Studies of the metabolism of lipoprotein(a) in humans

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

ii

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



2.3 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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2.4 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



2.5 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

x

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:

xvii

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:

xix

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

xx

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

xxii

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

xxiii

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.



Other authors

40

60

Conduct of the study Candidate 100

Collection of blood samples Candidate 100

Laboratory analyses Candidate 100


Other authors

70

30

Kinetics analyses Candidate 100


Other authors

70

30

Manuscript drafting Candidate

Other authors

70

30

Manuscript review Candidate

Other authors

50

50

xxiv

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.



Other authors

50

50

Conduct of the study Candidate 100

Collection of blood samples Candidate 100

Laboratory analyses Candidate 100


Other authors

70

30

Kinetic analyses Candidate 100


Other authors

60

40


Other authors

70

30


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.



Other authors

20

80


Other authors

70

30

Kinetic analyses Candidate 100


Other authors

60

40


Other authors

70

30


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

1

Chapter 1

Literature Review

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).

54

Chapter 2

Anatomy of the Thesis

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



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.


61


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.



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.

64

Chapter 3

Subjects, Materials and Methods

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


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


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


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).

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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

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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%.

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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.

95

Chapter 4

Study 1: Comparison of Methods for

Measuring Apolipoprotein(a)

Concentration

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.

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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.

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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.

135

Chapter 8

Overview, Discussion and Future Directions

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.

147

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.

151

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APPENDICES

190

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,

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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

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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

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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

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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

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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


Triglyceride (mmol/L) 1.3 (1.1, 1.5)



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


Triglycerides (mmol/L) 1.2 ± 0.5



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).

234

APPENDIX III

Royal Perth Hospital Ethics

Committee approval form

271

APPENDIX IV

Patient information sheet and consent

form

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.

273

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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Signature Date

† A senior member of the research team must provide the explanation of, and information concerning, this research project.

Note: All parties signing the consent section must date their own signature.

Studies of the metabolism of lipoprotein(a) in humans

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