Click here to load reader

The physiology and pathophysiology of hepcidin · PDF file Chapter 3 Hepcidin Regulation in Human Physiology 3.0 Introduction 133 3.1 Hepcidin and human disease 133 3.1.1 Iron deficient

  • View
    0

  • Download
    0

Embed Size (px)

Text of The physiology and pathophysiology of hepcidin · PDF file Chapter 3 Hepcidin Regulation in...

  • 1

    The physiology and

    pathophysiology of

    hepcidin

    A thesis submitted for the degree of Doctor of Philosophy

    Imperial College London

    2012

    Mark Busbridge

    Section of Investigative Medicine

    Division of Diabetes, Endocrinology and Metabolism

    Department of Medicine

    Imperial College London

  • 2

    Abstract

    Hepcidin, the critical iron regulatory factor, is a small peptide produced by the hepatocytes in

    response to increased body iron and inflammation. Circulating hepcidin controls both

    intestinal iron absorption and the release of iron from macrophages into plasma via a

    negative iron feedback system.

    I developed a novel competitive immunoassay for hepcidin using a polyclonal antibody

    produced against synthetic hepcidin. I validated the immunoassay and determined it was

    able to discriminate between healthy controls and selected disease groups. I compared the

    immunoassay against another established method of measuring hepcidin. I established that

    plasma hepcidin has a diurnal rhythm and that plasma hepcidin increases in response to

    intravenous iron in anaemic patients.

    Elevated levels of hepcidin in renal failure may have a role in the erythropoietin resistance

    observed in renal anaemia. In haemodialysis patients, hepcidin levels were significantly

    elevated, but there was no correlation with inflammatory markers. Elevated hepcidin was

    associated with anemia, but erythropoietin dose was negatively correlated with hepcidin,

    suggesting that erythropoietin suppresses hepcidin levels. This was confirmed in patients

    when hepcidin levels significantly decreased after erythropoietin treatment.

    The association between plasma hepcidin and other iron parameters were also examined in

    healthy controls after erythropoietin administration and venesection. Profound hepcidin

    suppression was observed after an erythropoietin dose, with peak levels reduced by 73.2%,

    and then gradually recovering over the following two weeks. A similar but more gradual

    change in hepcidin was observed after reducing hematocrit by removal of 250 mL blood. The

    studies suggested that the marrow–hepcidin axis is regulated by factors other than those

    specifically investigated.

    In summary, I have developed and validated a novel immunoassay for hepcidin which will

    allow further investigation of the vital role of this peptide in iron homeostasis and human

    physiology.

  • 3

    Declaration of contributors

    The majority of the work described in thesis was performed by the author. Any collaboration

    and assistance is described below. All radioimmunoassays were carried out under the

    supervision of Dr R S Chapman (Division of Diabetes, Endocrinology and Metabolism,

    Department of Medicine, Imperial College London).

    Chapter 2

    Dr Maggie Chambers from Diagnostic Scotland immunised both rabbits and mice. Healthy

    human volunteers were bled by Medical Staff, Imperial College. The immunocytochemistry

    work was carried out in collaboration with Dr Susan Van Noorden. The healthy control

    samples were kindly supplied by Prof K Srai for the hepcidin method comparison study and

    Dr Chris Tselepis, Senior Lecturer in Cancer Biology, University of Birmingham for the

    SELDI-TOF-MS method comparison data. Prof D Swinkels from the Department of Clinical

    Chemistry, Radboud University Nijmegen Medical Centre, organised and co-ordinated the

    first international Round Robin for hepcidin methods.

    Chapter 3

    Patient samples were collected by Dr A Sangiawya and nursing staff in the Gastroenterology

    Department, Ealing Hospital Trust and by Dr D Ashby and renal staff from West London

    Renal Service, Hammersmith Hospital NHS Trust. Dr D Ashby and Dr D Gale also collected

    plasma samples for the diurnal study. Dr A Sangiawya collected plasma samples for the oral

    and intravenous iron administration study.

    Chapter 4

    Patient samples were collected by Dr D Ashby and renal staff from West London Renal

    Service, Hammersmith Hospital NHS Trust. Dr D Ashby and Dr D Gale also collected

    healthy control plasma samples for the erythropoietin administration and venesection study.

    The statistical analysis on the patient data in this chapter was conducted by Dr D Ashby.

  • 4

    Acknowledgements

    I would first like to thank Prof Steve Bloom and Dr Richard Chapman for the opportunity to

    carry out these studies, and their guidance during the years. I would also like to thank my

    supervisors, Dr. Kevin Murphy for his invaluable scientific advice, support and his patient

    reading of this thesis and many useful comments, and Dr Richard Chapman for his

    supervision, support and advice over the years. I would also like to thank Dr Maggie

    Chambers from Diagnostic Scotland for the rabbit and mouse immunisations and bleeds and

    Mr J Arnold, Consultant Gastroenterologist, Ealing Hospital Trust for his early ideas for this

    thesis.

    I would also like to thank the clinical staff in Clinical Biochemistry, for their help in collecting

    samples and the technical staff of Haematology for their help and advice in assaying patient

    samples

    Dr D Ashby, Dr P Gale and the staff of the Renal Unit, Hammersmith Hospital for the

    recruitment and collection of the CKD patient samples.

    Dr A Sangaiwaya, Mr J Arnold and staff of the Gastroenterology Unit, Ealing Hospital for the

    recruitment and collection of UC and IDA patient samples

    Finally, I would like to thank my parents Ann and Dennis for their help and support

    throughout the years and my other friends and family for all their support and ‗enthusiasm‘

    for my work.

    Finally I‘m particularly indebted to Sarah, my wife for her tireless support and unquestioning

    faith in my abilities and for putting up with all the stressful times.

  • 5

    For Sarah and my parents

  • 6

    Table of Contents

    Contents Page No.

    Abstract 2

    Declaration of contributors 3

    Acknowledgments 4

    Dedication 5

    Table of contents 6

    List of figures 11

    List of tables 14

    Abbreviations 15

    Chapter 1 Introduction

    1.0 Iron 21

    1.1 Iron: essential and harmful 21

    1.2 Maintenance of systemic iron homeostasis 22

    1.2.1 Iron absorption 25

    1.2.2 Iron distribution 26

    1.2.2.1 Transferrin 29

    1.2.2.2 Transferrin receptor 30

    1.2.2.3 Ferritin 30

    1.3 Iron regulation 32

    1.3.1 Cellular iron metabolism 32

    1.4 Hepcidin 34

    1.4.1 Hepcidin synthesis and structure 35

    1.4.2 Hepcidin interaction with Ferroportin 38

    1.5 Hepcidin regulation 39

    1.5.1 Hepcidin regulation by iron 40

    1.5.2 Hepcidin regulation by erythropoiesis 45

    1.5.3 Hepcidin regulation by inflammation 47

    1.5.4 Influence of other cellular regulators on systemic iron balance 49

    1.6 Physiology of hepcidin in humans 50

    1.6.1 Hepcidin kinetics 50

    1.6.2 Assessment of quantitative methods for hepcidin 51

    1.6.3 Reference intervals for hepcidin 53

    1.6.4 Disorders caused by hepcidin deficiency 54

  • 7

    1.6.4.1 Hepcidin in haemochromatosis 54

    1.6.5 Disorders caused by hepcidin excess 56

    1.6.6 Hepcidin in acquired disorders 57

    1.6.6.1 Hepcidin deficiency in acquired disorders 57

    1.6.6.2 Hepcidin excess in acquired disorders 58

    1.7 Therapeutic aspects of hepcidin 61

    1.7.1 Hepcidin modulating agents 61

    1.7.2 Hepcidin agonists 61

    1.7.3 Hepcidin antagonists 62

    1.8 Summary 63

    1.9 Hypothesis 64

    1.9.1 Aims 64

    Chapter 2 Hepcidin Immunoassay Development

    2.0 Introduction to immunoassays 66

    2.1 Principles of immunoassay 66

    2.1.1.1 Limited reagent assays (competitive) 67

    2.1.1.2 Excess reagent assays (non-competitive) 69

    2.1 Adaptive humoral immune response to the immunogen 70

    2.1.2 Antibodies 74

    2.1.2.1 Polyclonal and monoclonal antibodies 75

    2.1.2.2 Monoclonal antibody production 77

    2.2 Hypothesis 80

    2.3 Aims 80

    2.4 Methods 81

    2.4.1 Conjugation of hepcidin to a carrier protein 81

    2.4.2.1 Production of a rabbit polyclonal antibody to hepcidin 82

    2.4.2.2 Production of a sheep polyclonal antibody to hepcidin 82

    2.4.3 Isotopic labeling of hepcidin 83

    2.4.4.1 Screening rabbit bleeds for antibody response 84

    2.4.4.2 Screening sheep bleed for antibody response 85

    2.4.5 Antibody displacement assay 85

    2.4.6 Production of mouse monoclonal antibodies to hepcidin 86

    2.4.6.1 Myeloma cell lines 87