowers Lp(a) in statin-treated patients with high Lp(a) and ASCVD.
Methods: The effects of 12-week alirocumab treatment (150 mg every 2 weeks) on apo(a)
kinetics were studied in 21 patients with elevated Lp(a) concentration (>0.5 g/L). Apo(a)
fractional catabolic rate (FCR) and production rate (PR) were determined using
intravenous D3-leucine administration, mass spectrometry and compartmental modelling.
All patients were on long-term statin treatment.
Results: Alirocumab significantly decreased plasma concentrations of total cholesterol (-
39%), LDL-cholesterol (-67%), apoB (-56%), apo(a) (-25%) and Lp(a) (-22%) (P< 0.001 for
all). Alirocumab also significantly lowered plasma apo(a) pool size (-26%, P <0.001) and
increased the FCR of apo(a) (+28%, P< 0.001), but did not alter apo(a) PR, which
remained significantly higher relative to a reference group of patients on statins with
normal Lp(a) (P< 0.001).
Conclusions: In statin-treated patients, alirocumab lowers elevated plasma Lp(a)
concentrations by accelerating the catabolism of Lp(a) particles. This may be consequent
on marked upregulation of hepatic receptors (principally for LDL) and/or reduced
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competition between Lp(a) and LDL particles for these receptors; the mechanism could
contribute to the benefit of PCSK9 inhibition with alirocumab on cardiovascular outcomes.
Abbreviations: Apo: Apolipoprotein; ASCVD: Atherosclerotic cardiovascular disease;
BMI: body mass index; CAC: Coronary artery calcification; CT: Computed tomography;
ELISA: Enzyme-linked immunosorbent assay; FCR: Fractional catabolic rate; FH: Familial
hypercholesterolemia; HDL: High-density lipoprotein; KIV: Kringle IV; LDL: Low-density
lipoprotein; Lp(a): Lipoprotein(a); Mabs: Monoclonal antibodies ; MBGP: Magnetic beads
G-proteins; PCSK9: Proprotein convertase subtilisin/kexin type 9; PR: Production rate;
SDS: Sodium dodecyl sulfate
Keywords: Alirocumab, atherosclerotic cardiovascular disease, lipoprotein(a),
apolipoprotein (a) kinetics, proprotein convertase subtilisin/kexin type 9
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Highlights
• PCSK9 inhibition decreases Lp(a) and cardiovascular events in high risk patients
with elevated Lp(a).
• The effects of 12-week alirocumab treatment on apo(a) kinetics was studied in 21
patients with elevated Lp(a).
• Alirocumab significantly lowered plasma apo(a) pool size and increased the
fractional catabolic rate of apo(a), but did not alter apo(a) production rate.
• The mechanism could contribute to the benefit of PCSK9 inhibition with alirocumab
on cardiovascular outcomes.
• The findings support use of new therapies for lowering Lp(a) in targeting apo(a)
production.
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1. Introduction
Lipoprotein(a) (Lp(a)) is a low-density lipoprotein (LDL) particle additionally containing
apolipoprotein(a) (apo(a)) covalently linked to apoB-100 [1]. Genetic, epidemiological and
Mendelian randomization studies suggest that elevated plasma concentrations of Lp(a) are
strongly and causally linked with an increased risk of atherosclerotic cardiovascular
disease (ASCVD) [2, 3]. Elevated plasma Lp(a) concentration is principally due to
increased hepatic production of Lp(a) particles [4]. The accumulation of Lp(a) may
promote foam cell formation, oxidative stress, inflammation and thrombosis in the
subendothelial space [2, 3, 5].
Statins effectively lower LDL-cholesterol, but have no impact or may even increase plasma
Lp(a) concentrations [6]. Elevated plasma Lp(a) concentration may partly account for the
residual risk of ASCVD in statin-treated patients [7, 8]. Lowering Lp(a) in this group of
patients is a major therapeutic target that may partly be met by inhibiting proprotein
convertase subtilisin/kexin type 9 (PCSK9) [9]. A recent recommendation by an expert
group identified several knowledge gaps in Lp(a) research, underscoring the importance of
investigating the kinetic mechanism whereby PCSK9 inhibitors reduce Lp(a) levels [3].
PCSK9 is a secretory protease that regulates cell surface receptors, principally the LDL
receptor [10]. The role of PCSK9 in Lp(a) metabolism may predominantly involve
regulation of the LDL receptor, but there is also evidence that PCSK9 may control the
hepatic secretion of apo(a) [11]. PCSK9 monoclonal antibodies (mAbs) potently lower
plasma LDL-cholesterol by markedly upregulating the LDL receptor and hence the
catabolism of LDL-apoB [12-14]; these agents are also known to lower Lp(a)
concentrations [9, 13, 14]. In normolipidemic individuals, we previously reported that
inhibition of PCSK9 with evolocumab lowered plasma Lp(a) concentration by decreasing
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the production of Lp(a) particles as monotherapy, and by increasing the catabolism of
Lp(a) particles in combination with a high potency statin [15].
In the present study, we extend our previous study by undertaking the first investigation of
the kinetic mechanism of action of the inhibition of PCSK9 with alirocumab on the
catabolism and production of apo(a) patients with elevated Lp(a), who were receiving long-
term treatment with maximally tolerated statins in a clinical setting.
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2. Methods
2.1 Patients
Twenty-one patients (12 men and 9 women) aged 18-70 years were recruited from the
Lipid Disorders Clinics, Department of Cardiology, Royal Perth Hospital. All patients
selected had plasma Lp(a) concentrations > 0.5 g/L and were on maximally tolerated statin
therapy and aspirin (100mg daily), and had clinical or subclinical evidence of ASCVD.
Clinical ASCVD was defined as a definite history of either stable angina, coronary artery
bypass graft surgery, percutaneous transluminal coronary angioplasty, transient ischemic
attack, or peripheral arterial disease. Subclinical evidence of ASCVD was defined as the
presence of carotid artery plaques and/or a non-zero coronary artery calcium score.
Exclusion criteria included: diabetes, renal disease (plasma creatinine >130 µmol/L),
hepatic dysfunction (alanine aminotransferase >3 x upper limit of normal), anaemia
(haemoglobin <125 g/L), women of child bearing and potential pregnancy, haematological
disorders or ASCVD events in the past 6 months and inability to consent to study; none
were current smokers or taking medications potentially affecting Lp(a) metabolism other
than statins (e.g. hormonal replacement therapy, niacin). Six of the 21 patients with
elevated Lp(a) were recruited from a case-control study reported earlier [16].
To assess the kinetic abnormalities in Lp(a) particles in relation to treatment with
alirocumab, we also compared apo(a) kinetics of the patients with elevated Lp(a) with a
reference group of 15 patients of comparable age and gender selected with normal plasma
Lp(a) concentrations (≤ 0.3 g/l) from our earlier report (8 men and 7 women; mean+SD:
aged 55 ± 12 years, body mass index [BMI] 28 ± 5.0 kg/m2) [16]; this reference group was
also receiving treatments with statins and aspirin (Appendix Supplementary Table 1). The
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study was approved by a national ethics committee (Bellberry Ltd, Eastwood, South
Australia); all subjects provided informed consent.
2.1 Study design and clinical protocol
We carried out a single-sequence study of the effect of alirocumab on plasma lipid,
lipoprotein and apolipoprotein concentrations (12-weeks on-treatment and 16-weeks
follow-up post-treatment, respectively), within which was nested a kinetic study (before
and after treatment) of the effect of alirocumab on apo(a) kinetics. All patients entered a 2-
week run-in, diet-stabilizing period, at the end of which they were administered PCSK9-
mAb therapy for 12 weeks (alirocumab SC 150 mg every 2 weeks). Advice was given to
patients to continue an isocaloric diet and maintain medication and physical activity
constant until the end of the post-treatment period. Body weight and arterial blood
pressure were recorded at the beginning and end of alirocumab treatment and at follow-up.
2.2 Metabolic protocol
Eligible patients and controls were admitted to the site metabolic ward after a 14-hour fast.
Weight was measured to the nearest 0.1 kg in light clothing (no shoes). Height was
measured as the distance from the top of the head to the bottom of the feet (no shoes)
using a fixed stadiometer. BMI was calculated as the weight in kg divided by the squares
of the height in metres. Waist circumference was measured at the point halfway between
the lower costal margin and the iliac crest. Arterial blood pressure was measured using
Vital Signs Monitor 53NTO (Welch Allyn, Macquarie Park, NSW, Australia). They were
studied in a semi-recumbent position and allowed to drink only water. Venous blood was
collected for laboratory measurements, and plasma volume was determined by multiplying
body weight by 0.045. A single bolus of D3-leucine (5 mg/kg of body weight) was
administered intravenously within a 2-minute period into an antecubital vein. Blood
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samples were taken at baseline and at 5, 10, 20, 30, and 40 minutes and at 1, 1.5, 2, 2.5,
3, 4, 5, 6, 8, and 10 hours after injection of the isotope. Subjects were then given a snack
and discharged home. Additional fasting blood samples were collected in the morning on
the following 4 days of the same week [4, 15, 16]. All the procedures were repeated after
the 12-week intervention period with alirocumab, except in the reference group of patients
in whom only a single isotope infusion study was performed. In the follow-up study, venous
blood was collected for laboratory analysis in the semi-recumbent position after a 14-hour
fast.
2.3 Isolation and measurement of isotopic enrichment of Lp(a)
Lp(a) was isolated by an immunoprecipitation method, as previously described [4, 15, 16].
Briefly, Lp(a) was isolated from the LDL-HDL subfraction by sequential ultracentrifugation
at a density of 1.06-1.21 g/L. 250 μL human Lp(a) antibody (Advy Chemical Ltd, Mumbai,
India) were immobilised onto 80 μL magnetic beads G-proteins (MBGP) by incubation for
1.5 hours at room temperature. The magnetic beads coated with Lp(a) antibody-MBGP
were then incubated overnight with 80μL plasma at 4oC, thereby separating Lp(a) from
plasma as an Lp(a)-Lp(a) antibody-MBGP complex. Lp(a) was eluted from the beads and
then reduced by dithiothreitol in sample buffer. The apo(a) protein was separated by
sodium dodecyl sulfate gel (SDS) electrophoresis and transferred onto polyvinylidene
fluoride membrane by Western Blotting. The predominant apo(a) isoform with the highest
density was excised and hydrolysed with 200 μl 6M HCl at 110oC overnight. After
evaporation of HCl, the amino acids were derivatised using an oxazolinone method.
Isotopic enrichment was determined as described previously [4, 15, 16].
2.4 Biochemical analyses
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Plasma glucose, cholesterol, triglyceride, total apoB, HDL-cholesterol, LDL-cholesterol,
apoB, were determined before and after 12-week intervention and at the end of the follow-
up period by standard enzymatic methods (Architect c16000 Analyser, Abbott Diagnostics,
Abbott Laboratories, Abbott Park, IL) and LDL-cholesterol by the Friedewald calculation.
Plasma Lp(a) mass was determined by a routine immunoassay (Quantia assay, Abbott
Diagnostics) for the screening and selection of patients for the study [16]. The coefficient
of variation for all biochemical analyses was less than 5%. Plasma Lp(a) protein
concentration was measured as particle number in nmol/L at the Northwest Lipid
Metabolism and Diabetes Research Laboratories (NLMDRL) by a direct-binding double
MAb-based ELISA [17]. Briefly, the capture MAb (a-6) was directed to an epitope present
in apo(a) K4 type 2, and the detection antibody (a-40) directed to an epitope present in
apo(a) K4 type 9. This ELISA method has been demonstrated to be insensitive to apo(a)
isoform size heterogeneity and employs a calibrator from a single donor with an assigned
value in molar units. Apo(a) isoform size was determined by a high-resolution SDS-
agarose gel electrophoresis method coupled with immunoblotting, as previously described
[18]. Apo(a) isoform size was identified from the migration distance on the gel and
expressed as the number of apo(a) Kringle IV (KIV) repeats [18]. For statistical analyses,
in the case of heterozygous patients, the predominant isoform was used to describe
apo(a) size. Plasma apo(a) was also determined as particle number in nmol/L by LC-
MS/MS analyses that utilises a synthetic peptide (LFLEPTQADIALLK), as a standard, that
targets the proteolytic domain of Lp(a) (Proteomics International, Queen Elizabeth II
Medical Centre, Perth, WA) [4, 15,16].
2.5 Kinetic modelling
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A compartmental model was constructed using the SAAM II software (The Epsilon Group,
Virginia, USA) and fitted to the apo(a) tracer data [4, 15, 16]. Two linked models were used
to account for the leucine tracer data; plasma leucine and apo(a) leucine. The fractional
catabolic rate (FCR) of apo(a) was estimated after fitting the model to the apo(a)
tracer/tracee ratio data. Models were selected using the Akaike information criterion and
goodness of fit was tested by confirming that the residuals were randomly distributed
around mean zero. The hepatic production rate (PR) of apo(a) was calculated as the
product of FCR and pool size of apo(a). Pool size was derived by multiplying plasma
volume and the plasma apo(a) concentration (nmol/L).
2.6 Statistical analyses
Data were analysed using SPSS 21 software (SPSS, Chicago, USA). Data were
presented as mean ± SD unless otherwise indicated. The Shapiro-Wilk test was used to
determine whether variables were normally distributed. Differences in apo(a) kinetic
characteristics between patients with elevated and normal Lp(a) concentration were tested
with independent t-tests, after logarithmic transformation of skewed variables where
appropriate. Changes in clinical and biochemical parameters during the pre-treatment, on-
treatment and post-treatment phases were compared using repeated measures ANOVA. If
the P-value from ANOVA test was < 0.05, post-hoc t-test was performed between pre-
treatment and on-treatment values. The effects of alirocumab on apo(a) kinetic parameters
were evaluated using paired-t-test. We also assessed the impact of the type and dose of
statin on kinetic outcome variables using general linear modelling; for dose assessment,
all statins were converted to an equivalent dose of atorvastatin [19]. Associations were
examined by Pearson’s correlational analyses. Statistical significance was defined at the
5% level using a two-tailed test.
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3. Results
3.1 Patient characteristics
Of the twenty-six subjects screened, four patients did not meet the selection criteria and
one patient withdrew prior to intervention for personal reasons. Twenty-one patients with
elevated Lp(a) (12 men and 9 women) were eligible and completed the study. Of these,
one was found to be positive for a mutation causative of familial hypercholesterolemia (FH),
and 2 were classified as definite FH according to the Dutch Lipid Clinic Network
phenotypic classification [20]. Appendix Table 1 shows the clinical and biochemical
characteristics of patients with elevated Lp(a) concentrations. They were on average
middle-aged, overweight and normotensive (Appendix Table 1). Eleven patients had at
least one clinical ASCVD event (stable angina, n=1; percutaneous transluminal coronary
angioplasty, n=5; coronary artery bypass graft surgery, n=3; transient ischemic attack, n=2
and peripheral arterial disease, n=2); 10 patients had subclinical ASCVD (plaques on
cardiac ultrasound or CT angiography n=8 and coronary artery calcification score > 0, n=2).
The 10 patients with subclinical ASCVD were considered to be at high ASCVD risk by the
Australian risk calculator [21]. All patients were on maximally tolerated and stable statin
therapy as prescribed in the clinic: rosuvastatin (40 mg/day, n=11; 20 mg/day, n=1; 10
mg/day, n=1; 5 mg/day, n=2), atorvastatin (80 mg/day, n=1; 20 mg/day, n=2), pravastatin
(40 mg/day, n=1; 20 mg/day, n=1) and fluvastatin (80 mg/day, n=1); sixteen patients were
also on a stable dose of ezetimibe (10 mg). All 21 patients were on aspirin (100 mg) and
seven on anti-hypertensive medication.
3.2 Pre-treatment apo(a) kinetics in patients with elevated Lp(a)
Apo(a) isoform size and apo(a) kinetic parameters between the patients with elevated and
normal Lp(a) levels are shown in Appendix Supplementary Table 2. Patients with elevated
Lp(a) concentration had significantly smaller size of apo(a) isoform compared with patients
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with normal Lp(a) concentration (the reference group) (P < 0.001). The concentration and
PR of apo(a) were significantly higher in patients with elevated Lp(a) concentration
compared with the reference group (P < 0.001 for both). However, the FCR of apo(a) was
not significantly different between the two groups (P > 0.05). All aforementioned
differences in Lp(a) particle kinetics remained statistically significant after adjusting for
differences in the type and dose of statin (data not shown). Age, gender, body mass index,
systolic and diastolic blood pressures, use of medications, fasting glucose, lipid, lipoprotein
and apoB concentrations were not significantly different between the two groups (data not
shown; P >0.05 for all).
3.3 Effect of alirocumab on plasma lipid, lipoprotein and apolipoprotein
concentrations: pre-treatment, on-treatment and post-treatment study
Appendix Table 2 shows plasma lipid, lipoprotein and apolipoprotein concentrations and
clinical variables of the twenty-one patients with elevated Lp(a) during the pre-treatment,
on-treatment and post-treatment phases. Alirocumab significantly lowered plasma total
cholesterol (-39%), triglycerides (-23%), LDL-cholesterol (-67%), apoB (-56%) and apo(a)
(-25%) (P < 0.001 in all); Lp(a) concentration measured by the ELISA method at the
NLMDRL (geometric mean 95%CI) also fell significantly (P < 0.001) during the pre-
treatment (251 [210, 302] nmol/L), on-treatment (196 [164, 234] nmol/L) and post-
treatment phases (255 [215-305] nmol/L). The percentage change in apo(a) concentration
was significantly correlated with percentage changes in LDL-cholesterol, apoB and Lp(a)
concentrations (r = 0.714, 0.680 and 0.872, respectively, P < 0.01 for all). Removal of the
three FH patients from the analyses did not alter the principal findings in Appendix Table 2
(data not shown). Body weight, waist circumference, -body mass index, systolic and
diastolic blood pressures and fasting glucose concentration did not alter significantly
during the study period (P>0.05 in all). As seen, post-treatment plasma cholesterol,
204
triglycerides, LDL-cholesterol, apoB, Lp(a) and apo(a) concentrations were comparable to
the corresponding pre-treatment values (Appendix Supplementary Figure 1). There were
no adverse events related to alirocumab treatment, except in two patients in whom minor
injection site reactions were reported.
3.4 Effect of alirocumab on plasma concentration, pool size and kinetics of apo(a):
pre-treatment and on-treatment study
The isotopic enrichment of apo(a) after the infusion of D3-leucine in the subjects before
and after alirocumab treatment is shown in Appendix Figure 1. As seen, the mean peak
enrichment was higher and the maximum of the enrichment curve attained earlier with
alirocumab treatment, reflecting both a smaller pool size and a faster catabolic rate of
Lp(a) with this intervention. Appendix Table 3 shows the effects of alirocumab treatment
on apo(a) concentration, pool size and kinetic parameters in the patients with elevated
Lp(a) concentration. Alirocumab significantly reduced apo(a) pool size (geometric mean
change of -26%, P < 0.001) and increased the FCR of apo(a) FCR (+28%, P< 0.001), but
had no significant effect on the PR of apo(a); these findings were independent of the
adjusted dose of statin employed (data not shown). Removal of the three FH patients from
the analyses did not alter the principal findings in Appendix Table 3. Appendix Figure 2
shows the geometric mean (95%CI) of apo(a) pool size, FCR and PR in the patients with
elevated Lp(a) (before and after alirocumab treatment) and in patients with normal Lp(a)
concentration (reference group). On-treatment apo(a) pool size, FCR and PR were
significantly higher (P < 0.001 for all) than in the reference group. Appendix Figure 3
shows the absolute change in the plasma pool size, fractional catabolic rate and
production rate of apo(a) for individual patients following treatment with alirocumab. As
seen, there was a wide range in the pool size of apo(a) among individual patients. The
estimated FCR of apo(a) was less variable, however. Apo(a) PR showed a similar degree
205
of dispersion to that of the pool size, consistent with apo(a) PR as a major determinant of
Lp(a) particle concentrations. Individual on-treatment values for apo(a) pool size, FCR and
PR were all higher in the patient than in the reference group. The percentage change in
the plasma pool size, fractional catabolic rate and production rate of apo(a) for individual
patients following treatment with alirocumab.are shown in Appendix Supplementary Figure
2.
Pre- and on-treatment plasma apo(a) pool sizes were significantly and directly associated
with the corresponding apo(a) PRs (Appendix Supplementary Figure 3A, r= 0.913 and
0.908, respectively; P <0.001 for both). However, there was only a weak positive
association between plasma apo(a) pool size and FCR in the pre-treatment (r= 0.356, P=
0.113) and on-treatment periods (Appendix Supplementary Figure 3B, r= 0.356, P= 0.113
and r= 0.908, P= 0.038, respectively). A significant association was also found between
the FCR and PR of apo(a) before and after alirocumab treatment (r= 0.594 and r=0.662, P
<0.01 in both). The absolute change in plasma apo(a) pool size was significantly
correlated with change in apo(a) PR (Appendix Supplementary Figure 4A, r = 0.597, P <
0.01), which remained highly significant after adjusting for apo(a) isoform size, pre-
treatment plasma apo(a) pool size, or dose of statin. However, change in plasma apo(a)
pool size was not associated with change in apo(a) FCR (Appendix Supplementary Figure
4B, r = -0.212, P > 0.05). Neither apo(a) isoform size or pre-treatment plasma apo(a) pool
size were significantly associated with on-treatment percentage changes in plasma pool
size, FCR and PR of apo(a) (data not shown).
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4. DISCUSSION
We present the first investigation of the mechanistic effects of PCSK9 inhibition with a
monoclonal antibody on Lp(a) metabolism in a group of patients with elevated Lp(a),
receiving maximally tolerated statin therapy. The novel finding in these patients, who had
increased production of apo(a), was that inhibition of PCSK9 with alirocumab lowered
plasma Lp(a) concentration by increasing the catabolism of circulating Lp(a) particles with
no effect on the production of such particles.
4.1 Previous association studies on apo(a) kinetics
Previous studies using endogenous labelling with stable isotopes have examined Lp(a)
metabolism in normal and diabetic subjects [4, 22]. In normolipidemic men with a wide
range of Lp(a) concentration, we previously reported that plasma Lp(a) concentration was
predominantly determined by the rate of production of apo(a), irrespective of apo(a)
isoform size [4]. In a fat-loading study of statin-treated diabetic patients, we reported that
patients with elevated Lp(a) had higher apo(a) production compared with those with
normal Lp(a) concentration [22]. In the present study, we confirmed our former observation
that plasma apo(a) concentration is significantly and positively associated with the
production of Lp(a) particles in patients with high Lp(a) concentration treated with statins
[16]. However, we newly demonstrate that in statin-treated patients with high Lp(a) PCSK9
inhibition lowered plasma Lp(a) concentration by increasing the catabolism of Lp(a)
particles.
4.2 Previous intervention studies on apo(a) kinetics
Previous studies have shown that in dyslipidemic patients, niacin decreased plasma Lp(a)
concentrations chiefly by decreasing the production of apo(a) [22, 23]. The role of PCSK9
inhibition in the upregulation of LDL receptor activity is well recognised [10]. We and others
207
have previously reported that PCSK9 inhibition with mAbs profoundly lowered plasma
LDL-cholesterol by increasing clearance of LDL particles [13, 14]. A composite analysis of
clinical trials has suggested a discordance between LDL and Lp(a) clearance with
evolocumab [24].The precise mechanisms of action of PCSK9 inhibition in lowering Lp(a)
concentration remains unclear, however.
A tracer study in non-human primates found that alirocumab reduced plasma Lp(a)
concentration by lowering Lp(a)-apo(a) production rate [25]. Only two studies have
reported the effect of PCSK9 inhibition on Lp(a) metabolism in humans. Reyes-Soffer et al.
found that alirocumab reduced plasma Lp(a) concentration by increasing the fractional
catabolism of apo(a), but the findings were not statistically significant [13]. In a group of
normolipidemic subjects with a wide range of Lp(a) concentration, we reported that PCSK9
inhibition by evolocumab, as monotherapy, reduced plasma Lp(a) concentration by
decreasing the production rate of apo(a), and by increasing the FCR of apo(a) in
combination with high-dose atorvastatin [15]. None of these reports referred specifically to
patients with elevated Lp(a) concentration. We have extended previous studies by
investigating the effect of PCSK9 mAbs on apo(a) metabolism in a clinically realistic group
of patients with elevated Lp(a) concentration and ASCVD who were on treated with
maximal tolerated dose of statins.
4.3 Mechanism of action of alirocumab on apo(a) kinetics
While apoB is essential for the binding of LDL particles to the LDL receptor, the role of the
LDL receptor in the catabolism of Lp(a) particle remains controversial [3, 26]. Early
radioisotopic studies showed that the LDL receptor is not required for normal catabolism of
Lp(a) [27]. PCSK9 mAbs could impact LDL receptor activity and/or decrease the
competition between LDL and Lp(a) particles for clearance by this receptor. We have
208
previously demonstrated that upregulation of LDL receptors with statins alone did not
increase the catabolism of Lp(a) particles [15]. Given that the circulating number of LDL
particles exceeds Lp(a) particles, the former are preferentially taken up by LDL receptors
under normal physiological conditions. In the presence of marked upregulation of the LDL
receptor (e.g. statin and PCSK9 mAb combination), we previously demonstrated a direct
correlation between the fractional catabolism of Lp(a) and LDL-apoB particles [15],
implying a role of increased uptake of Lp(a) by the LDL receptor pathway. We speculate
that Lp(a) particles may compete better with low plasma concentrations of LDL particles
for uptake by LDL receptors. Consistent with an earlier report [9], we found that the
percentage change in apo(a) concentration was correlated with percentage changes in
LDL-cholesterol and apoB, implying the operation of a common mechanism for the
clearance of Lp(a) and LDL particles. We specifically demonstrated that alirocumab
effectively reduced the plasma concentration of apo(a) by increasing the FCR of apo(a)
against a background of statin therapy in our patients with elevated Lp(a) concentration.
The effects of alirocumab on other cellular receptors, including very-low-density lipoprotein
receptor, LDL receptor-related protein, scavenger receptor class B type 1 and
plasminogen receptor, on Lp(a) catabolism merits further investigation [28].
Experimental studies suggest that PCSK9 mAbs may decrease the transcriptional
regulation, intracellular availability, and/or extracellular release of the apo(a) protein [11,
28]. This evidence concurs with kinetic data showing that PCSK9 mAbs lower Lp(a)-apo(a)
production rate in humans and non-human primates [15, 25]. In our study, we found no
significant effect of alirocumab on the production rate of apo(a), however. It is possible that
an effect of alirocumab on apo(a) secretion could have been ablated by statin therapy
owing to increase in LPA mRNA or marked upregulation of LDL receptors [6, 15]; these
209
effects could increase intra-cellular availability of apo(a) for production, potentially
offsetting the effect of alirocumab on apo(a) secretion.
In the present study, we found that the FCR and PR of apo(a) were positively correlated
before and after treatment with alirocumab. This association may reflect a balancing, feed-
forward mechanism in response to elevated production of apo(a) in these patients.
Consistent with this, we have previously found similar positive associations between the
FCR and PR of VLDL-apoC-III and HDL-apoA-I in patients with dyslipidaemia [29, 30].
While we found no significant effect of alirocumab on apo(a) production, the change in
apo(a) pool size was statistically correlated with variation in apo(a) production. We
previously showed a similar association across a wide range of pre-treatment plasma
Lp(a) concentrations with the combination of evolocumab plus atorvastatin (80mg) [15]. As
shown in the Supplementary data (Appendix Supplementary Figure 4A), apo(a) PR
remains a significant predictor of apo(a) pool size before and after treatment with
alirocumab despite the increase in apo(a) FCR (Appendix Supplementary Figure 3B). We
have previously postulated that the upregulation of the clearance of Lp(a) particles may
drive a balancing, feed-forward mechanism on the hepatic production of apo(a), the major
determinant of Lp(a) concentration [15]. Balancing feedback is well recognised to be
involved in the response of complex systems, such as lipoprotein metabolism, to
interventions [31, 32]
4.4 Study strengths and limitations
Our sample size was relatively small, but comparable to previous kinetic studies of Lp(a)
metabolism [13, 15, 22]. We did not employ a placebo controlled design to study the
effects of alirocumab on Lp(a) kinetics, but we demonstrated in the follow-up study that on-
210
treatment plasma Lp(a) and apo(a) concentrations returned to pre-treatment levels. Hence,
our findings are unlikely to be confounded by a regression-to-the-mean effect. We also
studied patients receiving treatment in a realistic clinical setting, some of whom could not
tolerate maximal dose of high potency statins. We did not measure the kinetics of both
apo(a) isoforms because our gel separation method was not sensitive for isolating minor
apo(a) isoforms. Hence, the estimation of apo(a) kinetics based on the predominant
isoform may not reflect the full spectrum of the kinetics of Lp(a) particles. We did not
simultaneously measure LDL-apoB kinetics in the present study, but would anticipate
similar findings to those reported earlier [13, 14]. We also studied the kinetic effect of
alirocumab against background use of statins, agents that have been recently suggested
to increase plasma Lp(a) concentration and production by an average of 10% [6], a
potential effect which is unlikely to be clinically significant [33]. The levels of LDL-
cholesterol before statin treatment were not available in the individual patients. Whether
this will impact an individual’s response to alirocumab in regard to Lp(a) metabolism
requires further investigation. The background use of different types and doses of statins
may have a differential impact on Lp(a) kinetics [6]. However, we found that the effects of
alirocumab on apo(a) concentration and kinetics were independent of the dose of statin
employed; we were not statistically powered to examine the impact of different types of
statins on apo(a) metabolism. Whether statins significantly increase Lp(a) production in
patients with high Lp(a) and a high rate of production of apo(a) merits further investigation,
but we anticipate that the effect would be minor. The catabolism of Lp(a) appears to be
tightly regulated and operates within a relatively narrow range compared with its
production, particularly when LDL receptor expression and activity are upregulated with
statin treatment. Hence, the interpretation of the correlational analyses pertaining to the
intervention with alirocumab needs to be cautious. The lack of significant association
between the change in apo(a) pool size and FCR with alirocumab does not fully exclude
211
the possibility that incremental changes in apo(a) FCR determine incremental changes in
Lp(a) particle concentrations. Future studies should also examine the postabsorptive and
postprandial effects of alirocumab on Lp(a) kinetics in patients with high Lp(a).
4.5 Clinical implications
The FOURIER (Further Cardiovascular Outcomes Research With PCSK9 Inhibition in
Subjects With Elevated Risk) and ODYSSEY Outcomes (Evaluation of Cardiovascular
Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) trials
suggest that effectiveness of PCSK9 mAb in reducing ASCVD events may be most
pronounced in patients with high Lp(a) and that the reduction in Lp(a) could partly mediate
the benefit [34-36]. These findings emphasize the importance of apo(a) as a target for new
therapies. We undertook this study in patients with high Lp(a) with clinical or subclinical
ASCVD, and for most of whom Lp(a) lowering with PCSK9 mAb would be clinically
indicated [37-39]. Treatment with both PCSK9 mAb and niacin are the only effective and
practicable means for lowering Lp(a) in high risk patients, noting that both agents operate
through different mechanisms of action [15, 22, 39, 40]. However, this therapeutic
approach has not been fully evaluated and is not included in current clinical guidelines [37,
38, 39, 41].
Our study suggests that while inhibition of PCSK9 lowers Lp(a) via upregulation of the LDL
pathway, further reduction in Lp(a) concentration to prevent ASCVD requires targeting the
production of Lp(a) particles in order to fully address the residual risk attributed to lowering
LDL-cholesterol alone [42, 43]. To this end, our findings foreshadow the value of apo(a)
antisense oligonucleotides or small interfering RNAs for lowering Lp(a) in targeting apo(a)
production against a background of statin and PCSK9 mAb therapy [44, 45].
212
5. Conclusions
In patients with elevated Lp(a) concentration receiving statins, alirocumab increases the
hepatic clearance of Lp(a) particles. This mechanism, which may relate to marked
upregulation of hepatic receptors (principally for LDL) and/or reduced competition between
Lp(a) and LDL particles for these receptors, could contribute to the benefit of PCSK9
inhibition with alirocumab on cardiovascular outcomes.
Funding: Regeneron Pharmaceuticals Inc funded this study.
Conflict of interest: GFW has received honoraria for advisory boards and speakers
bureau or research grants from Amgen Inc., Sanofi, Regeneron, Arrowhead, Kowa and
Gemphire; JP was supported by a WAHTN Early Career Fellowship and the Australian
Government’s Medical Research Future Fund. DCC, LM, QY, SA, SMM and PHRB have
no disclosures.
Contributions of Authors GFW, DCC and PHRB designed the study. GFW, DCC and SA
conducted the study and drafted the manuscript. DCC, JP, LM, QY and SMM analyzed the
data. DCC, LM and PHBR undertook mathematical modeling of Lp(a) kinetics. All authors
reviewed and approved the manuscript.
213
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Legends
Appendix Table 1. Clinical and biochemical characteristics of the 21 patients with elevated
Lp(a) concentration at recruitment
Appendix Table 2. Plasma lipid, lipoprotein and apolipoprotein concentrations and clinical
variables of the patients during the pre-treatment, on-treatment and post-treatment periods
with alirocumab
Appendix Table 3. Plasma molar concentration, pool size, fractional catabolic rate and
production rate of apo(a) in the patients pre- and on-treatment with alirocumab
Appendix Figure 1. Isotopic enrichment for apo(a) with D3 leucine in the patients before
and after alirocumab treatment
Appendix Figure 2. Effect of alirocumab on the plasma apo(a) pool size (A), fractional
catabolic rate (B) and production rate (C) in the patients , with the reference group of
patients with normal plasma levels of lipoprotein(a) shown for comparison.
Appendix Figure 3. Absolute change in the plasma pool size (A), fractional catabolic rate
(B) and production rate (C) of apo(a) for individual patients following treatment with
alirocumab.
220
Appendix Table 1. Clinical and biochemical characteristics of the 21 patients
with elevated Lp(a) concentration at recruitment
Data are presented as mean ± SD or geometric mean (95% confidence interval); Apo: apolipoprotein; ASCVD: atherosclerotic cardiovascular disease; HDL: high-density lipoprotein; LDL: low-density lipoprotein; Lp(a): lipoprotein(a); * measured by Abbott Quantia Lp(a) assay at screening
Age (years) 56 ± 11
Male (n) 12
Body mass index (kg/m2) 29 ± 4.3
Systolic blood pressure (mmHg) 128 ± 9.0
Diastolic blood pressure (mmHg) 79 ± 9.1
Symptomatic ASCVD (n) 11
Asymptomatic ASCVD (n) 10
Glucose (mmol/L) 4.9 2± 0.44
On statin (n) 21
Rosuvastatin 15
Atorvastatin 3
Simvastatin 0
Pravastatin 2
Fluvastatin 1
On ezetimibe (n) 16
Total cholesterol (mmol/L) 4.3 ± 0.72
Triglyceride (mmol/L) 1.3 (1.1, 1.5)
HDL-cholesterol (mmol/L) 1.3 ± 0.31
LDL-cholesterol (mmol/L) 2.3 ± 0.56
ApoB (g/L) 0.89 ± 0.20
Lp(a) mass (g/L)* 1.28 (1.10-1.48)
221
Appendix Table 2. Plasma lipid, lipoprotein and apolipoprotein concentrations and clinical variables of the patients during the pre-treatment, on-treatment and post-treatment periods with alirocumab
Data are presented as mean ± SEM or geometric mean (95% confidence interval); Apo: apolipoprotein; HDL: high-density lipoprotein; LDL: low-density lipoprotein; Lp(a): lipoprotein(a) *P-values refer to repeated measures ANOVA comparing pre-treatment, on-treatment and off-treatment levels with alirocumab † P <0.001 compared with pre-treatment levels ‡ Apo(a) concentration was measured by a liquid chromatography-mass spectrometry method (references 14 and 15)
Pre-treatment On-treatment Post-treatment P-value*
Total cholesterol (mmol/L) 3.8 ± 0.14 2.3 ± 0.10† 4.1 ± 0.21 <0.001
Triglycerides (mmol/L) 1.1 (0.94, 1.4) 0.85 (0.73, 0.97)† 1.2 (0.92, 1.4) <0.001
HDL-cholesterol (mmol/L) 1.2 ± 0.07 1.3 ± 0.06 1.2 ± 0.06 0.145
LDL-cholesterol (mmol/L) 2.1 ± 0.11 0.7 ± 0.06† 2.2 ± 0.15 <0.001
ApoB (g/L) 0.79 ± 0.04 0.35 ± 0.02† 0.83 ± 0.04 <0.001
Apo(a) (nmol/L)‡ 184 (150, 226) 137 (112, 166)† 182 (148, 223) <0.001
Weight (kg) 83 ± 4.1 83 ± 4.2 83 ± 4.2 0.687
Body mass index (kg/m2) 28 ± 0.92 28 ± 0.93 29 ± 0.98 0.719
Systolic blood pressure (mmHg) 125 ± 1.5 124 ± 1.5 124 ± 1.7 0.880
Diastolic blood pressure (mmHg) 76 ± 1.7 78 ± 1.5 78 ± 1.9 0.464
Glucose (mmol/L) 4.9 ± 0.09 5.0 ± 0.10 5.0 ± 0.10 0.282
222
Appendix Table 3. Plasma molar concentration, pool size, fractional catabolic rate and production rate of apo(a) in the patients pre- and on-treatment with alirocumab
Data are presented as geometric mean (95% confidence interval); Apo: apolipoprotein; FCR: fraction catabolic rate; PR: production rate *Apo(a) concentration was measured by a liquid chromatography-mass spectrometry method (references 14 and 15)
Pre-treatment On-treatment Change (95% CI) P-value
Apo(a) concentration (nmol/L)* 184
150, 226)
137
(112, 166)
-54
(-74, -35)
<0.001
Apo(a) pool size (nmol) 675
(526, 867)
498
(392, 632)
-213
(-291, -134)
<0.001
Apo(a) FCR (pools/day) 0.46
(0.44, 0.49)
0.59
(0.56, 0.63)
0.13
(0.11, 0.16)
<0.001
Apo(a) PR (nmol/kg/day) 3.9
(3.1, 4.9)
3.6
(2.9, 4.5)
-0.21
(-0.55, 0.13)
0.178
223
Appendix Figure 1. Isotopic enrichment for apo(a) with D3 leucine in the patients before and after alirocumab treatment
224
Appendix Figure 2. Effect of alirocumab on the plasma apo(a) pool size (A), fractional catabolic rate (B) and production rate (C) in the patients , with the reference group of patients with normal plasma levels of lipoprotein(a) shown for comparison.
Data are presented as geometric mean (95% confidence interval); Apo(a): apolipoprotein(a); FCR: fraction catabolic rate; PR: production rate
*P<0.001 compared with pre-treatment values;
†P<0.001 compared with the reference group (i.e. patients with normal Lp(a) concentrations)
225
Appendix Figure 3. Absolute change in the plasma pool size (A), fractional catabolic rate (B) and production rate (C) of apo(a) for individual patients following treatment with alirocumab. P-values refer to on-treatment group changes compared with pre-treatment values ------- depicts reference values for group with normal Lp(a); see Appendix Supplementary Table 1 for details.
226
Appendix Supplementary Table 1. Clinical and biochemical characteristics of the 15 statin-treated patients with normal Lp(a) concentration
Data are presented as mean ± SD or geometric mean (95% confidence interval); HDL: high-density lipoprotein; LDL: low-density lipoprotein
Normal Lp(a)
(n=15)
Age (years) 55 ± 12
Male (%) 53
Body mass index (kg/m2) 28 ± 5.0
Systolic blood pressure (mmHg) 128 ± 11
Diastolic blood pressure (mmHg) 73 ± 5.8
Glucose (mmol/L) 5.5 ± 0.4
On ezetimibe (%) 67
Total cholesterol (mmol/L) 4.4 ± 1.3
Triglycerides (mmol/L) 1.2 ± 0.5
HDL-cholesterol (mmol/L) 1.3 ± 0.4
LDL-cholesterol (mmol/L) 2.5 ± 0.9
Total apolipoprotein B (g/L) 0.8 ± 0.3
Lipoprotein(a) (g/L) 0.10 (0.06-0.16)
227
Appendix Supplementary Table 2. Comparison of apo(a) isoform size and apo(a) metabolic parameters between patients with elevated and normal Lp(a) concentrations
Data are presented as mean ± SD or geometric mean (95% confidence interval); Apo: apolipoprotein; KIV: Kringle-IV * Apo(a) concentration was measured by a liquid chromatography-mass spectrometry method (see text for details)
Elevated Lp(a)
(n=21)
Normal Lp(a)
(n=15)
P-value
Predominant apo(a) isoform (KIV repeats)
17 (16, 19) 23 (20, 26) 0.003
Apo(a) concentration (nmol/L)*
184 (150, 226) 13.0 (8.0, 22) <0.001
Apo(a) fractional catabolic rate (pools/day)
0.46 (0.44, 0.49) 0.48 (0.42, 0.56) 0.445
Apo(a) production rate (nmol/kg/day)
3.9 (3.1, 4.9) 0.24 (0.14, 0.40) <0.001
228
Appendix Supplementary Figure 1. Plasma concentrations of cholesterol, triglyceride, LDL-cholesterol, apoB, Lp(a) and apo(a) concentrations in the patients during the on-treatment and post-treatment periods relative to the pre-treatment period with alirocumab
Data are
presented as mean ±SD; Apo: apolipoprotein; LDL-C: low-density lipoprotein-cholesterol; Lp(a): lipoprotein(a); TC: total cholesterol; TG: triglycerides
*P <0.001 compared with pre-treatment values
229
Appendix Supplementary Figure 2. Percentage change in the plasma pool size (A), fractional catabolic rate (B) and production rate (C) of apo(a) for individual patients following treatment with alirocumab.
230
Appendix Supplementary Figure 3. Association between plasma pool size and production rate (A) and fractional catabolic rate (B) of apo(a) during pre-treatment and post- treatment with alirocumab.
231
Appendix Supplementary Figure 4. Association between absolute change in the plasma pool size and production rate (A) and fractional catabolic rate (B) of apo(a) following treatment with alirocumab.
232
APPENDIX II
Plasma concentration measured by
NLMDRL method and production rate
of apo(a) in patients with elevated and
normal plasma Lp(a) concentrations.
233
Appendix Table 4. Plasma concentration measured by NLMDRL method and production
rate of apo(a) in patients with elevated and normal plasma Lp(a) concentrations.
Elevated Lp(a)
concentration group
Normal Lp(a)
concentration group
p-value
Apo(a) concentration,
nmol/L
290 (241, 348) 12 (6.3, 22) <0.001
Apo(a) production
rate, nmol/kg/d
4.75 (3.46, 6.53) 0.21 (0.11, 0.39) <0.001
Data are presented as geometric mean (95% CI).
272
Participant Information Sheet/Consent Form School of Medicine and Pharmacology, Royal Perth Hospital
Title Lipoprotein(a) metabolism in subjects at high risk of cardiovascular disease (Study acronym: LIPA)
Short Title Studies of Lp(a) metabolism Protocol Number REG 2014-002 Project Sponsor National Health and Medical Research Council of
Australia
Coordinating Principal Investigator/ Principal Investigator
Winthrop Professor Gerald Watts
Winthrop Professor Hugh Barrett, Dr Esther Ooi, Dr Dick Chan, Dr Theodore Ng Associate Investigator(s)
(if required by institution)
Clinical Associate Professor Timothy Bates
Location (where CPI/PI will recruit) UWA School of Medicine and Pharmacology, Royal Perth Hospital Unit and Royal Perth Hospital Lipid Disorders Clinic.
Part 1 What does my participation involve?
1 Introduction
You are invited to take part in this research project, ‘Lipoprotein(a) metabolism in subjects at high risk of cardiovascular disease’. This is because you are either at moderate-to-high risk of heart disease, OR considered to be healthy and eligible to participate. This research project aims to study the metabolism of lipoprotein(a) or Lp(a).
What is Lp(a)? Lp(a) is a particle that is similar to low-density lipoprotein (LDL; bad cholesterol-carrying particle) found in your blood and at high levels is major cause of heart disease. This is an inherited metabolic condition that is presently very difficult to treat.
This Participant Information Sheet/Consent Form tells you about this research project. It explains the tests and research involved. Knowing what is involved will help you decide if you want to take part in this research.
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Please read this information carefully. Ask questions about anything that you do not understand or want to know more about. Before deciding whether or not to take part, you might want to talk about it with a relative, friend or your local doctor.
Participation in this research is voluntary. If you do not wish to take part, you do not have to. You will receive the best possible care whether or not you take part.
If you decide you want to take part in this research project, you will be asked to sign the consent section. By signing it you are telling us that you:
• Understand what you have read • Consent to take part in this research project • Consent to the tests and research that are described • Consent to the use of your personal and health information as described
You will be given a copy of this Participant Information and the signed Consent Form to keep.
2 What is the purpose of this research?
Heart disease is a major health problem in our community. It is estimated that 3.5 million Australians have chronic heart disease conditions and of these, 40% have abnormal blood fats (cholesterol and triglyceride) levels. Cholesterol-lowering medications called “statins” are used to treat the abnormal blood fat metabolism found in individuals at moderate-to-high risk of heart disease. However, 70% of these people remain at significant heart disease risk despite having their blood fats treated with the highest statin dose they can tolerate. New evidence suggest that this may be caused by having high levels of lipoprotein(a) [Lp(a)] in the blood. People with high Lp(a) levels have 60% higher risk of dying from a heart attack compared with the general population. Lp(a) is an LDL (bad cholesterol)-like particle linked to a very large protein molecule called apo(a). The size of apo(a) can vary between individuals and a higher proportion of smaller sized apo(a) has been linked to higher heart disease risk. Although statins are used to treat high lipid levels they have very little effect on Lp(a) levels.
Therefore, the purpose of this research is to study and compare how Lp(a) is processed in the liver and the body in three different groups of Caucasian men and women.
• Group 1: people with high levels of Lp(a) at moderate-to-high risk of heart disease and on statin therapy
• Group 2: people with low levels of Lp(a) at moderate-to-high risk of heart disease and on statin therapy
• Group 3: people with low levels of Lp(a) at low risk of heart disease and not on statin therapy
To carry out this study, we propose to use a harmless substance called a stable, non-reactive isotope (D3-Leucine), which traces the speed at which Lp(a) are released into the bloodstream by the liver and the speed at which they are broken down. This isotope has been widely used to study blood fat regulation in humans and the investigators involved with this study have been conducting similar studies using this stable isotope for more than 20 years.
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This approach will give new understanding to the formation and breakdown of Lp(a) and can contribute to development of new medical treatment to lower Lp(a), which is currently lacking.
This research has been initiated by the Principal Medical Investigator, Professor Gerald Watts and is funded by National Health and Medical Research Council of Australia
3 What does participation in this research involve?
If you decide to participate, you will be asked to attend for 7 visits to the Research Studies Unit located on Level 3 of the Medical Research Foundation Building, 50 Murray Street, Perth. We will also contact you by telephone two weeks after your last clinic visit. Your total period of involvement in this study will be approximately 7 weeks.
The visits will vary in time, from 30 minutes to 12 hours and we will ask you to fast (not consume any food or drink other than water) for 12 hours prior to all visits. The total time commitment in this study is approximately 18 hours over 7 weeks.
This study involves three periods.
• A 4 week weight ‘maintenance period’ (to ensure your medications and weight are stable)
• A 1 week ‘metabolic study period’ • A 2 week ‘follow-up period’ when study staff will telephone you to ask about your health If you decide to participate, you will be provided with a detailed list of your visits, including dates, the time of your appointments and any specific instructions. The study staff will also inform your local doctor or GP about your participation in this study.
Detailed description of study visits:
Visit 1: the screening visit will take approximately 3 hours at which the following will occur:
• The informed consent discussion will be conducted.
If you agree to participate following this discussion, you will be asked to sign the consent form and you will be given a copy of the consent form to take home with you.
The following screening procedures will be undertaken after the consent form is signed.
• A health and medical history review, including medications taken, to confirm your eligibility to participate in this study.
• Your weight and height will be measured and your body mass index (BMI) calculated and your waist to hip circumference will be measured to determine your waist to hip ratio.
• Body weight and fat mass will be measured by bioelectrical impedance method using a Tanita Body Composition Analyzer. This involves standing on a small platform in bare feet
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while the instrument measures your body fat, body water and weight. This procedure is very safe and will not cause any discomfort.
• An electrocardiogram (ECG) will be performed to record the rate, regularity and electrical activity of your heartbeats. This is not an invasive procedure, but the removal of the sticky electrode patches to your chest, arms and legs, may cause some discomfort. Removing the patches is like pulling off a small bandage. Some people who are allergic to tapes or adhesives may develop a skin rash in the area.
• A physical examination including blood pressure and pulse will be performed. • Fasting blood samples will be taken to check your health and to measure blood fats
(cholesterol, triglyceride) and Lp(a) levels, as well as glucose and insulin levels, which must be within a particular range for you to participate in this study.
• A urine sample will be tested to check that your kidneys are functioning normally. • If you are a woman and still of child bearing potential, you will have a urine pregnancy test.
Alternatively you will have a hormone test to check that you are post-menopausal if there is any uncertainty.
• A blood sample will also be taken for DNA extraction to test for a particular gene called apoE2, which is involved in cholesterol metabolism. This DNA test is mandatory and your consent to the test must be obtained before you are allowed to proceed with this study. If you have two apoE2 genes you will be ineligible to enter this study as you will have a different blood fat metabolism response and this could affect the results of this study. If you have had this gene test previously, you will not have to have this test again.
Additional optional storage of DNA: If you are required to have this gene test, your study doctor will ask you to consider signing an additional Consent Form, to allow your remaining DNA sample to be stored at Royal Perth Hospital and to be used further to help study genes involved in blood fat metabolism. If information from this exploratory research is found in future to have a direct relevance to you, then the principal investigator will make sure this information is passed on to you. The principal investigator will hold a document that links your unique participant number to your other study details, so that he/she may arrange to discuss any relevant findings with you if they become available. This document will be stored in a locked office in a secured floor at the Medical Research Foundation Building. If you do not consent to further testing of your DNA sample, the sample will be destroyed after testing for the apoE2 gene is complete. If you decide not to allow storage and further testing of your DNA sample you can still take part in this study.
Aspirin: Aspirin is a relatively safe and commonly used medication in heart disease prevention, and is prescribed at doses between 75mg and 300mg orally daily, with 100mg being the usual standard dose. Most people who are at high risk of heart disease are prescribed aspirin. Therefore, it is likely that people at risk of heart disease participating in this study will be taking 75-100mg aspirin daily. In order to reduce the variability of our study results, if you do not currently take aspirin daily, you will be asked that you start taking aspirin 75-100mg (low dose) daily with your evening meal from Visit 1 until the evening of Visit 6.
Visit 2: will take approximately 1 hour
Following 4 weeks of stable diet, exercise and aspirin treatment, we will ask you to return to the clinic to have further blood tests. At this visit, we will also measure your body weight, fat mass, waist and hip circumference to check that your weight has remained constant. You will be given a 3-day Food and Drink Diary and Exercise Questionnaire to take home in which you will be asked to list all the food and alcohol you consume and any exercise activities you undertake over a 3-day period, usually 2 week days and 1 weekend day, in the week prior to Visit 3.
Visit 3: is a long day visit; approximately 12 hours
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If you are eligible and agree to continue in this study, you will be asked to attend the clinic for a full day visit (approximately 12 hours). This visit will usually occur on a Monday. You will be asked to fast (water allowed) for 12 hours prior to this study and you will need to continue to fast until the last blood sample is collected on this day. The total duration of the fasting period of this visit is approximately 23 hours. You will be provided with a substantial meal at the end of the fasting period.
You will be asked to arrive at the clinic at approximately 7:00-7:30am and remain in the clinic until early evening.
• Your health, medical history and medication since the last visit will be reviewed and your blood pressure and pulse will be measured.
• Your weight, height, waist and hip circumference will be measured and BMI calculated. • Your body weight and fat mass will be measured by bioelectrical impedance method using
a Tanita Body Composition Analyzer. • Your completed 3-day Food and Drink diary and Exercise Questionnaire will be reviewed • Fasting blood samples will be taken to check your health and to measure blood fats
(cholesterol, triglyceride) and Lp(a) levels, as well as glucose and insulin levels.
You will have an intravenous cannula placed into a vein of the arm close to the elbow. Blood samples will be obtained from this cannula 16 times over a period of 10 hours. A second smaller needle will be placed into the vein of your other arm and you will be given a single dose of the stable isotope through this needle after which it will be removed. Over the whole day, a total of 252ml of blood will be collected (17 tablespoon).
During this visit you will be asked to rest quietly, you may read, watch TV or a DVD, listen to music or use a laptop if you prefer. After the last blood sample has been collected at the end of the 10-hour period (approximately 6:00pm), you will be given a substantial meal and a taxi will be arranged for your transport home.
Visits 4, 5, 6 and 7 (short visits; approximately 30 minutes each):
We will ask you to visit the clinic between 7:30-8:30am on the 4 consecutive days after the long study day to have a blood sample taken. Some of these days may fall on a weekend. You will need to fast overnight (water allowed) prior to each visit. A further 20ml (4 teaspoons) of blood will be obtained on Visits 4 - 6 and 35ml (5 teaspoons), on Visit 7. You will be offered a light breakfast at the end of each visit before leaving the clinic.
A total of 387ml of blood will be collected (31 tablespoon) over 5 weeks of this study. This amount is less than a single Red Cross blood donation collection, which is approximately 500ml in total.
A summary of the visits for this study and what will usually occur at each visit is detailed in the table in the next page of this form.
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Assessment Schedule Week 0
Screening
(Visit 1)
Week 4
(Visit 2)
Week 5
(Visit 3)
Week 5
(Visit 4-6)
Week 5 (Visit 7)
Week 7
(End)
Informed consent X
Medical history X
Physical examination X X
Vital signs X X X
ECG (12 lead) X X
Body weight, height, body fat, waist and hip circumference
X X X
Routine hematology and chemistry
Fasting bloods samples will be collected
X X X X
E2/E2 genotyping
Pregnancy test (urine, if female and of child bearing age)
X X X
Hormone test (if not certain of postmenopausal status)
X
3-day Food and Drink Diary and Exercise Questionnaire
X
(explain)
X
(review)
Stable isotope intravenous administration
X
Metabolic Study (long day)
Fasting blood samples will be collected at 16 time points over 10 hours.
X
Metabolic Study (short days)
Fasting blood samples collected at the four
X X
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consecutive days after long day.
Assessment of adverse events
X X X X
Assessment of medications
X X X X X
Follow-up phone call X
4 What do I have to do?
In order to get good quality results, we will ask you to maintain a constant weight and level of physical activity for the duration of this study.
If you are on statin and aspirin treatments, we will ask that you maintain your regular statin and aspirin treatments throughout this study. If you do not currently use aspirin daily, we will ask that you start taking aspirin 75-100mg (low dose) daily with your evening meal from Visit 1 until the evening of Visit 6.
If you are female and you are sexually active, you will be required to use appropriate birth control from the screening visit (Visit 1) until 2 weeks after Visit 7. Further information on appropriate birth controls can be found in the Section 9: What are the possible risks and disadvantages of taking part?
We will ask that you refrain from donating blood during this study and for 3 months after your participation in this study is finished.
You will not be paid to participate in this research. Taxi transport to and from the clinic and your evening meals will be provided on Visit 3 [12-hour visit day]. You will be reimbursed for any reasonable travel and parking expenses associated with the other clinic visits. We will reimburse the cost of your aspirin for the duration of this study if aspirin is not part of your usual medical treatment.
5 Other relevant information about this research project
We anticipate that 90 people will take part in this project, with 30 people in each of the groups being studied.
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This research project has been designed to make sure the researchers interpret the results in a fair and appropriate way and avoids jumping to conclusions. The doctors and nurses who conduct your clinic visits will know what treatment you are on and what group you are in but the laboratory staff who analyse the samples and other study staff will not.
6 Do I have to take part in this research project?
Participation in any research project is voluntary. If you do not wish to take part, you do not have to. If you decide to take part and later change your mind, you are free to withdraw from the project at any stage by contacting study staff.
Your decision whether to take part or not to take part, or to take part and then withdraw, will not affect your routine treatment, your relationship with those treating you or your relationship with Royal Perth Hospital and the University of Western Australia.
7 What are the alternatives to participation?
You do not have to take part in this research project to receive treatment at Royal Perth Hospital. Other options for the treatment of high lipid levels are available; these include referral to the Royal Perth Hospital Lipid Disorders Clinic. Your study doctor will discuss these options with you before you decide whether or not to take part in this research project. You can also discuss these options with your local doctor.
8 What are the possible benefits of taking part?
We cannot guarantee or promise that you will receive any benefits from this research. However possible benefits may include gaining useful information about your state of health and whether you carry two apoE2 genes. In addition, this study will provide important information about the formation and breakdown of Lp(a) and blood fats. These results will be useful for doctors who are looking to find ways to treat elevated Lp(a) levels and other blood fat disorders. It may also contribute to the development of new therapies that in the future may be used to help treat people with high levels of Lp(a).
9 What are the possible risks and disadvantages of taking part?
Risk and Discomfort: Fasting (having nothing to eat or drink except water before each visit) could cause dizziness, headaches, and stomach discomfort and rarely, fainting. You will be monitored by study staff throughout this study. While procedures used in this study are generally safe, you may have brief and/or minor discomfort, such as pain or bruising, associated with the required blood tests and intravenous cannula insertion. Rarely, fainting and local infection can also occur when blood is drawn.
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Aspirin treatment: Aspirin is commonly used for prevention of heart disease, at a dose range of 75 to 300mg daily, with 100mg being the usual standard dose. You may have none, some or all of the effects listed below, and they may be mild, moderate or severe. If you have any of these side effects, or are worried about them, talk with your study doctor. Your study doctor will also be looking out for side effects.
These side-effects of aspirin have been observed in 1% of the population:
• Heart burn or indigestion • Mild to moderate abdominal or stomach cramps, pains or discomfort
The following side-effects do not happen very often, but they could lead to serious problems if you do not seek medical attention:
• Bruising more easily • Confusion • Dizziness • Fainting
• Nausea or vomiting • Pain, buzzing or ringing
in the ears • Unusual tiredness or
weakness
• Severe or continuing abdominal or stomach pain, cramping or burning
Stop taking the medication and seek immediate medical attention if any of the following occur:
• Any loss of hearing • Bleeding • Signs of stomach bleeding, such as bloody or black, tarry stools • Swelling of the mouth and throat • Symptoms of an allergic reaction, e.g.:
o Difficulty breathing o Hives o Itchy skin rash
There may be side effects that the researchers do not expect or do not know about and that may be serious. Tell your study doctor immediately about any new or unusual symptoms.
Many side effects go away shortly after treatment ends. However, sometimes side effects can be serious, long lasting or permanent. If a severe side effect or reaction occurs, your doctor may need to stop your treatment. Your doctor should discuss the best way of managing any side effects with you.
Pregnancy / Birth Control: If you are female and you are sexually active, you will be required to use appropriate birth control from the screening visit (Visit 1) until 2 weeks after Visit 7. If you are already using a method of birth control, please check with the study staff to make sure it is considered acceptable for this study.
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Examples of acceptable methods of birth control are:
• Oral contraceptives • Implantable or injectable
contraceptives • Intrauterine device (IUD) • Condoms with contraceptive foam
• Diaphragm with spermicidal gel • Surgical sterilization • True abstinence or surgical sterilization
of partner
If you are pregnant or trying to become pregnant, or breast-feeding, you should not participate in this study. If you do become pregnant while participating in this study, you should advise your study doctor immediately. Your study doctor will withdraw you from this research project and advise on further medical attention should this be necessary. You must not continue in the research if you become pregnant.
10 What will happen to my test samples?
Your research samples will be used to study blood fat metabolism and if you have consented, your DNA will be stored at Royal Perth Hospital and used to study the genes involved in blood fat metabolism. Some of your blood samples will be used for routine hematology and biochemistry tests to check that it is safe for you to participate. These routine test samples will be destroyed after 7 days according to PathWest standard procedures.
Your research samples will be stored in a specially designated freezer in the School of Medicine and Pharmacology, Royal Perth Hospital Unit for 15 years after the completion of this study. Your research samples are stored for additional analyses related to this project (e.g. to study other lipid molecules, proteins or enzymes that may affect the formation and breakdown of Lp(a) and blood fats).
If you have consented to storage and use of your DNA, your samples will be stored in a specially designated freezer that will be kept locked at Royal Perth Hospital indefinitely. The freezer will be monitored by PathWest staff. Only study staff associated with this study will have access to your stored samples. All samples will be coded with your unique participant number and not your name or any other identifying features.
11 What if new information arises during this research project?
On receiving new information, your study doctor might consider it to be in your best interests to withdraw you from this research project. If this happens, he/she will explain the reasons and arrange for your regular health care to continue.
12 Can I have other treatments during this research project?
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It is important to tell your study doctor and the study staff about any treatments or medications you may be taking, including over-the-counter medications, vitamins or herbal remedies, acupuncture or other alternative treatments. Examples of treatments and medication include fish oils, plant sterol supplements (e.g. Flora Pro-active Margarine), blood pressure lowering medications, or blood thinning medications. You should also tell your study doctor about any changes to these during your participation in this research project.
13 What if I withdraw from this research project?
If you decide to withdraw from this study, please notify the study staff before you withdraw. If you do withdraw during this study, the study staff will not collect any additional personal and health information about you, although information already collected about you will be retained to ensure that the results of this study can be measured properly and to comply with law. If you do not want them to do this, you must tell them before you join this research project.
14 Could this research project be stopped unexpectedly?
It is unlikely that this study will be stopped unexpectedly. However, if this does occur, the study doctors or nurses will explain the reasons to you.
15 What happens when this research project ends?
When this study ends, the project results will be summarized in a manuscript and submitted to an appropriate peer-review medical journal for publication. The project results may also be communicated in, a balanced manner, to the medical and lay communities at international, national or local meetings. A lay summary of the project results will be sent to you.
Part 2 How is this research project being conducted?
16 What will happen to information about me?
By signing the Consent Form you consent to the study doctor and relevant study staff collecting and using personal and health information about you for this research project. Any information obtained in connection with this research project that can identify you will remain confidential. All individuals who handle your information will adhere to traditional standards of confidentiality and will also comply with all the relevant privacy legislation (in Australia, this is the Privacy Act 1988).
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Information about you may be obtained from your health records held at this and other health services for the purpose of this research. By signing the consent form you agree to the study staff accessing health records if they are relevant to your participation in this research project.
Your health records and any information obtained during this research project are subject to inspection for the purpose of verifying the procedures and the data. This review may be done by the relevant authorities and authorised representatives of the Sponsor, the National Health and Medical Research Council of Australia, Royal Perth Hospital and the University of Western Australia, or as required by law. By signing the Consent Form, you authorise release of, or access to, this confidential information to the relevant research personnel and regulatory authorities as noted above.
If you are a patient at Royal Perth Hospital, information about your participation in this research project will be recorded in your health records. Other information about you will be recorded in a clinic file that will be maintained for this research study and stored in a locked cabinet in a lock office in the research unit when not in use. Only de-identified information about you will be recorded in the research database for analysis. De-identified information about you might be used or released for other purposes without asking you again. Any publication or presentation arising from this research will not identify you or any other participant by name.
After completion of the study, the clinic files and other study files will be archived and sent to secure storage facility to be stored for 15 years in accordance with current research guidelines after which they will be destroyed.
In accordance with relevant Australian privacy and other relevant laws, you have the right to request access to the information collected and stored by the study staff about you. You also have the right to request that any information with which you disagree be corrected. Please contact the study staff members named at the end of this document if you would like to access your information.
17 Complaints and compensation
If you suffer any injuries or complications as a result of this research project, you should contact the study staff as soon as possible and you will be assisted with arranging appropriate medical treatment. If you are eligible for Medicare, you can receive any medical treatment required to treat the injury or complication, free of charge, as a public patient in any Australian public hospital. The Human Research Ethics Committee of Royal Perth Hospital and the University of Western Australia have approved this study on the basis (among others) that the reported risk of such an event is small. No provisions have been made in this study to offer participants who suffer an adverse reaction monetary compensation, but the absence of such a provision does not remove your rights to seek compensation under common law.
18 Who is organising and funding the research?
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This research project is being conducted by the Principal Medical Investigator, Professor Gerald Watts.
University of Western Australia School of Medicine and Pharmacology, Royal Perth Hospital Unit, will receive a payment from the National Health and Medical Research Council of Australia for undertaking this research project. No member of the research team will receive a personal financial benefit from your involvement in this research project (other than their ordinary wages).
You will not benefit financially from your involvement in this research project even if, for example, your research or DNA samples (or knowledge acquired from analysis of your samples) prove to be of commercial value to the University of Western Australia School of Medicine and Pharmacology, Royal Perth Hospital Unit.
In addition, if knowledge acquired through this research or use of your DNA sample leads to discoveries that are of commercial value to University of Western Australia School of Medicine and Pharmacology, Royal Perth Hospital Unit, the study investigators or their institutions, there will be no financial benefit to you or your family from these discoveries.
19 Who has reviewed this research project?
All research in Australia involving humans is reviewed by an independent group of people called a Human Research Ethics Committee (HREC). The ethical aspects of this research project have been approved by the HREC of Royal Perth Hospital and University of Western Australia.
This project will be carried out according to the National Statement on Ethical Conduct in Human Research (2007). This statement has been developed to protect the interests of people who agree to participate in human research studies.
20 Further information and who to contact
The person you may need to contact will depend on the nature of your query.
If you want any further information concerning this project or if you have any medical problems which may be related to your involvement in the project (for example, any side effects), you can contact:
• Your study doctor: Professor Gerald Watts on 0404 894 338 .
• The research nurses: on 08 9224 0380 or 9224 0317 (weekdays)
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This study has been approved by the Human Research Ethics Committee of Royal Perth Hospital and the University of Western Australia. If you have any questions about your rights as a research participant or the conduct of this study, you can contact:
• The Chairman of the Royal Perth Hospital Human Ethics Committee: Professor Frank van Bockxmeer on (08) 9224 2244
• The Administrative Officer of the Royal Perth Hospital Research, Ethics and Governance Office: Ms Amy Rickard on (08) 9224 2292
• Please quote this study approval number REG 14-002
Consent Form
Title Lipoprotein(a) metabolism in subjects at high risk of cardiovascular disease (Study acronym: LIPA)
Short Title Studies of Lp(a) metabolism Protocol Number REG 2014-002 Project Sponsor National Health and Medical Research Council of
Australia
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Coordinating Principal Investigator/ Principal Investigator
Winthrop Professor Gerald Watts
Winthrop Professor Hugh Barrett, Dr Esther Ooi, Dr Dick Chan, Dr Theodore Ng Associate Investigator(s)
(if required by institution)
Clinical Associate Professor Timothy Bates
Location (where CPI/PI will recruit) UWA School of Medicine and Pharmacology, Royal Perth Hospital Unit and Royal Perth Hospital Lipid Disorders Clinic.
Declaration by Participant
I have read the Participant Information Sheet.
I understand the purposes, procedures and risks of the research described in the project.
I have had an opportunity to ask questions and I am satisfied with the answers I have received.
I freely agree to participate in this research project as described and understand that I am free to withdraw at any time during the project without affecting my future health care.
I understand that I will be given a signed copy of this document to keep.
I understand that I will have DNA extracted from my blood for to determine if I carry the E2/E2 genotype and that this sample will be disposed of unless I sign a separate consent form for storage of my DNA for research purposes.
I understand that my research samples are stored for 15 years after the completion of this study for future analyses related to this project.
I understand that de-identified information might be used or released for other purposes without asking me again.
Name of Participant (please print) Signature Date
Declaration by Study Doctor/Senior Researcher†
I have given a verbal explanation of this research project, its procedures and risks and I believe that the participant has understood that explanation.
Name of Study Doctor/
Senior Researcher† (please print)
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