HAPTOGLOBIN AND OTHER BIOMARKERS OF CORONARY ARTERY DISEASE IN YOUNG ADULTS

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HAPTOGLOBIN AND OTHER BIOMARKERS OF CORONARY ARTERY DISEASE IN YOUNG ADULTS

WITH HYPERTENSION AND ACUTE MYOCARDIAL INFARCTION

BY

NORBAIYAH MOHAMED BAKRIM

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy (Medical Sciences)

Kulliyyah of Medicine

International Islamic University Malaysia

JUNE 2020

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ABSTRACT

Acute myocardial infraction (AMI) is the most common clinical manifestation of coronary artery disease (CAD). Young age is no longer considered a protective factor since the incidence of young adults with AMI is increasing. Hypertension is an important risk factor for CAD in young adults. Prehypertension without proper management is also associated with an increased risk of CAD. Hence, the identification of CAD biomarkers in young hypertensive and prehypertensive adults is necessary to improve risk stratification of premature AMI in these cohorts. The main objective of this study was to compare protein expression profiles of young adults with AMI to control subjects for the identification of proteins (candidate biomarkers) that are differentially expressed in AMI patients. This study also aimed to determine the plasma concentrations of the candidate biomarkers in young adults with normotension, prehypertension, hypertension and AMI and evaluate the relationship between AMI and potential CAD biomarker/s in young hypertensive and prehypertensive subjects. This study comprised of two phases; discovery and verification. In the discovery phase, proteins in the pooled plasma samples from young male adults (10 AMI patients and 10 controls) aged 18 to 45 years were separated by two-dimensional gel electrophoresis (2-DE). The protein spots that were differentially expressed in AMI patients relative to the controls were identified via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. In the verification phase, the plasma concentrations of the identified proteins were measured using enzyme-linked immunosorbent assay (ELISA) in 40 plasma samples of control, prehypertensive, hypertensive and AMI groups. In the discovery phase, haptoglobin (Hp), apolipoprotein AI (Apo AI) and apolipoprotein AIV (Apo IV) were significantly upregulated in AMI patients in comparison to the controls (p < 0.05). Meanwhile in the verification phase, the plasma concentration of Hp was significantly higher in AMI patients in comparison to the control, prehypertensive and hypertensive subjects (290.63±99.90 vs. 170.02±108.11 vs.

175.05±108.11 and vs. 208.47±112.97 ng/ml, p < 0.006) respectively. The plasma concentrations of Apo AI and Apo AIV were also elevated in AMI patients, yet the increases were not significant compared to the other groups (p > 0.05). Plasma concentration of Hp was significantly associated with young AMI (OR: 1.019, 95%

CI: 1.006-1.033, p = 0.003) after adjusting for other known CAD risk factors. There was also a significant association between AMI and plasma concentration of Hp in hypertensive and prehypertensive subjects (OR: 0.985, 95% CI: 0.973-0.997, p = 0.017 and OR: 0.981, 95% CI: 0.969-0.993, p = 0.002) respectively, independent of other known CAD risk factors. Plasma Hp concentration was significantly correlated with high sensitivity C-reactive protein hs-CRP (r = 0.370, p < 0.001). In Conclusion, consistent upregulation of Hp in discovery and verification phases reflect its potential role as a biomarker of CAD in young adults. Hp is also a potential CAD biomarker that could be utilized as AMI predictor in young adults with hypertension and prehypertension. The significant correlation between Hp and hs-CRP indicates the potential role of these proteins as inflammatory markers in the establishment of CAD in young adults.

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iii

ثحبلا ةصلاخ

داز ٍثهق مشف ٍخارنا ةهقنا ضزًن اػىُش زثكلأا حَزَزظنا زهاظًنا ٍه

لاياػ ازكثي ٍظنا ذؼَ ىن .

حتاصلإا حثظَ دادشذ ثُز اُئاقو ٍُتاصًنا ٍُغناثنا باثشنات

ت وذنا ظغض عافذرإ ذَذسذ ىذ .داز ٍثهق مشف

زَىطذ ٍف ىهي زطخ مياؼك

ٍخارنا ةهقنا ضزي ظغض عافذرإ ظثذزَ .جزكثي ٍط ٍف

جرادإ ٌود وذنا

ـت حتاصلإا زطخ جداَشت اًضَأ حثطاُي

ٍخارنا ةهقنا ضزي حُخىنىُثنا خايلاؼنا ذَذسذ ٌئف ، ٍنارناتو .

ةهقنا ضزي

ٍخارنا ٍُظسرن ٌروزض وذنا ظغض عافذرا مثق ايو وذنا ظغض عافذرا راغصنا ٍُغناثنا ٍف

ذَذسذ ٍف حقَزط مضفأ ِراثرػات ٍُُذوزثنا مُهسرنا ذازرقا ىذ .جاىفلأا ِذه ٍف داز ٍثهق مشف ؤثُرنا مثي مياىؼنا جدذؼري ضازيلأا ٍف حُخىنىُثنا خايلاؼنا

ٍخارنا ةهقنا ضزي فذهنا ٌاك :فذهنا .

لولأا

ًهػ جزطُظهن داز ٍثهق مشف غي ٍُغناثنا باثشنا ٍي ٍُذوزثنا زُثؼرنا ريلاي حَراقي ىه حطارذنا ِذه ٍي مشف ًضزًنا ٍف فهرخي مكشت اهُػ زُثؼرنا ىرَ ٍرنا حسشزًنا حُخىنىُثنا خايلاؼنا ذَذسرن خاػىضىًنا ات حصاخنا ايسلاثنا خاشُكزذ ذَذسذ ىه ٍَاثنا فذهنا ٌاك .داز ٍثهق ٍُغناثنا ٍف حُخىنىُثنا خايلاؼن

ىُُقرن داز ٍثهق مشف و وذنا ظغض عافذرا مثقايو وذنا ظغض عافذراو ٍؼُثطنا ظغضنات ٍُتاصًنا راغصنا حُخىنىُثنا حيلاؼناو داز ٍثهق مشف ٍُت حقلاؼنا

ٍخارنا ةهقنا ضزي عافذرا خاػىضىي ٍف مًرسًنا

ذ :حقَزطنا .وذنا ظغض عافذرا مثق ايو وذنا ظغض .ققسرناو فاشركلاا :ٍُرهززي ًهػ حطارذنا ِذه فنأر

( ٍُغناثنا باثشنا ٍي حؼًدًنا ايسلاثنا خاُُػ ٍف خاُُذوزثنا مصف ىذ ، فاشركلاا حهززي ٍف ٠١

ًضزي

و داز ٍثهق مشف ٠١

ٍُت ىهراًػأ ذوازرذ )ىكسذ زصاُػ ٠١

ًنإ مصفنا ساهخ حططاىت اًياػ ٥٤

صاُق ىذ ، ققسرنا حهززي ٍف .حفىفصًنا حططاىت ٍرنا ٍُذوزثنا غقت ذَذسذ ىذ .داؼتلأا ٍئاُث ٍئاتزهكنا ٍف ٍػاًُنا صاصريلإا صاُقي واذخرطات جدذسًنا خاُُذوزثهن ايسلاثنا خاشُكزذ ٥١

ايسلاثنا ٍي حُُػ

ذنا ظغض عافذرا مثقايو وذنا ظغض عافذرا ، ىكسرهن حهززي ٍف :حئارُنا .داز ٍثهق مشف ًضزيو ، و

ٍُذوزتىثُنىتأ ، ٍتىهغىرتاه ىُظُذ ىذ ، فاشركلاا

ٍُذوزتىثُنىتأو ، AI AIV

ًضزي ٍف زُثك مكشت

( ىكسرنا زصاُؼت ًحَراقي داز ٍثهق مشف ع

ءاشرزا داز ٍثهق مشف اػىُش زثكلأا حَزَزظنا زهاظًنا ٍه

ضزًن ةهقنا ٍظنا ذؼَ ىن .)ٍخارنا داز ٍثهق مشف ًضزي ٍف اًُهضافذ اهُػ زُثؼرنا ىذ ًلاياػ ًازكثي

دادشذ ثُز ًاُئاقو ىػذًنا رشُهنات ٍَأرنا / ذاَزهن ٍهركنا فُطنا صاُق للاخ ٍي ىكسرنا زصاُؼن حثظُنات ىهي زطخ مياؼك وذنا ظغض عافذرا ذَذسذ ىذ .داز ٍثهق مشف ـت ٍُتاصًنا ٍُغناثنا باثشنات حتاصلإا حثظَ

ذ ٍف زَىط

ٍخارنا ةهقنا ضزي ظغض عافذرا ظثذزَ .جزكثي ٍط ٍف

١٠١٤ <

، ققسرنا حهززي ٍف اًُُت .

داز ٍثهق مشف ًضزي ٍف ظىسهي مكشت ًهػأ ٍتىهغىرتاه ـن ايسلاثنا شُكزذ ٌاك غُضاىًنات ًحَراقي.

( وذنا ظغض عافذرا مثقايو وذنا ظغض عافذرلا حقتاظنا ٣٢١٠٩٢

. ± ٢٢٠٢١ متاقي

٠٠٤٤١٣ ±

٠١١٤٠٠

متاقي ٠٠٤٤١٤ ±

٠١١٤٠٠ متاقي و

٣١١٤٥٠ ±

٠٠٣٤٢٠

< ع ، مي /ng ١٤١١٩

دَاك .ٍناىرنا ًهػ )

ٍُذوزتىثُنىتأ ٍي ايسلاثنا خاشُكزذ ٍُذوزتىثُنىتأ وAI

داز ٍثهق مشف ًضزي ٍف اًضَأ حؼفذزيAIV

ع( يزخلأا خاػىًدًنات حَراقي جزُثك ٍكذ ىن خاداَشنا ٍكن ، ١٤٢٣=

= ع و ١٤٢٤٠ ثذرا .)

شُكزذ ظ

( باشنا داز ٍثهق مشف غي زُثك مكشت ٍتىهغىرتاه ـن ايسلاثنا :حُسخرلاا حثظَ

٠٤١٠٢ ، :حقثنا لادي

٠٤١١٩ - ٠٤١٢٢

، = ع ١٤١١٢ ٌاك .داز ٍثهق مشف ـن يزخلأا حفوزؼًنا زطخنا مياىؼن مَذؼرنا ذؼت )

ف ٍتىهغىرتاه ـن ايسلاثنا شُكزذو داز ٍثهق مشف ٍُت ىهي طاثذرا اًضَأ كاُه ظغض عافذرا خاػىضىي ٍ

( ظغضنا حضفاخو وذنا :حُسخلاا حثظَ

١٤٢١٤ ،

= ع ١٤٠٠

، : حقثنا لادي ١٤٢٠٢

- ١٤٢٢٠ ٢٤

⁒ ، = ع

١٤١٠٠ و

: حُسخرلاا حثظَ

١٤٢١٠

، :حقثنا لادي ١٤٢٩٢

- ١٤٢٢ ٢٤ ٣

، ع = ١٤١١٣ ٍناىرنا ًهػ )

ايسلاثنا شُكزذ ظثذرا .يزخلأا حفوزؼًنا زطخنا مياىػ ٍػ حهقرظي ،

ٍتىهغىرتاه غي زُثك مكشت

هػافرنا ٍط ٍُذوزثنا ٍي حُناػ حُطاظز

ٍ r ١٤٢٠١= ع،

<

١٤١١٠ قظرًنا واظرَلاا ضكؼَ :حصلاخنا .)

ٍتىهغىرتاه ـن زَىطرن ٌىُز زشؤًك مًرسًنا اهرود ققسرناو فاشركلاا مزازي ٍف

ٍخارنا ةهقنا ضزي

حُخىنىُت حيلاػ اًضَأ ٍتىهغىرتاه ذؼذ .باثشنا ٍف قنا ضزي

ٍخارنا ةه ٍف اهياذخرطا ٍكًَ حهًرسي

زُثك زطخن ٌىضزؼرَ ٍَذناو ، وذنا ظغض عافذرا مثقايو وذنا ظغض عافذرات ٍُتاصًنا ٍُغناثنا ذَذسذ زَىطرت حتاصلإا ٍي

ٍخارنا ةهقنا ضزي ٍهػافرنا ٍط ٍُذوزثنا و ٍتىهغىرتاه ٍُت واهنا طاثذرلاا زُشَ .

ءاشَإ ٍف حُتاهرنلاا خاراظًهن واهنا روذنا ًنإ

ٍخارنا ةهقنا ضزي .راغصنا ٍُغناثنا ذُػ

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APPROVAL PAGE

The thesis of Norbaiyah Mohamed Bakrim has been approved by the following:

_____________________________

Aszrin Abdullah Supervisor

_____________________________

Norlelawati A. Talib Co-Supervisor

______________________________

Jamalludin Ab Rahman Co-Supervisor

______________________________

Noraslinda Muhammad Bunnori Co-Supervisor

______________________________

Abdul Razak Kasmuri Internal Examiner

_____________________________

Abdul Latiff Mohamed External Examiner

_____________________________

Rafidah Hanim Mokhtar External Examiner

_____________________________

Ahmad Aidil Arafat Zulkarnain Chairman

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DECLARATION

I hereby declare that this thesis is the result of my own investigation, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Norbaiyah Mohamed Bakrim

Signature………. Date …...

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INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA DECLARATION OF COPYRIGHT AND AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

HAPTOGLOBIN AND OTHER BIOMARKERS OF CORONARY ARTERY DISEASE IN YOUNG ADULTS WITH HYPERTENSION

AND ACUTE MYOCARDIAL INFARCTION

I declare that the copyright holder of this thesis is International Islamic University Malaysia

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below.

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Norbaiyah Mohamed Bakrim

……..……..……… ………..

Signature Date

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ACKNOWLEDGEMENT

Alhamdulillah, all praises be to Allah under whose blessing and guidance that has made it possible for me to complete this PhD journey. He provided me with the opportunity to have worked with great and helpful people who have contributed in various ways to my PhD work that deserve special mention.

First and foremost, I wish to acknowledge my parents, Hj Mohamed Bakrim Bin Sani and Hjh Siti Rohaniah Marzuki who raised me up to be able to reach this level. My parents-in-law; Dr.

Hj Alias Othman and Hjh Naaimah Ramli who have shown their support and understanding of my decision to undertake this journey. The greatest appreciation to my supportive and respected husband; Dr. Mohd Afzal Alias who has been my backbone, source of motivation and courage, to keep me moving forward during the ups and downs of research. To my beloved children, Ameerul Muttaqeen, Taufeeq Muttaqeen, Himmah Aleeyah, Dzatiah Aleeyah and my PhD baby, Thoreeq Muttaqeen - thank you for being the main reason for me to keep going despite any difficulties I faced. I hope my perseverance would inspire all of you to be the best in anything that you do in the future.

Secondly, it was not possible for this work to come to light in this form, without the experience and confident guidance of my supervisor Asst. Prof. Dr. Aszrin Abdullah, who has shown constant interest, wholehearted commitment and pain staking supervision to ensure this research was conducted competently. Thank you Dr Aszrin, for the qudwah, mentorship and sisterly bonding that always inspire me to give the best as a student. My heartfelt gratitude goes to my experienced co-supervisor Assoc. Prof. Dr. Norlelawati A. Talib, for her endless assistance and essential guidance to ensure the best outcome for the study. I am also greatly indebted to all other experts in the supervisory team; Prof. Dr. Azarisman Shah Mohd Shah for his valuable clinical insight, Assoc. Prof. Dr. Jamalludin bin Ab Rahman for his enlightening statistical input, Asst. Prof. Dr. Noraslinda Muhammad Bunnori for her comprehensive guidance in proteomic and Asst. Prof. Dr. Aida Sharini Mohd Shah for her assistance in facilitating the recruitment of patients. My deepest gratitude to En Yusri Idorus, the Researcher Officer of Proteomic Laboratory at Institute of Medical Molecular Biotechnology (IMMB) UiTM Sungai Buloh for his important role as an advisor for the proteomic laboratory works. As a beginner, I was so fortunate to have him as a very helpful and skillful assistant.

A great deal of credit goes to Prof. Dr Mohd Azmi Md Nor (Dean on Kulliyyah of Medicine), Prof. Dr Imad Deen (Head of Department of Basic Medical Sciences) for their role in providing constructive advices and technical support in completion of my PhD. My greatest appreciation flows to all the helpful lecturers and staff at Department of Basic Medical Sciences (Kulliyyah of Medicine) who contributed their knowledge and experiences in improving my work. I would like to thank the Director of the Hospital Tengku Ampuan Afzan (HTAA), the Head and the staff of Emergency Department and Cardiology Department of HTAA and Jabatan Kesihatan Negeri Pahang for allowing me to use their facilities for subjects recruitment. I would also like to thank the IIUM Research Management Centre for the research initiative grant scheme (RIGS 15-076-0076) and Ministry of Education, Malaysia for the Fundamental Research Grant Scheme (FRGS 16-056-0555) to finance the study.

Last, but not least, my utmost appreciation to all my other family members and friends for all their support and prayers. I wish this piece of work will be beneficial to the ummah and will encourage other great work in the future.

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LIST OF TABLES

Table 2.1 Third Universal Definition of acute myocardial infarction. 14 Table 2.2 Epidemiological studies of acute myocardial infarction in

young adults.

20 Table 2.3 Age group of AMI patients who underwent PCI from 2013

till 2016.

23 Table 2.4 Framingham cardiovascular risks score for men. 24 Table 2.5 Studies on risk fator profiles of acute myocardial infarction in

young adults.

25 Table 2.6 Risk factors for young and elderly with AMI in Malaysia. 28 Table 2.7 Classification of blood pressure according to JNC 7. 30 Table 2.8 Advantages and disadvantages of proteomic techniques. 42 Table 2.9 Plasma proteomic studies reporting potential proteomic CAD

biomarkers.

48

Table 3.1 List of recruitment sites. 59

Table 3.2 Reagents for preparation of 1X Tris/glycine (TGS) running buffer.

71 Table 3.3 Reagents for preparation of stacking and resolving gel. 72 Table 3.4 Three-step protocol for isoelectric focus points. 80 Table 3.5 Reagents for preparation of Equilibrium Buffer I and II. 81 Table 3.6 Reagents for preparation of 12 % resolving gels. 82 Table 3.7 Serial dilution of standard working solution for ELISA. 90 Table 4.1 Baseline characteristics of all subjects in the discovery phase. 96 Table 4.2 Clinical presentation of young adults with AMI. 103 Table 4.3 Baseline characteristics of all subjects in the verification

phase.

106

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Table 4.4 Proteins that were differently expressed in young adults with AMI in comparison to the controls as identified by MALDI TOF MS.

115

Table 4.5 Association between haptoglobin and AMI. 122 Table 4.6 Association between haptoglobin in prehypertensive and

hypertensive subjects and AMI.

124

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LIST OF FIGURES

Figure 1.1 Conceptual framework. 11

Figure 2.1 The coronary blood vessels. 12

Figure 2.2 Risk stratifications for cardiovascular major events in 10 years according to blood pressure.

35 Figure 2.3 Pathophysiological links between hypertension and

coronary artery disease.

38 Figure 2.4 Procedures of plasma proteomic profiling using two-

dimensional electrophoresis (2-DE).

46

Figure 3.1 Study design. 57

Figure 3.2 Flow chart of subject recruitment for all groups and sample collection.

66 Figure 3.3 Stacking and resolving gel for one-dimensional of sodium

dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

73

Figure 3.4 The extracted protein sample at the end of clean up procedure (white pallet).

76 Figure 3.5 Electrophoresis using a PROTEAN IEF cell (Bio Rad) 80 Figure 3.6 Sodium dodecyl sulphate polyacrylamide gel

electrophoresis (SDS-PAGE).

83 Figure 3.7 Gel scanning using UMAX POWERLOOK 1000 image

scanner.

85 Figure 3.8 Scheme describing analysis gel images using PD Quest

Version 2.7.0 image analysis software.

86 Figure 3.9 Representative diagram of dilution method for reference

standard of haptoglobin protein.

90 Figure 3.10 Enzyme-linked immunosorbent assay (ELISA) plate wells

containing protein-antibody-HRP conjugate complex and

92

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xv substrate solution.

Figure 3.11 Enzyme-linked immunosorbent assay (ELISA) plate wells as the enzyme-substrate reaction was ceased.

93 Figure 4.1 Age distribution of young adults with acute myocardial

infarction.

98 Figure 4.2 Racial differences of young adults with acute myocardial

infarction.

99 Figure 4.3 Household income distribution of young adults with acute

myocardial infarction.

100 Figure 4.4 Type of occupations of young adults with acute

myocardial infarction

100 Figure 4.5 Cardiovascular risk factor profiles of young adults with

acute myocardial infarction

101 Figure 4.6 One-dimensional (1-D) of sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE) analysis

108 Figure 4.7 Two-dimensional electrophoresis (2-DE) gel image of

protein map in control plasma using a pH range 3 to 10

109 Figure 4.8 Two-dimensional electrophoresis (2-DE) gel images of

plasma from patients with acute myocardial infarction (AMI) and age-matched control subjects using a pH range 4 to 7 (Set 1).

110

Figure 4.9 Two-dimensional electrophoresis (2-DE) gel images of plasma from patients with acute myocardial infarction (AMI) and age-matched control subjects using a pH range 4 to 7 (Set 2).

111

Figure 4.10 Two-dimensional electrophoresis (2-DE) gel images of plasma from patients with acute myocardial infarction (AMI) and age-matched control subjects (Set 3) using a pH range 4 to 7 (Set 3).

111

Figure 4.11 Representative of two-dimensional electrophoresis (2-DE) gel image showing the location of proteinspots that were expressed differently (p<0.05) in AMI patients in

comparison to the controls using Independent Student‘s T- test as analysed by PD Quest 7.2.0 Software.

112

Figure 4.12 Representative patterns of protein spots SSP 2501, SSP 1601 and SSP 7201.

113

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Figure 4.13 Peptide mass spectra of A (SSP 2501), B (SSP 1601), and C (SSP 7201) as per Matrix-assisted Laser Desorption/

Ionization Time of Flight (MALDI-TOF) mass spectrometry analysis.

114

Figure 4.14 Graph of standards for haptoglobin. 116

Figure 4.15 Graph of standards for apolipoprotein AI. 117 Figure 4.16 Graph of standards for apolipoprotein AIV. 118

Figure 4.17 Plasma concentration of haptoglobin. 119

Figure 4.18 Plasma concentration of apolipoprotein AI. 120 Figure 4.19 Plasma concentration of apolipoprotein AIV. 121

Figure 4.20 Plasma concentration of hs-CRP. 125

Figure 5.1 Haptoglobin protein structure. 135

Figure 5.2 Apolipoprotein AI (Apo AI) protein structure. 138 Figure 5.3 Apolipoprotein AIV (Apo AIV) protein structure. 141

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LIST OF ABBREVIATIONS

ACS Acute coronary syndrome AMI Acute myocardial infarction Apo AI Apolipoprotein AI

Apo AIV Apolipoprotein AIV

APS Ammonium persulfate

BMI Body mass index

CAD Coronary artery disease

CABG Coronary artery bypass surgery

CI Confidence interval

CK-MB Creatinine kinase-myocardial band CRF Case record form

CTU Clinical trial unit CVD Cardiovascular disease DBP Diastolic blood pressure

1-DE One-Dimensional electrophoresis 2-DE Two-Dimensional electrophoresis ECG Electrocardiography

ELISA Enzyme-linked immunosorbent assay EDTA Ethylenediaminetetraacetic acid FBS Fasting blood sugar

HCl Hydrochloric acid HDL High density lipoprotein

Hs-CRP High sensitivity C-reactive protein Hs-cTn High sensitivity cardiac troponin HTAA Hospital Tengku Ampuan Afzan

Hp Haptoglobin

HRP Horseradish Peroxidase IEF Iso electric focus

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xviii IPG Immobolized pH gradient

iTRAQ Isobaric tags for relative and absolute quantification JNC Joint National Committee

kDa Kilodalton

LDL Low density lipoprotein

mRNA Messenger RNA

mmHg Milimetre per mercury

MALDI TOF Matrix-assisted laser desorption/ionization-time of flight MOH Ministry of Health

MS Mass spectrometry

mM Milimolar

ml Mililitre

MREC Medical Review and Ethics Committee NCVD National Cardiovascular Database NMRR National Medical Research Register NHMS National Health and Morbidity Survey NSTEMI Non-ST Elevated myocardial infarction

OD Optical density

OR Odd ratio

PCI Percutenous coronary intervention

RR Relative risk

SBP Systolic blood pressure

SDS PAGE Sodium dodecyl supfate polyacrylamide gel eletrophoresis SMCs Smooth muscle cells

STEMI ST-elevated myocardial infarction

TC Total cholesterol

TEMED Tetramethylenediamine USD United State Dolar

vCAM-1 Vascular cell adhesion molecule 1

Vs. Versus

WHO World Health Organization

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1

CHAPTER ONE INTRODUCTION

1.1 BACKGROUND AND JUSTIFICATION OF RESEARCH

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality that accounts for almost one third of deaths worldwide. In the United States, approximately 2200 Americans die of CVD each day, roughly one death in every 40 seconds (Mozaffarian, Benjamin, Go, Arnett, Blaha, Cushman, et al., 2016). The healthcare cost to manage CVD was about USD$ 272 billion (approximately RM 1132 billion) in 2010 and estimated to triple to USD$ 818 billion (approximately RM 3405 billion) in 2030 (Tran, Ohinmaa, Thanh, & Welsh, 2017). Unfortunately, nowadays CVD is not merely a disease in developed communities, but it has also affected developing countries. In fact, Southeast Asia was predicted to have the highest percentage increase in CVD-related deaths by 2030 (World Health Organization [WHO], 2017).

In Malaysia, CVD is the most common cause of death and accounted for 23%

of total hospital mortality (Ministry of Health, 2015). The most prevalent CVD is coronary artery disease (CAD), specifically acute coronary syndrome (ACS) which includes acute myocardial infarction (AMI) and angina. The hospitalisation cost for AMI patient requiring percutaneous coronary intervention (PCI) is approximately RM 12,117 in public hospitals and RM 16,289 in teaching hospitals (Lee, Azman, Ahmad, Low, Liau, Anchah, et al., 2017). The economic burden due to AMI is expected to increase as the prevalence of AMI is shifting to younger Malaysian adults. The mean age of AMI patients in Malaysia is 58 years old (Wan Ahmad, 2017), about seven

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years younger compared to AMI patients in general populations (Sorbets, Greenlaw, Ferrari, Ford, Fox, Michal, et al., 2017).

It is an established fact that an increasing age is a non modifiable risk factor for AMI (Agostino, Vasan, Pencina, Wolf, Cobain, Massaro, et al., 2008; Hajar, 2017).

Unfortunately, young age is no longer protective as the incidence of AMI in young adults is currently increasing. Over the past four decades, there has been a decline in AMI admissions among the general population (Bhatnagar, Wickramasinghe, Wilkins,

& Townsend, 2016). However, there has been no concomitant reduction in AMI admissions for patients below the age of 55 (Gupta, Wang, Spertus, Geda, Lorenze, Nkonde-Price, et al., 2014). The reported incidence of young patients diagnosed with AMI varied from 3% to 10% in high income populations, and 10% to 20% of total CAD admissions in low to middle income populations (Joshi, Islam, Pais, Reddy, Dorairaj, Kazmi, et al., 2007; Shah, Kelly, Cox, Wong, & Soon, 2016).

According to the Malaysia National Cardiovascular Disease Database (NCVD) Registry Report from 2007 to 2009, approximately 16% of the AMI patients who underwent PCI in tertiary hospitals were less than 45 years for male and 55 years for female (Zuhdi, Mariapun, Mohd Hairi, Wan Ahmad, Abidin, Undok, et al., 2013). A more recent NCVD-PCI Registry in 2016 reported the percentage of AMI patients aged less than 50 years old had increased from 22.1% between 2013 till 2014 to 23.7%

between 2015 till 2016 (Wan Ahmad, 2017b). Meanwhile, NCVD-ACS Registry Report in 2016 documented that approximately 25% of ACS admissions from 2014 to 2015 were patients aged less than 50 years old (Wan Ahmad, 2017a). Apparently, AMI remains a principal cause of deaths among male Malaysian adults (13.2%) in the

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past five years as reported by the Department of Statistics Malaysia in 2017 (Health Fact Ministry of Health, 2017)

The diagnosis of AMI at a prime age during family and career establishment, leads to significant adverse effects on the physical well-being and mental state of the patients, their families and the community. Furthermore, AMI at young age is more prevalent in males, who are usually the main breadwinners for the families. AMI at this productive age contributes to a greater socioeconomic impact due to the loss of vital human capital, increased family financial burden and excessive usage of public health care facilities. It has been reported that approximately 15% of young patients were not able to return to work following AMI episode due to deterioration of health condition that compromised their performances at the workplace (Dreyer, Xu, Zhang, Du, Strait, Bierlein, et al., 2016).

In the light of significant health and economic burden of AMI in young adults, identification of those with a higher risk of developing CAD at a young age is crucial to improving disease preventative strategies. Undeniably, the traditional risk factors such as hypercholesterolemia, hypertension, smoking, diabetes and family history have important role in identifying those at high risk of developing CAD. However, the risk of CAD in young adults is usually underestimated, as the young age is considered as a main protective factor (Agostino et al., 2008; Lakatta & Levy, 2003).

Therefore, with the increasing trend in the prevalence of premature AMI, additional biomarkers are required to improve the risk stratifications of CAD among young adults.

Apart of smoking that is found to be the most prevalent risk factor for young adults with AMI in general population, hypertension remains an important risk factor

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among young Malaysian AMI patients (Hoo, Foo, Mohd, & Lim, 2016; Zuhdi et al., 2013). Unlike smoking which is an obvious high-risk behaviour, hypertension particularly in young adult is more challenging to be recognized due to to lower level of health awareness in this age group (Abdul-razak, Daher, Ramli, Ariffin, Mazapuspavina, Ambiga, et al., 2016).

Consequently, young patient with the first episode of AMI was more likely to have untreated hypertension compared to the elderly AMI patients (OR: 2.99; 95%

CI: 2.00-4.46, p < 0.001) (Chan, Woo, Wong, Chia, Sutandar & Tan, 2006). Poor management of high blood pressure (BP) may lead to acceleration of CAD development. It has been shown that uncontrolled hypertension in young adult was associated with higher relative risks (RR) for CVD mortality than older hypertensive patients; RR: 4.1, 95% CI 3.7-4.6, RR: 2.6, 95% CI 2.4-2.9 and RR: 1.9, 95% CI 1.8- 2.0 at ages 35-59, 60-69 and 70-79 years respectively (Lewington, Lacey, Clarke, Guo, Kong, Yang, et al., 2016).

Besides hypertension, there is increasing evidence that individuals with prehypertension (BP 120-139/80-89 mm Hg) is almost twice as likely to develop CVD as normotensive individuals (Huang, Wang, Cai, Mai, Hu, Tang & Xu, 2013). The risk of CVD is higher in individuals with Stage 2 prehypertension (BP 130-139/85-89) than those in Stage 1 prehypertension (BP 120-129/80-84 mm Hg) (Huang et al., 2013). Therefore, accurate CVD risk assessement and effective blood pressure control are not only crucial at hypertension stage, individuals with prehypertension should also be identified and managed accordingly to reduce the risk of CVD development.

Current management for hypertension with low CVD risk and prehypertension is risk factor modification as recommended by the Malaysia 5^th Edition of Clinical

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Practice Guideline for Management of Hypertension (2018). Unfortunately, risk factor modification strategies seem less likely to benefit the young people due to a false sense of good health, which cause delays in seeking medical advice and poor compliance (Hulsegge, Looman, Daviglus, Schouw, & Verschuren, 2016). A survey performed among 3501 young AMI patients showed that only half of patients believed that they were at risk of heart disease prior to the acute event (Leifheit-Limson, D'Onofrio, Daneshvar, Geda, Nueno, Spertus & Krumholz, 2015). These patients also had never discussed risk factors modification with their health care providers, despite nearly all of them having more than one risk factor, and 64% having more than three risk factors.

Apparently, CAD risk among young hypertensive and prehypertensive adults are commonly ineffectively assessed. Thus, the role of biological markers in identifying young adults who are at risk of CAD in these cohorts has grown in importance. The identification of CAD biomarkers among young adults with elevated blood pressure will improve predictive accuracy of CAD and enhance clinical decision for blood pressure management in the low CVD risk group.

High-sensitivity C-reactive protein (hs-CRP) is one of the most promising protein markers in predicting the occurrence of CAD in a clinical setting (Torres &

Ridker, 2003; Wang, Tan, Han, Bai, He & Liu, 2017). However, hs-CRP is a general inflammatory marker, thus it is less specific as it may be elevated in other inflammatory reactions. Consequently in CAD, the use of multi-biomarkers approach was proposed to produce a more accurate predictive value, instead of depending on a single biomarker (Bogavac-stanojevic & Jelic, 2010).

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The emergence of ‗omic‘ technologies such as genomic, proteomic and metabolomics have allowed more research opportunities in the discovery of new CAD biomarkers. Of these, proteomic analysis is proposed to be the best method to study multifactorial disease such as CAD, which is a result of interactions between genetic abnormalities and environmental influences (Singh, Aikawa, & Aikawa, 2016).

Protein is the main functional component in any cell or tissue. Thus, modifications in protein expression at various stages of the disease development are potential candidates for risk, diagnostic or prognostic markers.

CAD is a silent disease and the development of underlying atherosclerosis occurs many years prior to the acute presentation of AMI. Several pathophysiological mechanisms such as inflammation, lipid dysregulation, oxidative stress and coagulation have been proposed to play important roles in the establishment of atherosclerotic plaques (Ambrose & Singh, 2015). Each process is associated with different proteins expressions. Hence, investigation on the alterations of protein expression in diseased tissues may reflect the pathophysiological changes that occur, and lead to the discovery of novel biomarkers as well as therapeutic target proteins.

Plasma proteomic study specific to young AMI patients is essential to discover potential CAD biomarkers that could improve the risk stratification, diagnosis, and prognostication of the disease in younger population. Apart from predicting CAD in healthy individuals, the new biomarker might be utilized as a predictor of CAD in young prehypertensive and hypertensive adults. Such discovery is important to improve clinical decision for blood pressure control in young adults. Additionally, plasma proteomic study specific to young adults may help unearth the reason behind the acceleration of atherosclerotic process which leads to the early emergence of AMI.

HAPTOGLOBIN AND OTHER BIOMARKERS OF CORONARY ARTERY DISEASE IN YOUNG ADULTS

HAPTOGLOBIN AND OTHER BIOMARKERS OF CORONARY ARTERY DISEASE IN YOUNG ADULTS

WITH HYPERTENSION AND ACUTE MYOCARDIAL INFARCTION

BY

NORBAIYAH MOHAMED BAKRIM

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy (Medical Sciences)

Kulliyyah of Medicine

International Islamic University Malaysia

JUNE 2020

ABSTRACT

ثحبلا ةصلاخ

APPROVAL PAGE

DECLARATION

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA DECLARATION OF COPYRIGHT AND AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

HAPTOGLOBIN AND OTHER BIOMARKERS OF CORONARY ARTERY DISEASE IN YOUNG ADULTS WITH HYPERTENSION

AND ACUTE MYOCARDIAL INFARCTION

ACKNOWLEDGEMENT

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER ONE INTRODUCTION