Amylin Peptide: Dissertation - White Rose University...

Amylin Peptide: An Association with Feed Intolerance in Preterm Neonates and Infants of

Diabetic Mothers

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Dr Venkatesh Ramulu Kairamkonda

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A Dissertation Submitted to the Faculty of Medicine in Partial Fulfilment of the

Requirements for the Degree of Doctor of Medicine at the University of Sheffield

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This Research is conducted at Departments of Neonatal Intensive Care Unit

and Academic Unit of Reproductive and Developmental Medicine, Jessop Wing,

Royal Hallamshire Hospital and Neonatal Intensive Care Unit, Doncaster

General Hospital

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November 2012

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Declaration

I hereby declare that my dissertation entitled “Amylin peptide: An association

with feed intolerance in preterm neonates and infants of diabetic mothers” is the

result of my own work and includes nothing which is the outcome of work done

in collaboration except as declared in the Preface and specified in the text. It is

not substantially the same as any that I have submitted, or, is being

concurrently submitted for a degree or diploma or other qualification at the

University of Sheffield or any other University or similar institution except as

declared in the Preface and specified in the text. I further state that no

substantial part of my dissertation has already been submitted, or, is being

concurrently submitted for any such degree, diploma or other qualification at the

University of Sheffield or any other University of similar institution except as

declared in the Preface and specified in the text. Where other sources of

information have been used, they have been referenced and/or acknowledged.

Dr Anton Mayer explored the role of amylin in critically ill paediatric patients with

feed intolerance that provided the basis to investigate the role of amylin in

neonatal population. He also contributed significantly towards this project as a

study collaborator. Myself and Drs Robert Coombs and Robert Fraser helped

formulate hypotheses and overall study methodology in the neonatal setting. As

a principle investigator I was responsible for ethics application and coordination

of the three studies at Jessop Wing, Royal Hallamshire Hospital. I was also

responsible for arranging funding, consenting, blood collection, data collection

and completion of case report forms, analyses, presentation and report writing.

Dr Anjum Deorukhkar was the principle investigator responsible for ethics

application and coordination of study C at the neonatal unit, Doncaster General

Hospital. Neonatal nurses at Jessop Wing and Doncaster General Hospital

routinely performed and documented prefeed gastric residual volumes. Blood

samples were also collected by junior doctors and colleague registrars.

Analytical expertise including sample size calculations for the individual study

was provided by the statisticians at this University. All blood samples were

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assayed by Mrs Christine Bruce, technician at the academic unit of reproductive

and developmental medicine, Jessop Wing, Royal Hallamshire Hospital.

Name : Dr Venkatesh Ramulu Kairamkonda Date: November 2012

Signature:

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Acknowledgements

I would like to thank my supervisor, Mr Robert Fraser, Reader at the University

of Sheffield and Consultant Obstetrician, section of Reproductive &

Developmental Medicine at Jessop Wing, Royal Hallamshire Hospital. His

constant enthusiasm, assistance, guidance, patience and advice helped me to

complete the project successfully.

I am indebted to Dr Anton Mayer, Consultant Paediatric Intensivist, Royal

Hallamshire Hospital, Sheffield and Dr Robert Coombs, Consultant

Neonatologist, Jessop Baby Unit, Sheffield for their advice, encouragement,

assistance and support towards the initiation and publication of this research.

I thank Mrs Christine Bruce for her invaluable contribution in the analyses of the

blood samples, but most of all for her continual encouragement and kindness.

I would like to thank the infants and their parents whose trust and co-operation

enabled this work to be completed. I would also like to thank the Clinical staff

and Nursing staff of the Neonatal Intensive care unit at Jessop Baby Unit and

Doncaster General Hospital for their patience and assistance.

I would like to thank the Baby Fund at the Jessop Wing, Royal Hallamshire

Hospital for providing the financial support for this project.

Finally, I would like to thank my wife Manjula and daughter Coral for their

encouragement, understanding and support.

3

Abstract

Title: Amylin peptide: An association with feed intolerance in preterm neonates

and infants of diabetic mothers

Introduction: Delayed enteral nutrition due to feed intolerance is common in

preterm infants and infants of mothers whose pregnancy was complicated by

diabetes mellitus (IMDM). Amylin, a 37 amino-acid polypeptide hormone, is a

potent inhibitor of gastric emptying that may play a role in the patho-physiology

of feed intolerance in these infants.

Aims and Objectives: To determine serum amylin levels (i) at birth (umbilical

cord) and postnatal day 5 (Guthrie) in healthy preterm and term infants-Study A,

(ii) at birth (umbilical cord) and postnatal day 5 (Guthrie) in preterm and term

IMDM-Study B, and (iii) in preterm infants experiencing feed-intolerance (nTOL)

and feed-tolerance (TOL)-Study C.

Hypothesis: Serum amylin levels are raised in (i) IMDM and (ii) preterm infants

with increased gastric residual volumes (GRV); which may explain their feed

intolerance.

Methods and Material: Blood samples were analysed for total amylin

immunoreactivity using monoclonal antibody based sandwich immunoassay.

Results: Serum amylin concentrations (median (interquartile range)) in healthy

term infants at birth (n=138) and postnatal day 5 (n=14) were 6.10 (3.30-9.70)

pmol/L and 5.65 (3.10-8.20) pmol/L respectively. Similarly, the amylin

concentrations in healthy preterm infants at birth (n=43) and postnatal day 5

(n=25) were 4.60 (1.90–8.30) pmol/L and 6.9 (2.75–9.50) pmol/L respectively.

The amylin concentrations were significantly raised in both term IMDM at birth

[n=17, 34.30 (28.35-50.00) pmol/L, p<0.0001] and postnatal day 5 [n=4, 25.20

(22.20-48.75) pmol/L, p<0.0001] and preterm IMDM at birth [n=14, 32.0 (18.65-

44.27) pmol/L, p<0.0001] and postnatal day 5 [n=9, 23.4 (15.37-46.57) pmol/L,

p<0.0001].

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The amylin concentration was significantly elevated in nTOL group [n=30, 47.9

(21.4-79.8) pmol/L, p<0.0001)] compared to TOL group [n=30, 8.7 (5.7-16)

pmol/L].

Conclusions: Amylin by virtue of its inhibitory effect on gastric emptying may be

responsible in delaying establishment of enteral nutrition in preterm infants and

infants of mothers whose pregnancy was complicated by diabetes mellitus.

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Outline of Dissertation

The structure of this dissertation is based on the suggested MD dissertation by

the University of Sheffield. Chapter 1 introduces to the research project, study

rationale, hypotheses, aims and objectives. Chapters 2 to 6 present literature

review relevant to the dissertation. Chapters 7 and 8 to 10 outline the project

plan and describe in detail methodology, results, discussion and conclusions of

individual study. Chapters 11 and 12 conclude the dissertation with emphasis

on suggested future studies and work. Chapters 13 and 14 provide publications

arising out of this project and appendices respectively. Finally, chapter 15

outlines bibliography relevant to the dissertation.

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Table of Contents

Chapter 1- Overview & Background.............................................................22

1.1 Foreword..............................................................................................22

1.2 Study Rationale....................................................................................23

1.3 Study Hypotheses................................................................................23

1.4 Study Aims...........................................................................................24

1.5 Study Objectives..................................................................................24

1.5.1 Primary Objectives.........................................................................24

1.5.2 Secondary Objectives....................................................................24

Chapter 2- Literature Review: Amylin Peptide..............................................26

2.1 Introduction & Background...................................................................26

2.2 Amylin Synthesis, Sources, Release & Metabolism.............................26

2.3 Amylin Structure, Gene & Relation to CGRP Superfamily...................30

2.4 Feto-Maternal Passage of Amylin........................................................32

2.5 Synthetic Analogue ‘Pramlintide’..........................................................32

2.6 Amylin Receptors & Signal Transduction Mechanisms........................34

2.7 Amylin Receptor Antagonist.................................................................35

2.8 Biological Functions of Amylin..............................................................35

2.9 Mechanisms of Amylin Action at Molecular level..................................39

2.10 History of Discovery of Amylin..........................................................42

2.11 Association of Amylin with Islet Amyloid Deposits in Diabetes.........43

2.12 Amyloidosis and Composition of Amyloid Deposits..........................43

2.13 Laboratory Assays............................................................................48

Chapter 3- Literature Review: Gastric Emptying..........................................50

3.1 Anatomy and Physiology of Gastric Motor Function.............................50

3.2 Regulation of Gastric Emptying............................................................51

3.3 Role of Other Gastrointestinal Peptides on Gastric Emptying..............52

3.4 Diabetes and its Effects on Gastric Emptying......................................53

3.5 Methods of Measurement of Gastric Emptying in Humans..................55

3.6 Methods and Factors Influencing Gastric Emptying in Neonates.........58

Chapter 4- Literature Review: Feed Intolerance in Preterm Infants.............60

4.1 Scale of the Problem............................................................................60

4.2 Physiology of Gastric and Gastrointestinal Motility in Preterm Infants. 60

4.3 Definition of Feed Intolerance..............................................................61

4.4 Patho-Physiology of Feed Intolerance.................................................63

4.5 Management of Feed Intolerance........................................................65

Chapter 5- Literature Review: Gestational Diabetes Mellitus.......................69

5.1 Definition of Gestational Diabetes Mellitus...........................................69


5.3 Physiology of Insulin Secretion............................................................69

5.4 Physiology of Gestational Diabetes Mellitus........................................70

5.5 Strategies to Prevent Fetal and Neonatal Hyperinsulinaemia..............70

Chapter 6- Literature Review: IMDM............................................................72


6.2 Physiology of Fetal Hyperinsulaemia...................................................72

6.3 Feed Intolerance in IMDM....................................................................73

6.4 Proposed Patho-Physiology of Feed intolerance in IMDM...................74

Chapter 7- Overview of Project Plan............................................................75

7.1 General Design....................................................................................75

7.2 General Study Configuration and Methodology...................................78

7.3 Ethics Consideration............................................................................81

7.4 Data Handling and Record Keeping.....................................................83

7.5 Benefits and Risks...............................................................................84

7.6 Funding................................................................................................84

Chapter 8- Clinical Study A: Umbilical & Guthrie Amylin in Preterm and Term

Infants 85

8.1 Introduction..........................................................................................85

8.2 Methodology.........................................................................................85

8.2.1 Study Design & Setting..................................................................85

8.2.2 Consent.........................................................................................85

8.2.3 Inclusion Criteria............................................................................85

8.2.4 Exclusion Criteria...........................................................................86

8.2.5 Study Procedure............................................................................86

8.2.6 Study End Point.............................................................................87

8.2.7 Sample Collection & Storage.........................................................88

8.2.8 Sample Analysis............................................................................88

8.2.9 Sample Size..................................................................................89

8.2.10 Statistical Analysis......................................................................89

8.2.11 Data Collection & Case Report Form.........................................90

8.2.12 Study Summary..........................................................................90

8.3 Results.................................................................................................92

8.4 Discussion..........................................................................................116

8.5 Conclusions........................................................................................119

Chapter 9- Clinical Study B: Umbilical and Guthrie Amylin in Preterm and

Term IMDM 120

9.1 Introduction........................................................................................120

9.2 Methodology.......................................................................................120

9.2.1 Study Design & Setting................................................................120

9.2.2 Consent.......................................................................................120

9.2.3 Inclusion Criteria..........................................................................120

9.2.4 Exclusion Criteria.........................................................................120

9.2.5 Study Procedure..........................................................................121

9.2.6 Study End Point...........................................................................121

9.2.7 Sample Collection & Storage.......................................................121

9.2.8 Sample Analysis..........................................................................121

9.2.9 Sample Size................................................................................121

9.2.10 Statistical Analysis....................................................................122

9.2.11 Data Collection & Case Report Form.......................................122

9.2.12 Study Summary........................................................................122

9.3 Result.................................................................................................125

9.4 Discussion..........................................................................................144

9.5 Conclusions........................................................................................147

Chapter 10- Clinical Study C: Amylin Levels in Preterm infants with Feed

Intolerance 148

10.1 Introduction.....................................................................................148

10.2 Methodology...................................................................................148

10.2.1 Study Design & Setting............................................................148

10.2.2 Consent....................................................................................148

10.2.3 Inclusion Criteria.......................................................................148

10.2.4 Exclusion Criteria.....................................................................149

10.2.5 Study Procedure.......................................................................149

10.2.6 Definition of feed intolerance....................................................150

10.2.7 Definition of feed tolerance.......................................................151

10.2.8 Study End Point........................................................................151

10.2.9 Sample Collection & Storage....................................................151

10.2.10 Sample Analysis.......................................................................151

10.2.11 Sample Size.............................................................................151

10.2.12 Statistical Analysis....................................................................151

10.2.13 Data Collection & Case Report Form.......................................152

10.2.14 Study Summary........................................................................152

10.3 Results............................................................................................155

10.4 Discussion.......................................................................................166

10.5 Conclusions....................................................................................173

Chapter 11- Conclusions of the Dissertation................................................174

Chapter 12- Implications for Future Research..............................................176

Chapter 13- Publications arising from this Project........................................179

13.1 Amylin peptide levels are raised in infants of diabetic mothers. Arch Dis Child. 2005 Dec; 90(12):1279-82...........................................................179

13.2 Amylin peptide is increased in preterm neonates with feed intolerance. Arch Dis Child Fetal Neonatal Ed. 2008 Jul; 93(4):F265-70.....184

Chapter 14- Appendices...............................................................................190

Chapter 15- Bibliography..............................................................................228

Key to Figures

Figure 1. Amylin is the second beta cell hormone.............................................26

Figure 2. Twenty four hour plasma profile of amylin and insulin in healthy adults

..........................................................................................................................28

Figure 3. Amylin response after a liquid sustacal meal in patients with type 1 &

type 2 diabetes mellitus.....................................................................................29

Figure 4. Representative concentration profiles of amylin (A), total amylin

immunoreactivity (TAI) (B) and insulin (C).........................................................30

Figure 5. Alpha helix structure of human amylin...............................................31

Figure 6. Comparison of aminoacid sequence of amylin and pramlintide.........33

Figure 7. Role of amylin in regulation of postprandial glycemia........................36

Figure 8. Amylin binding sites in rat brain..........................................................39

Figure 9. Location of amylin action within circumventricular organs in rat brain41

Figure 10. Amyloid deposit in pancreatic islet...................................................44

Figure 11. Amyloid deposit in lymph node showing birefringence.....................45

Figure 12. Extended cross-beta structure of amyloid........................................46

Figure 13. Superpleated beta structure of amyloid fibrils..................................47

Figure 14. Region responsible for amyloid formation........................................48

Figure 15. Site of synthesis of main gut hormones influencing appetite and

gastric emptying................................................................................................53

Figure 16. Project plan Gantt chart....................................................................75

Figure 17. Study plan Gantt chart......................................................................79

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Figure 18. General study methodology.............................................................80

Figure 19. Study A Gantt chart..........................................................................92

Figure 20. Study A flow diagram of participants and blood samples.................93

Figure 21. Box-and-whisker plot of umbilical cord amylin levels in healthy term

infants................................................................................................................96

Figure 22. Box-and-whisker plot of Guthrie amylin levels in healthy term infants

..........................................................................................................................98

Figure 23. Notched box-and-whisker plot of amylin levels at birth and postnatal

day 5 in healthy term infants...........................................................................100

Figure 24. Box-and-whisker plot of umbilical cord amylin levels at birth in

healthy preterm infants....................................................................................102

Figure 25. Box-and-whisker plot of amylin levels at postnatal day 5 in healthy

preterm infants................................................................................................104

Figure 26. Notched box-and-Whisker plot of amylin levels at birth and postnatal

day 5 in healthy preterm infants......................................................................106

Figure 27. Comparison Box-and-Whisker plot of amylin level in term and

preterm infants at birth....................................................................................108

Figure 28. Comparison Box-and-Whisker plot of serum amylin level in term and

preterm infants on postnatal day 5..................................................................109

Figure 29. The correlation graph showing line of best fit to assess the degree of

association between paired umbilical cord venous amylin (x axis) and umbilical

cord arterial amylin (Y axis).............................................................................110

Figure 30. Bland Altman method comparison plot of paired venous and arterial

levels...............................................................................................................111

Figure 31. Bland Altman method comparison, percentage difference plot, of

paired venous and arterial levels.....................................................................112

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association between paired umbilical cord insulin (x axis) and umbilical cord

amylin (Y axis) in healthy preterm and term infants........................................114


association between gestational age at birth (x axis) and umbilical cord amylin

(Y axis)............................................................................................................115


association between birth weight (x axis) and umbilical cord amylin (Y axis)..116

Figure 35. Study B Gantt chart........................................................................124

Figure 36. Study B-flow diagram of participants and blood samples...............125

Figure 37. Box-and-whisker plot of umbilical cord amylin levels in term IMDM

infants..............................................................................................................127

Figure 38. Box-and-whisker plot of Guthrie amylin levels in term IMDM infants

........................................................................................................................129

Figure 39. Comparison Box-and-Whisker plot of amylin levels at birth and

postnatal day 5 in term IMDMs........................................................................131

Figure 40. Box-and-whisker plot of umbilical cord amylin levels in preterm IMDM

infants..............................................................................................................133

Figure 41. Box-and-whisker plot of Guthrie amylin levels in preterm IMDM

infants..............................................................................................................135

Figure 42. Comparison box-and-whisker plot of amylin concentration at birth

and postnatal day 5 in preterm IMDM.............................................................137

Figure 43. Comparison box-and-whisker plot of amylin concentration in

umbilical cord and Guthrie blood in term and preterm IMDM..........................138

Figure 44. Comparison box-and-whisker plot of amylin levels at birth and

postnatal day 5 between term IMDM and healthy term infants (controls)........140

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postnatal day 5 between preterm IMDM and healthy preterm infants (controls)

........................................................................................................................142


association between umbilical cord insulin (x axis) and umbilical cord amylin (Y

axis) from paired umbilical cord blood samples of healthy and IMDM preterm

and term infants...............................................................................................143

Figure 47. Study C Gantt chart........................................................................154

Figure 48. Study C-Flow diagram of participants and blood samples.............155

Figure 49. Box and whisker plot of amylin levels (y-axis) in feed-intolerant

(nTOL) and feed-tolerant (TOL) neonates (x-axis)..........................................158

Figure 50. Box and whisker plot of percentage GRV (y axis) in feed-intolerant

(nTOL) and feed-tolerant (TOL) neonates (x axis)..........................................159

Figure 51. The box and whisker plot of days to reach full feeds (Y axis) in feed-

intolerant (nTOL) and feed-tolerant (TOL) neonates (X axis)..........................160

Figure 52. Correlation graph showing line of best fit to assess the degree of

association between amylin concentration (y axis) and % GRV (x axis) in feed-

intolerant (nTOL) group...................................................................................161


association between amylin concentration (y axis) and days to reach full enteral

feeds (x axis) in nTOL group...........................................................................162


association between days to discharge (x axis) and amylin concentration (Y

axis) in the feed-intolerant (nTOL) group.........................................................163


association between % GRV (x axis) and amylin levels (y axis) in feed-tolerant

(TOL) group.....................................................................................................164

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association between days to full enteral feeds (x axis) and amylin levels (Y axis)

in feed-tolerant (TOL) group............................................................................165


association between days to discharge (x axis) and amylin levels (Y axis) in the

feed-tolerant (TOL) group................................................................................166

Figure 58. Proposed pathway of amylin action................................................168

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Key to Tables

Table 1. Study Objectives & Outcome Variables...............................................25

Table 2. Study A Summary...............................................................................91

Table 3. Summary statistic of amylin concentration and demographic

characteristics at birth in healthy term infants...................................................95


characteristics at postnatal day 5 in healthy term infants..................................97

Table 5. Demographic characteristics of healthy term infants at birth and

postnatal day 5..................................................................................................99


characteristics at birth in healthy preterm infants............................................101


characteristics at postnatal day 5 in healthy preterm infants...........................103

Table 8. Demographic charateristics of healthy preterm infants at birth and

postnatal day 5................................................................................................105

Table 9. Comparison of demography and amylin levels in healthy term and

preterm infants at birth and postnatal day 5....................................................107

Table 10. Study B summary............................................................................123


characteristics at birth in term IMDM infants...................................................126


characteristics on postnatal day 5 in term IMDM infants.................................128

Table 13. Demographic characteristic of term IMDM at birth and postnatal day 5

........................................................................................................................130

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characteristics at birth in preterm IMDM infants..............................................132


characteristics at postnatal day 5 in preterm IMDM infants.............................134

Table 16. Demographic characteristics of preterm IMDM at birth and postnatal

day 5................................................................................................................136

Table 17. Demographic characteristics and serum amylin levels of term healthy

controls and term IMDM at birth and postnatal day 5......................................139

Table 18. Demographic characteristics and serum amylin levels of preterm

healthy controls and preterm IMDM at birth and postnatal day 5....................141

Table 19. Study C summary............................................................................153

Table 20. Baseline characteristics at the time of blood sampling of preterm

neonates defined as feed-intolerant (nTOL) and feed-tolerant (TOL).............156

Table 21. Serum amylin levels, % GRV, days to reach full feeds and length of

stay in feed-intolerant (nTOL) and feed-tolerant (TOL) neonates....................157

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Key to Appendices

Appendix 1.Basic principle of luminescent ELISA...........................................190

Appendix 2. Human Amylin (Total) ELISA KIT and Assay Procedure.............193

Appendix 3. Characteristics of Human Amylin (Total) ELISA Kit.....................196

Appendix 4. Research protocol for ethics approval, South Sheffield Research

Ethics Committee............................................................................................199

Appendix 5. Research protocol for ethics approval, Doncaster Research Ethics

Committee.......................................................................................................205

Appendix 6. Child participation information leaflet (Study A & B)....................210

Appendix 7. Child participation information leaflet (Study C) Royal Hallamshire

Hospital...........................................................................................................213

Appendix 8. Child participation information leaflet (Study C) Doncaster Royal

Infirmary..........................................................................................................216

Appendix 9. Study A & B Consent form...........................................................219

Appendix 10. Study C Consent form...............................................................220

Appendix 11. Advert for Study A.....................................................................221

Appendix 12. Advert for Study B.....................................................................222

Appendix 13. Advert for Study C.....................................................................223

Appendix 14. Case report form Study A..........................................................224

Appendix 15. Case report form Study B..........................................................225

Appendix 16. Case report form Study C..........................................................226

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Key to Abbreviations

AC 66 Amylin antagonist, also known as

salmon calcitonin

AC187 Amylin antagonist

ACSA Antral cross sectional area

ADP Adenosine diphosphate

AMY 1, AMY 2 and AMY 3 Amylin receptors

ATP Adenosine triphosphate

BMI Body Mass Index

CPAP Continuous positive airway pressure

CT Calcitonin

CTR Calcitonin receptor

CGRP Calcitonin gene related peptide

CV Coefficient of variation

C.F.U

CCK

Colony forming unit

Cholecystokinin

CRF Case Report Form

DBS Dried blood spot

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EGG Electrogastrography

ELISA Enzyme linked Immunosorbent Assay

ELBW Extremely low birth weight infants

FDA Food and drug administration, USA

4-MUB 4-Methylumbelliferyl Phosphate

GDM Gestational diabetes mellitus

GIP Glucagon inhibitory peptide

GLP-1 Glucagon like peptide 1

GLP-2 Glucagon like peptide 2

GRV Gastric residual volumes

GLUT 1 and GLUT 2 Glucose transporters

GSK 3 Glycogen synthase kinase 3

IGF Insulin like growth factor

IGFBP Insulin like growth factor binding

protein

IMDM Infant of mothers whose pregnancy

was complicated by diabetes mellitus

IAPP Islet amyloid polypeptide

KATP ATP sensitive potassium channel

MMC Migrating motor complex20

NEC Necrotising enterocolitis

nTOL Preterm infants with feed intolerance

PC2 Protein convertase 2

PK-PD Pharmacokinetic-Pharmacodynamic

PYY Peptide YY

RAMP Receptor activity modifying proteins

RFU Relative Fluorescent Unit

RIA Radioimmunoassay

sCT Salmon calcitonin, amylin antagonist

TBS Tris Buffered Saline

TOL Preterm infants without feed

intolerance

TPN Total parenteral nutrition

VLBW Very low birth weight infants

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Chapter 1-

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Chapter 1 - Overview & Background

Chapter 2- Overview & Background

2.1 Foreword

Neonatologists often struggle to attain full enteral nutrition in majority of preterm

infants with very low birth weight due to common occurrence of feed intolerance

(Reddy, Deorari et al. 2000; ElHennawy, Sparks et al. 2003; Patole, Rao et al.

2005) characterised by large gastric residual volumes (GRV) with or without

associated clinical manifestations (Jadcherla and Kliegman 2002; Mihatsch, von

Schoenaich et al. 2002; Patole 2005). Consequently feeds are either reduced or

withheld and frequently these neonates are subjected to investigations and

treatments for presumed necrotising enterocolitis (NEC). Reliance on total

parenteral nutrition (TPN) during this period is usually not enough to maintain

protein and lipid intake (Grover, Khashu et al. 2008; Hay 2008) and this often

leads to problems with infection and hepatic damage (Donnell, Taylor et al.

2002; Kaufman, Gondolesi et al. 2003). These infants may lag behind in protein

and calorie intake by up to 6-8 weeks by the time they are ready to go home

(Grover, Khashu et al. 2008) which may have adverse effects on their postnatal

growth and neurodevelopment (Frank and Sosenko 1988; Hack, Breslau et al.

1991; Ehrenkranz, Younes et al. 1999; Hack, Schluchter et al. 2003; Cooke,

Ainsworth et al. 2004; Hay 2008). The resultant increased morbidity and

hospital stay increases the cost of treatment (Reddy, Deorari et al. 2000). The

patho-physiology of feed intolerance in these high risk infants is poorly

understood, limiting the therapeutic options. Delayed gastric emptying, intestinal

immaturity, ileus of prematurity, and gastro-esophageal reflux may all play a

role. It is not clear whether one or all of these mechanisms contribute to the

observed feed intolerance.

Thirty-seven per cent of infants born to mothers whose pregnancy was

complicated by diabetes mellitus (IMDM) (Kitzmiller, Cloherty et al. 1978) also

experience feed intolerance immediately after birth. This is in addition to the

other well-known morbidities in this population of infants. These apparently

healthy looking babies often require admission to the special care or transitional

care unit until establishment of oral feeding which invariably leads to

prolongation of parent-infant separation and hospital stay (Kitzmiller, Cloherty et

23


al. 1978; Lee-Parritz 2004), and consequently cost of treatment. It is reasonable

to acknowledge that clinical practise has significantly changed in the last three

decades for diabetic mothers and their infants and thus the actual incidence of

feed intolerance or poor feeding may not be as high as previously reported. This

may also explain the apparent absence of published literature on feed

intolerance in IMDM. However with increasing incidence of diabetes in the

general population, the clinical burden of IMDM and associated illnesses

including feed intolerance may also increase. Therefore there is a need to

understand the aetiology and pathophysiology of feed intolerance in this group

of infants to formulate appropriate therapeutic interventions.

Amylin is a 37 amino acid peptide hormone belonging to the calcitonin gene

related peptide super family that is co-secreted with insulin from the pancreatic ß

cells in response to enteral nutrient intake (Rink, Beaumont et al. 1993). In

conjunction to playing a role in glucose homeostasis, it is recognised as a

potent inhibitor of gastric emptying (Macdonald 1997). Amylin has been shown

to delay gastric emptying in adult diabetic patients (Hoppener, Ahren et al.

2000) and critically ill children with feed intolerance (Mayer, Durward et al.

2002).

There are no published data at the time of writing this dissertation on amylin

and its role in the neonatal population. The focus of this dissertation is to

establish whether amylin is responsible for feed intolerance in preterm infants

and IMDM.

2.2 Study Rationale

Amylin is a potent inhibitor of gastric emptying that may be linked to the

pathophysiology of feed intolerance in the neonatal population. Understanding

its role in new-born population may help develop therapeutic interventions

which could lead to improved nutrition and outcomes.

24


2.3 Study Hypotheses

It is hypothesized that serum amylin levels are raised in (i) infants of mothers

whose pregnancy was complicated by diabetes and (ii) preterm infants with

increased gastric residual volumes; and this is likely to be the principal cause of

their feed intolerance.

2.4 Study Aims

The overall aims of this study were to determine serum amylin levels in (i)

healthy preterm and term infants (control population), (ii) preterm and term

IMDM and (iii) preterm infants with feed intolerance compared to preterm infants

without feed intolerance.

2.5 Study Objectives

2.5.1 Primary Objectives

The specific primary objectives of this study as detailed in (Table 1) were to

determine serum amylin concentration (i) at birth and in the postnatal period in

healthy preterm and term infants (ii) at birth and in the postnatal period in

preterm and term IMDM and (iii) in preterm infants admitted to neonatal

intensive care unit who subsequently experience feed intolerance compared to

those who do not.

2.5.2 Secondary Objectives

The specific secondary objectives of this study as detailed in (Table 1) were to

investigate (i) the relationship between amylin and birth weight / gestational age

(ii) if amylin and insulin were indeed co-secreted (iii) agreement of amylin levels

in paired umbilical cord arterial and venous blood (iv) the relationship of amylin

concentrations with gastric residual volumes (GRV), time to reach full enteral

feeds and length of hospital stay.

Rationale for secondary objective (iii): Umbilical venous sampling is technically

easy compared to umbilical arterial sampling and was chosen as the ideal route

for taking blood for this study. However experience of fellow researchers whose

studies involved blood sampling from umbilical cords suggested that

25


occasionally it was difficult to gain access to the umbilical vein necessitating

umbilical arterial sampling. Secondly umbilical venous blood is thought to

represent blood from the placenta whilst umbilical arterial blood to represent

baby’s blood. Hence the study team decided to investigate amylin levels in

paired samples wherever possible to investigate differences if any in amylin

levels to inform the current and future studies employing umbilical cord blood.

Table 1. Study Objectives & Outcome Variables

Primary Objective Primary Outcome Variable

To determine serum amylin

concentrations

1. at birth and postnatal period in

healthy preterm and term infants,

2. at birth and postnatal period in

preterm and term IMDM, and

3. in preterm infants with and without

feed intolerance.

Amylin (median and interquartile

range) concentration in pmol/L.

Secondary Objectives Secondary Outcome Variables

To investigate the relationship between

serum amylin concentrations and

gestational age and birth weight

Correlation Coefficient

To determine the relationship between

amylin and insulin levels to confirm co-

secretion


To determine agreement between

paired umbilical arterial and venous

amylin levels

Correlation Coefficient and Bland

Altman method comparison test

To investigate the relationship between

serum amylin concentrations and

influential covariates (gastric residual

volumes (GRV), time to full enteral


26


feeds and length of hospital stay).

27

Chapter 2 – Literature Review: Amylin Peptide

Chapter 3- Literature Review: Amylin Peptide

3.1 Introduction & Background

Amylin or Islet amyloid polypeptide (IAPP) was first discovered in 1987 and is a

second beta cell hormone that is co-located and co-secreted with insulin from

pancreas (Figure 1). The names ‘Amylin’ and ‘islet amyloid polypeptide’ are

often used interchangeably in current literature. The difference in nomenclature

is largely geographical; European researchers tend to prefer IAPP whereas

American researchers prefer Amylin. To maintain consistency ‘IAPP’ is referred

to as ‘Amylin’ throughout this dissertation.

Figure 1. Amylin is the second beta cell hormone

This slide shows pictures (on the left) demonstrating that amylin and insulin are

both present in the beta cells of islet of pancreas. A graphical representation of

the structure of the amylin molecule is shown on the right.

28


3.2 Amylin Synthesis, Sources, Release & Metabolism

Amylin is co-synthesized, co-packaged within the Golgi apparatus, and co-

secreted within the secretory granule by the islet beta cells in response to

elevations of plasma glucose following enteral nutrient intake (Rink, Beaumont

et al. 1993; Cooper 1994; Wimalawansa 1997). Although the major site of

amylin biosynthesis lies in the pancreatic islet beta cells, animal studies have

identified secondary sites in the endocrine cells of gastric mucosa (Mulder,

Lindh et al. 1994; Mulder, Ekelund et al. 1997), dorsal root ganglias (Ferrier,

Pierson et al. 1989; Fan, Westermark et al. 1994; Mulder, Leckstrom et al.

1995), and in the developing kidney (Wookey, Tikellis et al. 1998). Large

number of amylin-immunoreactive cells were also found in the pyloric antrum,

and a small number in the body of the stomach, duodenum and rectum in both

man and rat (Toshimori, Narita et al. 1990).

Amylin peptide may be prepared in vitro from diabetic pancreas by solubilisation

of amyloid and isolation of the peptide by gel filtration and reverse phase

chromatography. Amylin may also be produced by recombinant DNA

techniques using the disclosed nucleic acid sequences. However one of the

major problems with amylin is its spontaneous ability to deamidate and

aggregate (Nilsson, Driscoll et al. 2002) rendering it insoluble and toxic for

human administration.

Amylin release into the circulation is increased postprandially and is in

proportion to the amount of digested food (Bronsky, Chada et al. 2002). Oral

and intravenous dextrose loads also promote amylin release possibly due to co-

secretion with insulin in response to hyperglycemia. On the other hand, amylin

release is inhibited by both hypoglycaemia and somatostatin (Mitsukawa,

Takemura et al. 1990).

Amylin is co-released with insulin in a molar ratio of approximately 1 to 100 and

its diurnal profile corresponds to insulin in healthy non-diabetic subjects in

response to carbohydrate and protein containing meals (Figure 2) (Butler, Chou

et al. 1990; Mitsukawa, Takemura et al. 1990; Koda JE and JF 1995).

29


Figure 2. Twenty four hour plasma profile of amylin and insulin in healthy adults

Twenty-four hour plasma profile of amylin is similar to insulin in healthy adults

with low baseline levels that rapidly increase in association with increase in

plasma glucose concentrations. Image courtesy of (Koda JE and JF 1995).

In normal subjects plasma levels of amylin vary from fasting level of 5 pmol/L to

30 pmol/L postprandially (Bronsky, Chada et al. 2002). There is no difference in

the mean (SD) amylin concentration in lean subjects (3.4 ± 0.7 pmol/L) and

obese subjects (4.7 ± 0.9 pmol/L) with normal glucose tolerance (Hartter,

Svoboda et al. 1991; Reda, Geliebter et al. 2002). In contrast amylin secretion

following sustacal meal was observed to be absent in patients with type 1

diabetes and impaired markedly in patients with type 2 diabetes on insulin

therapy (Figure 3) (Fineman MS 1996).

30


Figure 3. Amylin response after a liquid sustacal meal in patients with type 1 &

type 2 diabetes mellitus

The graph shows almost absence of amylin secretion in patients with type 1

diabetes, and its secretion is markedly impaired in insulin-requiring patients with

type 2 diabetes. Image courtesy of (Fineman MS 1996).

In healthy adults during glucose stimulation, like insulin, amylin concentrations

exhibit high-frequency oscillatory pattern of nonglycosylated as well as

glycosylated forms (Figure 4). The amylin frequency (min/pulse), burst mass

(pM/burst), burst amplitude (pM/min), and basal secretion (pM/min) were 6.3 ±

1.0, 1.63 ± 0.88, 0.77 ± 0.4 and 0.04 ± 0.2. Whether basal amylin

concentrations also exhibit an oscillatory pattern is not known (Juhl, Porksen et

al. 2000).

31


Figure 4. Representative concentration profiles of amylin (A), total amylin

immunoreactivity (TAI) (B) and insulin (C)

This graph shows concentration profiles of amylin, total amylin immunoreactivity

and insulin during constant glucose infusion (2 mg/ kg / min). Image courtesy of

(Juhl, Porksen et al. 2000).

The mean removal time of amylin in plasma is longer than insulin and similar to

C-peptide (Clodi, Thomaseth et al. 1998), despite amylin having a faster

clearance rate than insulin at the kidney (Thomaseth, Kautzky-Willer et al.

1996).

3.3 Amylin Structure, Gene & Relation to CGRP Superfamily

Human amylin has 37 amino-acid sequences

KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY, with a disulfide bridge

between cysteine residues 2 and 7 and is characterised by a NH2-terminal

cyclic ring structure and an amidated COOH-terminus (Figure 1, Figure 6 &

Error: Reference source not found). Both the amidated C terminus and the

disulphide bridge are essential for the full biological activity of amylin (Cooper,

32


Day et al. 1989; Roberts, Leighton et al. 1989). The dynamic alpha helix

structure of human amylin is shown in Figure 5.

Figure 5. Alpha helix structure of human amylin

Image courtesy of http://wiki.verkata.com/en/wiki/Amylin

The human amylin gene is located on the short arm of chromosome 12

(Cooper, Day et al. 1989) and encodes the complete polypeptide precursor in

two exons which are separated by an intron of approximately 5 kb transcribing

an 89 amino acid precursor peptide (Nishi, Sanke et al. 1989). The proprotein

convertase PC2 is primarily responsible for amylin processing within the

secretory granule by converting the 89 aminoacid prohormone to the actively

secreted 37 amino acid amylin (Badman, Shennan et al. 1996).

Amylin belongs to the calcitonin gene related peptide superfamily (Rink,

Beaumont et al. 1993) which in addition consists of calcitonin (CT), calcitonin

gene-related peptide (CGRP) and adrenomedullin. CT and CGRP are derived

from the CT/CGRP gene, which is encoded on chromosome 11. Chromosome

12 is thought to be an evolutionary duplication of chromosome 11 as amylin has

20% sequence homology with calcitonin and adrenomedullin and 44% 33


homology with calcitonin gene-related peptide (Bronsky, Chada et al. 2002).

Importantly, a portion of the B-chain of insulin is strongly homologous to these

four peptides. Thus amylin, CGRP, CT, and adrenomedullin are related to the

insulin gene superfamily of peptides, which may all have diverged from a

common ancestral gene during evolution and therefore may share some of the

characteristics of insulin (Wimalawansa 1997).

3.4 Feto-Maternal Passage of Amylin

Information with regards to transplacental passage of amylin is lacking at the

time of writing this dissertation. The molecular weight of human amylin is 3904

Da compared to 5808 Da for human insulin. Published literature regarding

transplacental passage of insulin may shed some light on passage of amylin

across feto-maternal barrier. Early in vitro studies suggested that insulin does

not cross the human placenta (Keller and Krohmer 1968; Sabata, Frerichs et al.

1970). However a subsequent placental perfusion study demonstrated that a

small amount of human insulin, representing 1–5% of 59, 104, 448, and 1,198

μU/ml insulin concentration in the maternal artery (corresponding to

subcutaneous doses of insulin of approximately 14, 24, 104, and 278 units

respectively), was transferred from the maternal to the fetal circulation (Challier,

Hauguel et al. 1986). Similarly maternal insulin lispro concentrations of ≥580

μU/ml (corresponding to subcutaneous dose of insulin lispro of approximately

75 units) led to a small transfer of insulin lispro across the placenta (Boskovic,

Feig et al. 2003). In summary both human insulin and insulin lispro are unlikely

to transfer across term placenta at clinically relevant therapeutic doses. Thus it

may be assumed that endogenous amylin molecule has similar characteristic to

insulin with regards to the transplacental passage.

3.5 Synthetic Analogue ‘Pramlintide’

Human amylin is insoluble and potentially toxic for human administration as a

replacement in diabetic patients. Therefore synthetic, soluble, non-aggregating,

equipotent (Fineman, Weyer et al. 2002; Symlin 2005) amylin analogue

pramlintide acetate (Symlin, Amylin Pharmaceuticals) was developed by

substitution of 3 residues with proline at positions 25 (alanine), 28 (serine), and

34


29 (serine) as shown below in Figure 6. Pramlintide acetate (Symlin) is

formulated as a clear, isotonic, sterile solution for subcutaneous administration.

Figure 6. Comparison of aminoacid sequence of amylin and pramlintide

Image is courtesy of http://diabetesmanager.pbworks.com

Like amylin, pramlintide decreases post-prandial glucagon secretion, slows

gastric emptying, and increases satiety. In addition, pramlintide results in weight

loss, allows patients to use less insulin, lowers average blood sugar levels, and

substantially reduces blood sugar surges that occurs in diabetic patients

immediately after eating (Ratner, Want et al. 2002; Hollander, Levy et al. 2003;

Riddle, Frias et al. 2007). The absolute bioavailability of a single subcutaneous

dose of symlin is approximately 30 to 40% and approximately 40% of the drug

is unbound in plasma and plasma concentrations peaks at 20 minutes,

regardless of dose, and then declines over the subsequent 3 hours. It

undergoes little or no hepatic metabolism and is cleared mainly via the kidneys,

with a plasma half-life of 50 minutes (Weyer, Maggs et al. 2001). Pramlintide

has been approved for use by the US Food and Drug Administration (FDA) in

2005 in patients with type 1 and type 2 diabetes who use insulin (Ryan, Jobe et

35


al. 2005). Hypoglycemia, nausea, vomiting, and anorexia were the most

frequently reported adverse drug events associated with pramlintide therapy.

Personal communication with Amylin Pharmaceuticals provided information

regarding transplacental passage of pramlintide from studies performed on

perfused human placenta. Term human placenta from uncomplicated

pregnancy was obtained immediately after delivery. Pramlintide, at varying

concentrations was introduced into the maternal reservoir. The maternal side of

the placenta was perfused with constant concentration of pramlintide; the fetal

circulation was closed. Samples were drawn from both the maternal and fetal

circulations at regular intervals. The appearance of pramlintide in the fetal

circulation was analyzed by a specific ELISA. The resulting data demonstrated

that pramlintide had low potential (maternal to fetal ratio >1000/1) to cross the

maternal-fetal placental barrier (Amylin Pharmaceuticals 2003). These results

however cannot be extrapolated directly to the endogenous amylin and

therefore in vivo and in vitro studies are required to investigate transplacental

passage.

3.6 Amylin Receptors & Signal Transduction Mechanisms

The calcitonin (CT) family of peptides utilize two seven transmembrane G

protein-coupled receptors known as calcitonin receptor (CTR) and the calcitonin

receptor-like receptor (CRLR). These receptors can dimerize with three different

single transmembrane spanning proteins called Receptor activity-modifying

protein (RAMP). When associated with a calcitonin receptor, RAMP alters its

pharmacology from calcitonin-preferring to amylin-preferring. When co-

expressed with RAMP1, RAMP2, or RAMP3 (Hay, Christopoulos et al. 2004),

the CTR/RAMP complexes generate pharmacologically distinct amylin

receptors, AMY1, AMY2, and AMY3, respectively (Poyner, Sexton et al. 2002)

which bind with high affinity to amylin. The binding sites at nucleus accumbens

and subfornical organ are composed of CTR/RAMP1 (AMY1) whereas the

binding sites at area postrema are composed of CTR/RAMP3 (AMY3) (Young

2005). Despite pharmacological evidence of amylin sensitivity in several

peripheral tissues, selective amylin binding outside of the brain is observed only

in the renal cortex.

36


3.7 Amylin Receptor Antagonist

Peptides such as AC187, salmon calcitonin (sCT), AC66, CGRP [8-37], which

are typically analogues of truncated salmon calcitonin, have been developed as

potent and selective amylin antagonists and have been useful in identifying

amylinergic responses. Antagonism of a response with an order of potency of

AC187> AC66 > CGRP [8-37] is suggestive that it is mediated via amylin

receptors. Activation of a response with salmon calcitonin (sCT) > amylin >

calcitonin gene-related peptide (CGRP) > mammalian CT suggests activation

via the AMY1 receptor, while sCT = amylin >> CGRP > mammalian CT

suggests activation via AMY3 receptors (Young A, 2005).

AC-187 is particularly selective and potent at blocking amylin receptors (Young,

Gedulin et al. 1994) and most often cited in studies using amylin antagonists. It

also blocks amylin mediated excitatory effect on area postrema neurons and

subfornical organ neurons. In addition it blocks amylin induced c-Fos

expression in the area postrema (Riediger, Schmid et al. 2001).

3.8 Biological Functions of Amylin

Amylin has a number of biological functions both in animals and humans of

which inhibition of post prandial glucagon secretion, delaying gastric emptying,

and reduction of appetite and food intake are its most potent role. Amylin plays

a vital role in maintaining glucose homeostasis by virtue of its activity on the

stomach and glucagon secretion which is illustrated in Figure 7.

37


Figure 7. Role of amylin in regulation of postprandial glycemia

This slide demonstrates the actions of amylin in regulation of blood glucose in

fed state. Following ingestion of food the rise in blood glucose stimulates the

pancreas to secrete insulin along with amylin which proceeds through the

circulation to reach the brain where it binds to nuclei to slow gastric emptying

and decrease food intake both of which delay the delivery of nutrients to the

circulation. In addition amylin reduces hyperglucagonemia in the postprandial

period, thereby reducing the delivery of glucose from the liver into the

circulation. Thus blood glucose is maintained by fine tuning of the rate of

glucose appearance and glucose disappearance in the blood.

Following ingestion of nutrient intake, glucose is released into the blood stream

which stimulates the co-secretion of insulin and amylin from pancreatic beta

cells. Amylin regulates postprandial glycaemia by suppression of glucagon

release (Ludvik, Thomaseth et al. 2003), delaying gastric emptying (Kolterman,

Gottlieb et al. 1995; Young, Gedulin et al. 1995; Young 2005; Young 2005),

inhibition of glucose release and hepatic glucose production (Ludvik, Kautzky-

Willer et al. 1997) and possibly by noncompetitively antagonizing the effect of

38

The University of Sheffield, 25/04/12,

I still don’t think this figure is about Amylin!!


insulin and increasing the activity of the glycogen synthase kinase 3 (GSK 3)

(Abaffy and Cooper 2004).

The most reported role in literature of amylin and pramlintide is inhibition of

gastric emptying in animals (Young, Gedulin et al. 1995; Clementi, Caruso et al.

1996; Young, Gedulin et al. 1996), adult patients with type 1 and type 2

diabetes (Kong, King et al. 1997; Kong, Stubbs et al. 1998; Vella, Lee et al.

2002) and non-diabetic subjects (Samsom, Szarka et al. 2000). The effect of

amylin on gastric emptying is greater than that produced by other gut peptides,

such as gastric inhibitory peptide (GIP), cholecystokinin (CCK), and glucagon

like peptide (GLP-1) (Young, Gedulin et al. 1996). In particular, amylin is 15- to

20 fold more potent than GLP-1 and CCK (Gedulin 1996; Young, Gedulin et al.

1996). It is also known to inhibit gastric acid secretion and maintain gastric

mucosal integrity (Young 2005).

Amylin is also known to control the central mechanisms responsible for food

intake and for the feeling of satiety and thirst. Amylin decreases food intake

(Morley and Flood 1991; Lutz, Del Prete et al. 1994; Lutz, Geary et al. 1995;

Bronsky, Chada et al. 2002) mainly through reduction in meal size (Lutz, Geary

et al. 1995; Mollet, Gilg et al. 2004) and increasing satiety (Morley and Flood

1991; Lutz, Del Prete et al. 1994). Amylin appears to reduce food intake

centrally by activation of receptors in area postrema, a circumventricular organ

outside the blood brain barrier involved in regulating food intake and

peripherally through augmenting the actions of other gut peptides such as CCK,

glucagon, and bombesin, all of which inhibit food intake by stimulating

ascending vagal fibers and secretion of amylin. In addition amylin may decrease

food intake by inducing anorexia through its effect on brain by increasing the

transport of the precursor tryptophan into the brain to inhibit feeding and

increase satiation by serotonin action in the paraventricular nucleus, stimulating

histamine H1 receptors, inhibition of stimulation of feeding by potent

hypothalamic neuropeptide Y (NPY) and peripherally by inhibition of nitric oxide

which is a major regulatory agent in the gastrointestinal tract (Reda, Geliebter et

al. 2002). Amylin significantly increased water intake in euhydrated rats by

activating neurons in the subfornical organ of the brain suggesting amylin

release during food intake may stimulate prandial drinking (Riediger, Rauch et

al. 1999).39


Amylin has also been found to be a growth factor for cells involved in bone

metabolism (Cornish, Callon et al. 1995; Villa, Rubinacci et al. 1997; Cornish,

Callon et al. 2001), particularly for the proliferation of osteoblasts (Cornish,

Callon et al. 1995; Cornish, Callon et al. 1998) and differentiation of osteoclast

(Cornish, Callon et al. 2001) and inhibition of bone resorption (Bronsky, Chada

et al. 2002) which helps increase bone density and bone mass (Cornish and

Naot 2002). Amylin is found to be a growth factor for isolated fetal beta cells

which may have a role in the development of fetal pancreas (Karlsson and

Sandler 2001).

Amylin is a potent proliferation factor both during the development of proximal

tubules (Wookey, Tikellis et al. 1998) and in the proliferation of adult epithelial

cells (Harris, Cooper et al. 1997). Amylin at physiological doses has been

shown to stimulate renin activity in both rats (Wookey, Tikellis et al. 1996) and

man (Cooper, McNally et al. 1995) and demonstrates a modest pressor effect

(Cooper, McNally et al. 1995; Haynes, Hodgson et al. 1997) possibly mediated

by insulin-induced changes in rennin release (Ikeda, Iwata et al. 2001). In

addition renal amylin binding sites in proximal tubules could be correlated with

blood pressure in rat models of renal hypertension (Cao, Wookey et al. 1997;

Wookey, Cao et al. 1997; Wookey, Cao et al. 1998; Wookey and Cooper 1998).

Amylin has been reported to induce relaxation of aortic rings (Luk'yantseva,

Sergeev et al. 2001), inhibit CCK induced contraction of smooth muscle cells

(Ochiai, Chijiiwa et al. 2001), and decrease pulmonary vascular tone (Golpon,

Puechner et al. 2001). Amylin modulates insulin sensitivity of skeletal muscle

(Bronsky, Chada et al. 2002).

Amylin is vasoactive and produces hypotension and vasodilatation (Young, Rink

et al. 1993; Bronsky, Chada et al. 2002) probably by inhibiting the secretion of

the atrial natriuretic peptide through CGRP1 and CT receptors (Piao, Cao et al.

2004), and exerts anti-inflammatory activity (Hall and Brain 1999) possibly due

to cross reactions with CGRP receptors (Zhu, Dudley et al. 1991). Like CGRP

amylin may also play a role in maternal sepsis and shock (Parida, Schneider et

al. 1998).

40


This all looks like cut and paste!


3.9 Mechanisms of Amylin Action at Molecular level

Experimental investigations in rodents indicate that the gastrointestinal effects of

amylin are mediated via a central pathway that involves the area postrema and

visceral efferents of the vagus nerve. The area postrema in the brainstem which

contains a high density of amylin binding sites (Figure 7) is exposed to changes

in plasma amylin and specifically co-excited by glucose (Riediger, Schmid et al.

2002) because it does not have a blood-brain barrier (Gross 1992). As there is

no unique amylin receptor gene, the most likely mechanism of amylin action at

molecular level in the brain and elsewhere is likely to be through CTR and

RAMPs bestowing functionality to the amylin receptors and binding sites (Figure

8).

Figure 8. Amylin binding sites in rat brain

An audioradiograph of the rat brain demonstrating major binding sites

containing amylin receptors in nucleus accumbens, the dorsal raphe, and the

area postrema which are thought to be important in regulation of appetite as

well as sympathetic and parasympathetic tone.

41


In addition peripherally injected amylin strongly activates neurons of nucleus

tractus solitarius (Rowland and Richmond 1999; Riediger, Schmid et al. 2002)

which is neuronally interconnected with the area postrema (van der Kooy and

Koda 1983; Shapiro and Miselis 1985). Selective lesioning of the area postrema

and/or bilateral vagotomy abolishes the effect of amylin on gastric emptying

(Jodka 1996; Edwards 1998) demonstrating the importance of this central

pathway in mediating amylin’s physiological functions. In addition the effect of

amylin to slow gastric emptying is overridden in the presence of hypoglycaemia

(Gedulin and Young 1998). Amylin exerts its inhibitory action on glucagon by

mechanisms extrinsic to pancreas (Silvestre, Rodriguez-Gallardo et al. 2001).

Thus amylin contributes to metabolic control by regulating nutrient influx into the

blood resulting in increased glucose disposal and slowing nutrient delivery and

postprandial hyperglycemia.

Two main regions of the circumventricular organs in the rat brain (Figure 9)

namely subfornical organ and area postrema mediate peripheral variations in

amylin levels via receptors including drinking (Schmid, Rauch et al. 1998;

Riediger, Rauch et al. 1999; Riediger, Schmid et al. 1999) and feeding

behaviour (Lutz, Senn et al. 1998; Riediger, Schmid et al. 2001) respectively.

42


Figure 9. Location of amylin action within circumventricular organs in rat brain

The circumventricular region contains a rich capillary plexus and has a

fenestrated endothelium rendering the structures outside of the blood brain

barrier. It is made up of 7 organs namely median eminence, organum

vasculosum of the lamina terminalis, subfornical organ, choroid plexus, pineal

gland, and subcommissural organ and area postrema. Image courtesy of

http://www.endotext.org

Additionally peripherally applied amylin inhibits feeding through binding sites in

area postrema and nucleus accumbens (Sexton, Paxinos et al. 1994; van

Rossum, Menard et al. 1994; Lutz, Senn et al. 1998; Lutz, Mollet et al. 2001;

Bronsky, Chada et al. 2002) which may be processing these peripheral signals

(Baldo and Kelley 2001) and is independent of vagal afferents (Lutz, Del Prete

et al. 1995). Amylin may induce anorexia through stimulating H1 receptors

(Mollet, Lutz et al. 2001) and this effect is attenuated with dopamine D2 receptor

antagonists (Lutz, Tschudy et al. 2001). Amylin also inhibits stimulation of

feeding centrally by the potent hypothalamic neuropeptide Y (NPY) (Morris and

Nguyen 2001) and peripherally by inhibiting nitric oxide (Morley and Flood 1994;

Morley, Flood et al. 1995).

43


In summary amylin is a neuroendocrine hormone which helps in regulating

feeding behaviour by reducing meal size, increasing satiety and anorexia by

stimulating neurons particularly in the area postrema and hindbrain.

3.10 History of Discovery of Amylin

Although the presence of amyloid deposits within the islet was first described at

the beginning of the 20th century, it was not until 1987 that the structure of

amylin was discovered (Cooper, Willis et al. 1987; Westermark, Wernstedt et al.

1987; Cooper, Leighton et al. 1988).

The history leading to the discovery of amylin is an interesting story and began

in 1869 when Paul Langerhans (Langerhans 1869; Morrison 1937) described

the existence of acinar glandular cells and pairs or groups of small polygonal

cells with round nuclei in the interacinar spaces of rabbit’s pancreas. Oscar

Minkowski in 1889 observed the connection between pancreatic secretion and

diabetes on depancreatized dogs (Bliss 1982). It was Languesse E (Languesse

1893) in 1893 who named the clumps of cells observed by Paul Langerhans as

the ‘Islands or Islets of Langerhans’. Eugene Opie in 1889 was the first to

describe "hyaline degeneration of the islands of Langerhans" in the pancreas of

patients with hyperglycemia and in 1901 provided its pathological link to

diabetes (Opie 1901). It was not until 1922 however, that a Canadian team

comprising of Banting, Best, Macleod and Collip discovered insulin (Banting

1922). Thirty-three years later, in 1955, the protein structure of insulin was

completely sequenced by Frederick Sanger (Ryle, Sanger et al. 1955). The

amyloidal nature of the hyaline material described by Eugene Opie was

established by Ahronheim in 1943 and later confirmed by Ehrlick and Ratner in

1961 (Ahronheim 1943; Ehrlich and Ratner 1961). Westermark in 1973

demonstrated the fibrillar structure (Westermark 1973) of the hyaline staining

material on electron microscopy. Importantly, in 1987 he also discovered that it

was predominantly made up of a 37 amino acid monomer and referred to it as

“islet amyloid polypeptide” (IAPP). Interestingly in the same year Cooper from

another laboratory also discovered this 37 amino acid polypeptide in pancreatic

amyloid deposits and named it “amylin” (Cooper, Willis et al. 1987; Westermark,

Wernstedt et al. 1987; Cooper, Leighton et al. 1988).

44


3.11 Association of Amylin with Islet Amyloid Deposits in Diabetes

Amylin is associated with type 2 diabetes, a disease that has increased over the

past two decades and now afflicts an estimated 100 million worldwide (Hossain,

Kawar et al. 2007). At post-mortem 95% patients with type 2 diabetes stain

positive for amyloid deposits in the pancreas composed of mature, fibrillar

amylin (Hoppener, Ahren et al. 2000). Since 1987 there has been a rapid

increase in publications on amylin most probably due to the fact that islet

amyloid in type 2 diabetes mellitus is strongly associated with islet beta cell loss

(Hayden and Tyagi 2001; Jaikaran and Clark 2001). Additionally, it is also

acknowledged that amylin may be linked to the pathogenesis and therefore a

potential target for the treatment of diabetes (Schmitz, Brock et al. 2004).

3.12 Amyloidosis and Composition of Amyloid Deposits

Amyloidosis refers to group of disorders characterised by abnormal deposition

of amyloid proteins in organs and/or tissues. The clinical classification of

amyloidosis includes primary (de novo), secondary (a complication of a

previously existing disorder), familial, and isolated types. Out of the

approximately 60 amyloid proteins which have been identified so far (Mok,

Pettersson et al. 2007), at least 36 have been associated in some way with a

human disease (Pettersson-Kastberg, Aits et al. 2008) such as Bovine

spongiform encephalopathy (prion protein PrPSc), Alzheimer’s disease (beta-

amyloid), Parkinson’s (Alpha-synuclein) and Creutzfeldt-Jakob disease (prion

protein PrPSc). A characteristic feature of amyloid deposit histologically is the

positive staining with Congo red (Figure 10) (Satoskar, Burdge et al. 2007) and

birefringence on viewing with polarised light (Figure 11) (Hayden and Tyagi

2001).

45


Figure 10. Amyloid deposit in pancreatic islet

High power microscopic view showing a pancreatic islet with extensive

deposition of a pink homogenous material (Congo red stain) that has features

suggestive of amyloid surrounded by normal pancreatic acini. Image courtesy of

http://pathcuric1.swmed.edu/PathDem0/end6/end660.htm

46


Figure 11. Amyloid deposit in lymph node showing birefringence

Congo red staining of amyloid deposit in the lymph node demonstrating green

birefringence to polarised light. Image courtesy of

http://www.flickr.com/photos/euthman/377559955/

Deposition patterns of amyloid vary between patients but each disease is

characterized by a particular protein or polypeptide that aggregates in an

ordered manner to form insoluble amyloid fibrils that are deposited in the

tissues, where they have been associated with the pathology of the disease.

These different proteins and peptides, are characterised by common structural

features, whereby stacks of parallel peptide strands (Jayasinghe and Langen

2004) run perpendicular to the fibril axis hydrogen-bond (Sumner Makin and

Serpell 2004) to form extended beta-sheets (Error: Reference source not

found).

47


Figure 12. Extended cross-beta structure of amyloid

Extended cross-beta sheet amyloid structure consisting of layers of aminoacid

chains lying perpendicular to the fiber axis. Image courtesy of

http://www.vanderbilt.edu/vicb/

Similarly one of the major polypeptide components forming the monomeric unit

of polymerized, aggregated, and beta pleated sheet structure of islet amyloid in

type 2 diabetic patients is amylin (Figure 13) (Hoppener, Ahren et al. 2000;

Kajava, Aebi et al. 2005).

48


Figure 13. Superpleated beta structure of amyloid fibrils

The superpleated beta-structural model of amyloid fibrils of human amylin

viewed along the fibril axis. Image courtesy of (Kajava, Aebi et al. 2005)

Amylin is implicated in the pathogenesis of type 2 diabetes and Alzheimer’s due

to its ability to form amyloid deposits leading to destruction of cell membranes

(Green, Goldsbury et al. 2004; Sedman, Allen et al. 2005). Amylin receptors

may play an important role in the neurotoxic effects of amyloid beta and

therefore its inhibition could be promising in the treatment of Alzheimer’s

disease (Kurganov, Doh et al. 2004)

Sequences of human proamylin that may contribute to the processes of amyloid

formation include phenylalanine-23 (Azriel and Gazit 2001) and amino and

carboxy terminal regions (Jaikaran and Clark 2001; Jaikaran, Higham et al.

2001; Padrick and Miranker 2001). Amylin and its 10-residue fragment between

amino acids 20 and 29 (Error: Reference source not found) is known to

spontaneously deamidate, and the presence of less than 5% of deamidation

impurities leads to the formation of aggregates by the purified peptide that has

the hallmarks of amyloid (Nilsson, Driscoll et al. 2002).

49


Figure 14. Region responsible for amyloid formation

The primary structure of amylin showing region responsible for amyloid

formation. Image courtesy of http://www.joplink.net/prev/200507/02.html

3.13 Laboratory Assays

Studies investigating the biological roles of amylin in health and disease require

sensitive and specific assays capable of measuring amylin at the concentrations

at which it circulates in plasma. Some of the Radioimmunoassay’s (RIAs)

developed initially to measure human amylin were reported to be sensitive

enough to detect fasting concentration of amylin. However RIA is labour

intensive, time consuming taking up to 5 days, extraction and assay procedure

could potentially introduce variability into the results and required up to 5 ml

blood volume to obtain enough plasma before assay to extract amylin. This

blood volume would be unsafe and thus prohibitive especially in preterm and

very low birth weight population. To combat the problems highlighted with RIA,

two sandwich-type immunoassays were developed which are considered to be

rapid, sensitive, analyze large numbers of samples within a few hours and

capable of measuring 0.5 to 2 pmol/L amylin in 50 µL of plasma. Furthermore,

50


Is this your wording – not cut and paste



these two assays exhibited limited cross reactivity (< 0.01 %) with other related

peptides, detectable dynamic range of amylin concentration 2 to 100 pmol/L,

average intraassay coefficient of variations (CVs) of <10%, average interassay

CVs of <15%, and good linearity on dilution and recovery of added amylin. Thus

the availability of these assays made it possible to study plasma amylin

concentrations in various disease states in different age groups including

neonatal population.

51

Chapter 3 – Literature Review: Gastric Emptying

Chapter 4- Literature Review: Gastric Emptying

4.1 Anatomy and Physiology of Gastric Motor Function

An understanding of the pathogenesis of delayed gastric emptying may be

helped by revisiting the anatomy and physiology of normal gastric motor

function.

The stomach is divided into cardia, fundus, body and pylorus each with different

cells and functions. The oesophageal and pyloric sphincters help to keep the

contents of the stomach contained. In adult humans, the anatomic capacity of

stomach is about 45 ml which normally expands to hold about 1-3 litres of food.

The stomach volume in the fetus is approximately 10 ml which increases to 18-

21 ml by term (Nagata, Koyanagi et al. 1994; Sase, Miwa et al. 2005). The

anatomic capacity of new-born stomach depends on birth weight and varies

from 22-38 ml in 1.5-4.0+ kg infants which increases to 45 ml in 1 week and 75

ml in 2 weeks postnatal age. The physiologic capacity is less than the anatomic

capacity at birth, equals by day 5 and reaches 81 ml by day 10 in 2.0-4.0+ kg

infants (Scammon RE 1920).

The gastric motor function is controlled by parasympathetic and sympathetic

nervous systems, enteric brain neurons and smooth muscle cells. Extrinsic

neural controls by parasympathetic pathways are conveyed to the stomach and

upper intestine through the vagal efferents arising in the dorsal motor nucleus of

the vagus nerve, nucleus ambiguus and tractus solitaries which form

characteristic bead chain like terminals in the myenteric plexus throughout the

stomach and upper intestine but without directly innervating muscle (Berthoug

1993). The vagus nerve regulates the distribution of food between proximal and

distal regions of the stomach. The proximal stomach serves as a reservoir for

solid foods and induces tonic pressure to facilitate liquid emptying. At the distal

end of the stomach, the tone of the pylorus controls the rate at which the

contents of the stomach are emptied into the small intestine. During fasting

state both adults as well as term infants exhibit migrating motor complexes

(MMCs) which are peristaltic waves originating in the stomach that progress

through small intestine into colon approximately every 75-90 minutes and are

interrupted by a meal. As the distal stomach grinds ingested solid foods, phasic 52


and tonic pyloric pressures in conjunction with duodenal contractions slow

gastric emptying, which in turn disrupts MMC to allow for normal digestion and

absorption of nutrients (Chaikomin, Rayner et al. 2006).

In summary, normal gastrointestinal motor function is a complex sequence of

events that is controlled by an extrinsic nerve supply from the brain and spinal

cord, the complex plexi within the wall of the stomach and intestine, and the

effects of locally released amines and peptides that alter the excitability of the

smooth muscle of the intestine. Abnormalities in any of these locations can lead

to delayed gastric emptying.

4.2 Regulation of Gastric Emptying

Carbohydrate absorption is crucial to understanding the control of gastric

emptying. In the normal adult human, a moderately sized meal contains up to

50-100 g of glucose but the total amount of glucose in plasma is approximately

5 g (Young, Pittner et al. 1995). Thus without regulation of its absorption,

glucose could easily exceed the normal level found in plasma. The glucose

released into the blood stream following ingestion of food stimulates rapid

secretion of insulin and simultaneous suppression of glucagon secretion. These

reciprocal changes in islet hormones stimulate glucose uptake and suppress

hepatic glucose production. At the same time, glucose transport and disposal

are facilitated in peripheral tissues, mainly in muscle, and to some extent in

kidney and adipose tissue (Kelley, Mitrakou et al. 1988; Meyer, Dostou et al.

2002). As blood glucose concentrations rise, the decelerated rate of gastric

emptying contributes to maintenance of glucose homeostasis (Barnett and

Owyang 1988). Furthermore, in the presence of acute hyperglycaemia, proximal

gastric tone is diminished, frequency and propagation of antral pressure waves

are suppressed, and pyloric contractions are stimulated, all of which contribute

to the slowing of gastric emptying (Meyer, Dostou et al. 2002) as a protective

mechanism to control glucose efflux into the blood stream. Hence there seems

to be matching of fluxes via ‘entero-insular loop’ to ensure regulated

carbohydrate efflux from the stomach for absorption is approximately equal to

the regulated insulin-mediated uptake from the plasma into peripheral tissue.

This regulatory system which is essential for normal control of blood glucose is

disrupted in amylin-deficient state such as type-1 diabetes.

53


The rate of gastric emptying in adults is also influenced by a number of other

factors, including the size, composition (fat versus carbohydrate) and type

(liquid versus solid) of the meal (Moore, Christian et al. 1981; Collins, Horowitz

et al. 1983), and activity of the gut hormones (Macdonald 1997). Osmolarity and

temperature of feed, posture, and stress also impact the gastric emptying rate

(Hunt and Knox 1968; Blair, Wing et al. 1991). Small meals empty more rapidly

than large ones and a meal with a high fat content has a higher half time for

gastric emptying than a meal with high carbohydrate content (Sidery,

Macdonald et al. 1994). The rate of gastric emptying of liquids is faster than that

of solids (Collins, Houghton et al. 1991), and addition of soluble fiber to food

delays gastric emptying rate (Torsdottir, Alpsten et al. 1991; Frost, Brynes et al.

2003).

4.3 Role of Other Gastrointestinal Peptides on Gastric Emptying

Gastric emptying is modulated by feedback mechanisms arising from the

interaction of nutrients from food with the small intestine (Chaikomin, Rayner et

al. 2006) and intestinal peptides including glucagon-like peptide 1 (GLP-1),

CCK, secretin, peptide YY, gastrin-releasing peptide (bombesin), and amylin.

GLP-1 inhibits gastric emptying; its secretion stimulated by the intestinal nutrient

content and flow rather than by the plasma glucose concentration itself (Schirra,

Katschinski et al. 1996; Schirra, Leicht et al. 1998). On the other hand, amylin

inhibits gastric emptying secondary to release of insulin due to postpandrial

hyperglycemia with amylin concentrations increasing from 9 to 43 pmol/L

(Samsom, Szarka et al. 2000; Woerle, Albrecht et al. 2008). Of the reported

peptides known to inhibit gastric emptying, amylin is the most potent of the

endogenous peptides identified to date (Young, Gedulin et al. 1996). In contrast

to amylin, Ghrelin is synthesised predominantly by the stomach and is the

endogenous ligand for the growth hormone secretagogue receptor which is

expressed in brain stem and hypothalamic nuclei (Kojima, Hosoda et al. 1999).

Exogenous ghrelin causes large meals to be eaten (Tschop, Smiley et al. 2000;

Wren, Seal et al. 2001; Wren, Small et al. 2001) and is a most potent stimulator

of gastric contractions and emptying (Hellstrom, Gryback et al. 2006). Other gut

hormones such as gastrin, PP, CCK, secretin, GLP-1, GLP-2, oxyntomodulin,

54


and PYY also influence gastrointestinal motility and may potentiate amylin’s

inhibition of gastric emptying (Figure 15).

Figure 15. Site of synthesis of main gut hormones influencing appetite and

gastric emptying

These gut hormones may also potentiate amylin’s role in delaying gastric

emptying. CCK, cholecystokinin; PYY, peptide YY; GLP1, glucagon-like

peptide 1; GLP2, glucagon-like peptide 2. Image courtesy of (Druce and Bloom

2006)

4.4 Diabetes and its Effects on Gastric Emptying

Diabetes is frequently associated with symptoms due to disturbances of

gastrointestinal motility and/or sensation (Samsom, Roelofs et al. 1998; Bytzer,

Talley et al. 2001). Approximately 30%-50% of patients with long standing type

1 and type 2 diabetes show delayed gastric emptying (Horowitz, O'Donovan et

al. 2002; Samsom, Vermeijden et al. 2003) which may coexist with

gastroparesis due to diabetic autonomic neuropathy (Samsom, Akkermans et

al. 1997; Rayner, Samsom et al. 2001).

55


Type 1 diabetes patients have delayed gastric emptying rates in response to

hyperglycemia (Schvarcz, Palmer et al. 1997) including patients with autonomic

neuropathy probably due to changes in antroduodenal motility (Samsom,

Akkermans et al. 1997). On the contrary, gastric emptying rate is accelerated in

type 1 diabetics in insulin induced hypoglycaemia (Schvarcz, Palmer et al.

1993). Thus there is an inverse relationship between the rate of gastric

emptying and the blood glucose concentration, so that emptying is slower

during hyperglycaemia and faster during hypoglycaemia.

Delayed gastric emptying also occurs frequently in type 2 diabetes mellitus

partly due to increased plasma glucose concentrations (Ziegler, Schadewaldt et

al. 1996) and partly due to raised amylin levels. However unchanged (Kong,

King et al. 1999) or accelerated (Schwartz, Green et al. 1996) gastric emptying

rates have also been reported.

Although a review reports increase in amylin level in gestational diabetes

(Ludvik, Kautzky-Willer et al. 1997), literature on the effects of gestational

diabetes on gastric emptying is lacking.

Because amylin is co-secreted by β-cells, individuals with diabetes have both

insulin and amylin deficiency. Amylin deficiency is absolute in type 1 diabetics,

whereas in type 2 diabetics, amylin levels increase in the early stage due to

insulin resistance but decrease progressively in tandem with the decline in

insulin secretion (Edelman and Weyer 2002). However contrary to the

expectation of accelerated gastric emptying, even patients with type 1 diabetes

demonstrate delayed gastric emptying suggesting factors other than amylin play

a role in control of gastric motility (Heptulla, Rodriguez et al. 2008) in diabetic

patients. Delayed gastric emptying in type 2 diabetes patients (Hoppener, Ahren

et al. 2000) may be partly explained by raised amylin levels due to insulin

resistance syndrome.

Amylin’s synthetic analogue pramlintide slows liquid and solid gastric emptying

by 1–2 hours through vagal inhibition in both type 1 and type 2 diabetic patients

(Whitehouse, Kruger et al. 2002; Kellmeyer, Kesty et al. 2007).

Thus gastric emptying in diabetes is influenced particularly by glycaemia with

reciprocal relationship between the rate of gastric emptying and the blood

56


glucose concentration. Factors other than and partly due to amylin seem to be

responsible for the gastrointestinal symptoms associated with delayed gastric

emptying in patients with type 1and type 2 diabetes respectively.

4.5 Methods of Measurement of Gastric Emptying in Humans

A variety of different techniques are available to study the rate of gastric

emptying in humans. The majority of these tests are only applicable to adult

patients. However methodological details and review of individual tests may

help to establish relevance of these tests to the neonatal and paediatric setting.

Early methods employed to measure gastric emptying such as dye dilution and

single and double sampling gastric aspiration techniques (George 1968) are

now almost obsolete due to the availability of more advanced techniques.

Gastric emptying scintigraphy is considered gold standard as it is a physiologic,

quantitative, non-invasive test for evaluating gastric emptying for both solids

and liquids. It is validated in healthy subjects and also allows for assessment of

intragastric distribution (Tougas, Chen et al. 2000; Abell, Camilleri et al. 2008;

Abell, Camilleri et al. 2008). A radiolabelled 99mTc-sulphur colloid or 113mIn

DTPA or 67Ga EDTA meal is ingested under standardized ambient noise,

ambient light, and patient comfort. Imaging is performed while the patient is

positioned constant either standing or sitting. The passage of the meal from the

stomach to the small intestine is then followed by taking images at regular

intervals with a gamma camera (Macdonald 1997). The main disadvantages of

this procedure are its cost and exposure to a small amount of radiation which

would be a limitation for its use in neonates’ especially preterm infants.

The basis of the bio impedance technique (Savino, Cresi et al. 2004) is that

after the ingestion of fluids with a different conductivity from body tissues, the

impedance to an electrical current through the upper abdomen changes,

impedance increases as the stomach fills and decreases as it empties. The

slope of the plot of impedance versus time is used to calculate the time for

emptying half of the meal giving a value for impedance half-life. Epigastric

impedance patterns closely correlated with scintigraphy in the measurement of

gastric emptying in infants (Savino, Cresi et al. 2004).

57


The 13C-octanoate breath test is a non-invasive approach for measuring gastric

emptying, which correlates well with scintigraphy (Ziegler, Schadewaldt et al.

1996). When octanoic acid (with solid meal) or acetate (with liquid meal)

labelled with 13C is combined with different food substrates, these compounds

pass unabsorbed through the stomach to the duodenum but are quickly

absorbed in the small intestine and metabolized to 13CO2 in the liver.

Appearance of 13CO2 in the breath marks gastric emptying, absorption and

excretion of 13CO2. This methodology offers significant advantages over

scintigraphy and other methods in terms of cost, accessibility, and avoidance of

radioactive materials. Sensitivity and specificity of the 13C-octanoate breath test

for measuring gastric emptying has been shown to be 75% and 86%,

respectively (Ziegler, Schadewaldt et al. 1996). In addition, because of low

intraindividual variability, breath tests may be best applied to tracking gastric

emptying during therapeutic interventions. It is noteworthy that the 13C-

octanoate exhalation times correlate with gastric emptying but do not represent

real gastric emptying times and the results may depend on intact liver and

pulmonary function. 13C-Spirulina platensis breath test (Vella, Lee et al. 2002)

is a slight variation to 13C-octanoate breath test, has recently been validated

and deemed as reproducible as scintigraphy (Szarka, Camilleri et al. 2008).

Ultrasonography (Holt, Cervantes et al. 1986) allows real-time changes in

gastric motility and gastric emptying including transpyloric flow of liquid gastric

contents, and accommodation in the proximal stomach (Berstad, Hausken et al.

1996; Kim, Myung et al. 2000; Parkman, Hasler et al. 2004). Ultrasound

measurements of gastric emptying is well validated in healthy subjects and

showed a high correlation to scintigraphy in quantifying emptying including

liquids of both low and high nutrients (Benini, Sembenini et al. 1999). Although

ultrasound methodologies are relatively low cost and will not expose patients to

radioactive materials, their performance is operator-dependent and these

studies are reliable only for measurement of liquid emptying rates (Parkman,

Hasler et al. 2004). Also, it does not permit determination of the phase of the

meal that is being emptied from the stomach. Recently, 3D ultrasonography has

been shown to provide a valid measure of gastric emptying of liquid meals in

healthy subjects (Gentilcore, Hausken et al. 2006). Real time ultrasound

measurement of gastric antral cross sectional area (ACSA) was shown to be

58


reproducible in the assessment of gastric emptying in preterm infants (Ewer,

Durbin et al. 1994). The technique is based on serial measurements of ACSA

as it fills and empties during and after a feed. Ultrasound and scintigraphic

techniques showed 90% concordance in subjects varying from 15 day infants to

10 year old children. The 10% discordant results were related to overlapping of

duodenum and stomach during scintigraphy and shadowing of the gastric

antrum by air during ultrasonography (Gomes, Hornoy et al. 2003).

Magnetic resonance imaging is another non-invasive test that allows for

measurement of gastric motility and emptying and can differentiate among an

ingested meal, solid and liquid phases, gastric secretion, and air (Schwizer,

Maecke et al. 1992; Feinle, Kunz et al. 1999). This technique strongly correlates

with gastric emptying profiles obtained by scintigraphy for liquid and solid meals

(Schwizer, Maecke et al. 1992; Feinle, Kunz et al. 1999). Although magnetic

resonance imaging requires no exposure to radioactive materials, the procedure

cost is high and it requires significant time for interpretation of test results

(Parkman, Hasler et al. 2004).

The appearance of paracetamol in the systemic circulation is an indirect method

of determining the rate of gastric emptying. Because paracetomol after oral

administration is not absorbed from the stomach but is rapidly absorbed from

the small intestine, the area under the serum concentration curve (AUC) is

determined by the rate of gastric emptying (Heading, Nimmo et al. 1973).

Paracetamol absorption test (van Wyk, Sommers et al. 1995) is simple,

reproducible, and accurate bedside tool (Sanaka, Koike et al. 1997; Mayer,

Durward et al. 2002) and may be useful for screening purposes in large

populations. It is commonly used to measure gastric emptying in critically ill

adults (Heyland, Tougas et al. 1996; Heyland, Tougas et al. 1996) and even in

pregnant women (Stanley, Magides et al. 1995). The safety and reliability of the

test in neonatal population is not known.

The newest technology in measuring gastric emptying is a technique involving a

non-digestible capsule or SmartPill that can record luminal pH, temperature and

pressure during gastrointestinal transit, thus allowing measurement of gastric

emptying times (Kuo, McCallum et al. 2008). The data for gastric emptying

times obtained with the SmartPill correlated well with data obtained with

59


scintigraphy. The technology does not involve any radioactivity and has been

tested in healthy individuals.

In summary, a number of different methods are available for the measurement

of gastric emptying in humans, and all have some advantages and

disadvantages. The method of choice will depend on whether solid or liquid

meals are to be studied, level of precision required, degree of invasiveness that

the subject will tolerate, ethical considerations, and the facilities available.

4.6 Methods and Factors Influencing Gastric Emptying in Neonates

Until recently, few techniques to measure gastric emptying in neonates for

clinical and research purposes were non-invasive, safe and reliable. Most data

in preterm infants have been obtained using single aspiration technique

(Husband and Husband 1969; Husband, Husband et al. 1970) or modified

double sampling marker dilution technique (George 1968). Although the former

method assessed the pattern of gastric emptying for an individual feed, the

latter method however required repeated aspiration of gastric contents which

may be unphysiological and not well tolerated in preterm infants (Ewer, Durbin

et al. 1994) and thus obsolete due to availability of newer non-invasive

techniques. Radionuclide methods using radioactive low-dose markers such as 99mTc sulfur colloid or with 99mTc phytate (Reddy, Deorari et al. 2000) mixed with

milk is a reliable and reproducible technique to assess gastric emptying in

infants. However the costs of a gamma camera and exposure to ionising

radiation are major disadvantages in neonates and children.

Various factors influence gastric emptying in neonates. Using marker dilution

technique in newborns, Husband et al showed that 5% glucose and starch

emptied faster than 10% glucose and glucose solution of comparable energy

content (Husband and Husband 1969; Husband, Husband et al. 1970). Cavell

using the same technique in healthy preterm infants showed that half the

human milk and formula left the stomach in 25 and 51 minutes respectively

(Cavell 1979). Other researchers also used marker dilution technique and

demonstrated delayed gastric emptying to increased calorific density (Siegel,

Lebenthal et al. 1984) and carbohydrate composition of feeds (Siegel, Krantz et

al. 1985) and presence of respiratory distress syndrome (Victor 1975) and 60


congenital heart disease (Cavell 1981) in preterm infants. Feed osmolality

(Hunt, Antonson et al. 1982; Siegel, Lebenthal et al. 1982) and the temperature

of feeds (Costalos, Ross et al. 1979; Blumenthal, Lealman et al. 1980) did not

appear to influence gastric emptying. Whereas the effects of fat content of feed

(Sidebottom, Curran et al. 1983; Siegel, Krantz et al. 1985), phototherapy

treatment (Blumenthal, Lealman et al. 1980; Costalos, Russell et al. 1984), and

the posture of the infant (Victor 1975; Blumenthal and Pildes 1979) on gastric

emptying was less clear.

Using ultrasonography, Ewer et al confirmed Cavell’s finding that breast milk

emptied faster than formula milk in preterm infants (Ewer, Durbin et al. 1994).

The most likely reason for this observation was attributed to gut peptide content

and composition of breast milk (Lucas, Sarson et al. 1980). ElHennawy et al

failed to show change in gastric emptying rate, antroduodenal motor

contractions and gastrointestinal transit times to intragastric erythromycin in

feed intolerant preterm infants (ElHennawy, Sparks et al. 2003).

The gastric emptying rate measured by 13C-octanoate breath test in preterm

neonates was shown to be independent of milk amount (Pozler, Neumann et al.

2003). Similarly, gastric emptying was not affected by changes in feed

osmolarity, volume, or energy density individually in preterm infants in a study

by Ramirez et al. However reducing osmolality combined with increasing feed

volume increased gastric emptying (Ramirez, Wong et al. 2006). Notably right

lateral position increased gastric emptying in preterm infants (Omari, Rommel et

al. 2004).

Thus ultrasonography (Ewer, Durbin et al. 1994), (13)C-octanoic acid breath

test (Pozler, Neumann et al. 2003) and epigastric impedance (Savino, Cresi et

al. 2004) have gained popularity to monitor gastric emptying in premature

infants and children both in research and clinical settings mainly due to the non-

invasive nature of the technique and correlation with well-known techniques.

The conflicting data on gastric emptying in new-born and preterm infants may

be due to different methods of analysis and feed types used.

61

Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants

Chapter 5- Literature Review: Feed Intolerance in Preterm Infants

5.1 Scale of the Problem

Provision of adequate nutrition for growth and development is a cornerstone of

the care of preterm infants. However feed intolerance is a common problem and

a major hurdle in optimising enteral nutrition in preterm neonates undergoing

intensive care. Attempts to reach full enteral feeds in these infants are

frequently limited by increased residual feeds in the stomach with or without

other manifestations such as abdominal distension, bile stained aspirates, or

lack of stooling. These manifestations in preterm infants are not exclusive to

feed intolerance and can be experienced in other common clinical conditions

such as infection and necrotising enterocolitis. Additionally the symptoms and

signs of feed intolerance are difficult to quantify accurately and are prone to

subjective variability. Thus the quoted incidence of feed intolerance in literature

varies from up to 50% of ELBW infants (ElHennawy, Sparks et al. 2003) to 67%

of VLBW infants (Gross 1983; Gross and Slagle 1993; Premji, Wilson et al.

1997). Under nutrition at critical stages of development may lead to short

stature, organ growth failure, and later adverse behavioural and cognitive

outcomes (Hay 2008). Therefore establishing full enteral feeds quickly in high

risk preterm neonates has become a priority in neonatal intensive care (Patole,

Rao et al. 2005).

5.2 Physiology of Gastric and Gastrointestinal Motility in Preterm Infants

Mature gastrointestinal motility and migrating motor complexes depends on a

complex interaction of neural and hormonal control mechanisms which are

influenced by gestational age. By 34 weeks of gestation these MMCs are of

variable length, with clear intervals and being increasingly propagated and

except for the considerably shorter periodicity of 20-40 minutes, the MMC has

adult characteristics at term gestation including interruption by feeding (Bryant,

Buchan et al. 1982; Berseth 1989).

62


In new-born’s at birth cutaneous electrogastrography (EGG) (Abell and

Malagelada 1988; Familoni, Bowes et al. 1991) demonstrated absence of

normal slow waves and maturation process regulated by enteral feeding (Chen,

Co et al. 1997; Liang, Co et al. 1998; Riezzo, Indrio et al. 2000). A recent study

has shown mature EGG as early as 34 weeks gestation (Riezzo, Indrio et al.

2009). Fasting antral motor activity however is comparable in preterm and term

neonates and the degree of antroduodenal coordination improves

simultaneously towards term (Ittmann, Amarnath et al. 1992).

5.3 Definition of Feed Intolerance

There are no universally agreed-upon criteria to define feed intolerance in

preterm infants. Feed intolerance in preterm infants usually manifests as

residual feeds in the stomach prior to the next scheduled feed and may be

associated with vomiting, regurgitation, bile stained aspirates and abdominal

distension (Akintorin, Kamat et al. 1997; Kliegman 1999; Rayyis, Ambalavanan

et al. 1999; Dollberg, Kuint et al. 2000; Jadcherla and Kliegman 2002; Mihatsch,

von Schoenaich et al. 2002). Of these, most clinicians consider vomiting,

abdominal distension, and increased gastric residual volume as the ‘triad’ for

defining feed intolerance. The literature definitions of feed intolerance in preterm

infants mostly include an assessment of gastric residual volume (GRV) probably

because it may indicate rapidity of gastric emptying. Furthermore majority of

preterm and VLBW infants admitted to the neonatal unit need gastric tube

placement and it is common practice to check the gastric residuals prior to the

next scheduled feed. The significance of these GRV however may depend upon

various factors (both systemic and local) including feeding method (continuous

or bolus), feeding volume, feeding type (breast milk versus formula), feeding

duration and birth weight and gestation of the infant. GRV are also dependent

upon the technique of aspiration and body positions (Geraldo V 1997) and may

be subject to variability. It is therefore not surprising that the definition of feed

intolerance in preterm infants is varied in literature with several dynamic and

fixed definitions based on assessment of prefeed residual volumes, the colour

of these GRVs, and associated clinical manifestations such as abdominal

distension, vomiting, blood in stool, and apnoea with bradycardia (Akintorin,

Kamat et al. 1997; Rayyis, Ambalavanan et al. 1999; Dollberg, Kuint et al. 2000;

63


Mihatsch, von Schoenaich et al. 2002). Dynamic definitions of feed intolerance

include GRV of > 20% (Dollberg, Kuint et al. 2000) of the previous 4 hour feed

volume, > 50% (Akintorin, Kamat et al. 1997; Schanler, Shulman et al. 1999) or

20% (Rayyis, Ambalavanan et al. 1999; Dollberg, Kuint et al. 2000) of a 3 hour

bolus feed volume or > 30% (Currao, Cox et al. 1988) or 20% (Wang, Hung et

al. 1997; Dollberg, Kuint et al. 2000) of a 2 hour bolus feed volume, or as a total

GRV > 10% of the previous day feed volume (Slagle and Gross 1988). Fixed

definitions of feed intolerance include GRV > 2ml of undigested formula

(Silvestre, Morbach et al. 1996), > 2ml or any bilious coloured aspirate (Dunn,

Hulman et al. 1988), or > 3 ml/kg or any green coloured aspirate (Meetze,

Valentine et al. 1992).

Critical assessment of these publications revealed small sample size and

variable birth weights and gestations, arbitrarily developed definitions based on

consensus among the study staff and the attending neonatologists (Akintorin,

Kamat et al. 1997), use of variable dilutions of formula feeds and hypocaloric

feeds (Currao, Cox et al. 1988; Silvestre, Morbach et al. 1996), variable feed

types, duration, and method of feeding (all studies), and prolonged period of nil

by mouth and TPN use with variable feed advancement rate (Slagle and Gross

1988). All the studies used excessive GRV, either determined by % of the

previous feeding volume or an absolute volume, as a proxy for feeding

intolerance and the authors provided no physiological basis or demonstrated

correlation with the known measures of gastric emptying or gastrointestinal

motility or relevance to clinical outcomes. Even when definition seemed

comprehensive the clinical significance of each of the criteria for feeding

intolerance was not determined (Mihatsch, von Schoenaich et al 2002; Dollberg,

Kuint et al 2000; Rayyis, Ambalavanan et al 1999; Akintorin, Kamat et al 1997).

A study which did attempt to investigate the impact of high GRV and colour in a

cohort of ELBW infants showed that threshold GRV of 2ml/3ml and green,

milky, or clear GRV had no significant impact on feeding volume on day 14

(Mihatsch, von Schoenaich et al. 2002). However, the median feeding volume

of 103 (0-166) ml/kg birth weight on day 14 in the study was far below volumes

aimed for in current practice which may have contributed to gut hypomotility.

Additionally, the results cannot be generalised for infants fed at an incremental

rate > 12ml/kg/day or with a different formula.

64


On the other hand, threshold or % prefeed GRV was assessed against

objectives measures of gastric emptying in critically ill children and adults

(Mayer, Durward et al. 2002; ElHennawy, Sparks et al. 2003; Gomes, Hornoy et

al. 2003; Pozler, Neumann et al. 2003). Using GRV > 125% of previous 4 hour

feed volume or > 150 ml as indicative of feed intolerance, Mayer et al showed

correlation with delayed gastric emptying measured by paracetamol absorption

test. However the study sample sizes were small (Mayer, Durward et al. 2002;

ElHennawy, Sparks et al. 2003; Gomes, Hornoy et al. 2003; Pozler, Neumann

et al. 2003) and the study cohort was of wide ranging age and weight (Mayer,

Durward et al. 2002).

Thus the definition of feed intolerance in the literature varies and is arbitrarily

based mainly on an assessment of prefeed gastric residual volumes, the colour

of these GRVs, and the associated clinical manifestation. Although a high pre-

prandial GRV or green GR are generally regarded as significant markers of feed

intolerance, its clinical significance is yet to be determined. It is unclear if they

are predictive of serious disease such as necrotising enterocolitis, a delayed

time to achieve full enteric alimentation, or developmental physiologic

expectations when feeding the preterm infants.

5.4 Patho-Physiology of Feed Intolerance

Feed intolerance is rarely observed in well term infants most likely due to

controlled gastrointestinal motility which matures as gestational age increases

(Tomomasa, Itoh et al. 1985; Berseth 1989; Berseth 1990; Berseth 1996). The

gastrointestinal tract has formed and has completed its rotation back into the

abdominal cavity by 10 weeks of gestation. The fetus can swallow amniotic fluid

by 16 weeks and gastrointestinal motor activity is present before 24 weeks but

organized peristalsis is not established until 29-30 weeks. At 32-34 weeks

coordinated sucking and swallowing develops and by term the fetus swallows

about 150 ml/kg/day of amniotic fluid containing carbohydrates, protein, fat,

electrolytes, immunoglobulin’s and growth factors which plays an important role

in development of gastrointestinal function. Preterm birth interrupts this

development. In the postnatal period the lack of enteric intake leads to

decreased circulating gut peptides, slower enterocyte turnover and nutrient

transport, decreased bile acid secretion; even when nutrients are provided

65


parenterally. There is increased susceptibility to infection due to impaired barrier

function by intestinal epithelium, lack of colonization by normal commensal flora

and colonization by pathogenic organisms. Physiologic studies have shown a

lack of the propagative phase III of the MMC in the duodenum of preterm infants

less than 32 weeks gestational age (al Tawil and Berseth 1996). Additionally,

very immature infants also have poor gastroduodenal coordination and

excessive quiescence in motor activity (Jadcherla and Kliegman 2002) and non-

propagated contractions of the gastrointestinal tract. Furthermore, control

systems of gastrointestinal motility are immature in preterm animals because

interstitial cells of Cajal (ICC), nerve, and muscle control systems are not fully

differentiated (Daniel and Wang 1999; Jadcherla and Kliegman 2002).

Therefore immaturity of the gastrointestinal tract due to premature birth has

been proposed as the main cause of feed intolerance in preterm infants (Ng and

Shah 2008).

Prefeed gastric residual volumes (GRV) and bilious residuals may reflect poor

gastric emptying, duodeno-gastric reflux, and gastroduodenal hypomotility

(Jadcherla and Kliegman 2002). Although not proven, it is likely that colonic

motility is also slow and delayed in VLBW infants as supported by delayed

stooling pattern, and emesis in bowel obstruction or functional ileus (Jadcherla

and Kliegman 2002).

Although historically hypoglycaemia has been the clinical concern,

hyperglycaemia is also a well-documented problem in the perinatal period in

VLBW infants. This hyperglycaemia is a marker of insulin resistance and

relative insulin deficiency and may reflect the prolonged catabolism observed in

VLBW infants (Beardsall and Dunger 2007). Intrauterine growth retardation is

also associated with increased fasting insulin levels and insulin resistance

(Mericq 2006; Mericq 2006). These infants are unable to suppress glucose

production within a large range of glucose and insulin concentrations, insulin

secretory response is inappropriate, insulin processing is immature and there is

an increased ratio of the glucose transporters Glut-1/Glut-2 in fetal tissues,

which limits sensitivity and hepatocyte reaction to increments in glucose/insulin

concentration during hyperglycaemia (Mericq 2006). Although unproven, it is

plausible that relative insulin resistance in preterm VLBW may induce

66


hyperamylinemia which may be responsible for feed intolerance by delaying

gastric emptying.

In conclusion, prematurity may predispose to feed intolerance due to immaturity

of gastrointestinal motor activity (Bisset, Watt et al. 1988; Berseth 1989; Reddy,

Deorari et al. 2000; Patole, Rao et al. 2005), and delayed gastric emptying and

whole intestinal transit (Gupta and Brans 1978; Cavell 1982; Ernst, Rickard et

al. 1989). Relative insulin resistance and hyperamylinaemia may also be

responsible for feed intolerance in these neonates which is an emerging theory

and hypothesis and subject of investigation of this dissertation.

5.5 Management of Feed Intolerance

A range of management strategies currently exists for preventing and/or

minimizing feed intolerance in preterm neonates reflecting the dilemma

surrounding the definition and significance of signs of feed intolerance due to

gastrointestinal hypomotility on one end and the fear of NEC on the other end of

the spectrum. Additionally, the relationship of feed intolerance and gastro-

esophageal reflux is unclear and therefore often used interchangeably.

Furthermore due to the absence of robust diagnostic tests authors have

resorted to surrogate markers of gastrointestinal motility such as gastric

residuals and time to reach full enteral feeds to assess efficacy of therapeutic

interventions. The following paragraphs provide critical appraisal of current

literature on the management of feed intolerance in preterm infants.

Prokinetic agents are often used in an attempt to overcome the functional

immaturity by speeding up gastric emptying and increasing small intestinal

motility. Until July 2000, cisapride was the mainstay of therapy in preterm

infants with feed intolerance and/or gastro-oesophageal reflux. Due to reports of

prolongation of the QT interval with occasional fatal cardiac arrythmias and

sudden death, cisapride was taken off the market and restricted to a limited

access programme supervised by a paediatric gastroenterologist (Gounaris,

Costalos et al. 2010; Maclennan, Augood et al. 2010). Metoclopromide and

domperidone, dopamine antagonists, started to be widely prescribed soon after

(Clark, Bloom et al. 2006; Hibbs and Lorch 2006) although its efficacy was in

doubt. A systematic review of metoclopramide use did not show benefit but

67


reported the possibility of adverse effects such as irritability, dystonic reactions,

oculogyric crisis, drowsiness, apnea and emesis (Putnam, Orenstein et al.

1992; Hibbs and Lorch 2006). There have been no adequately powered

controlled trials of metoclopramide use in preterm neonate. Similarly efficacy

data of domperidone in preterm infants is lacking. The few studies with

domperidone are old and methodologically poor. In contrast the side effects

profile is similar to those of cisapride with prolonged QT interval, sudden death

and theoretical risk of neurological adverse effects. A recent small crossover

study of domperidone use in 22 preterm infants showed faster gastric emptying

rate measured by ultrasound antral cross sectional area half-value time (in

minutes) n the domperidone group (Gounaris, Costalos et al. 2010). However

the study did not report on important clinical correlates of efficacy and long term

safety. There have been no controlled trials of domperidone use in preterm

infants. A plethora of publications soon followed on erythromycin use in the

prevention and treatment of feeding intolerance in preterm infants. Erythromycin

is a motilin agonist that exerts its prokinetic effect by stimulating propagative

contractile activity in the interdigestive phase. Although previous studies

demonstrated variable efficacy, a meta-analysis (three prevention and seven

treatment studies) concluded that there is insufficient evidence to recommend

the use of erythromycin in low or high doses for preterm infants with or at risk of

feeding intolerance (Ng and Shah 2008). Furthermore the same author

concluded that although high dose regimens as a rescue therapy for

established gastrointestinal dysmotility have consistently shown clinical benefit,

further research is warranted to justify its widespread use due to the theoretical

risks of prolonged antibiotic use (Ng et al 2011) and hypertrophic pyloric

stenosis.

Clinical trials followed to investigate interventions to promote intestinal

maturation and enhance feeding tolerance and decrease time to reach full

enteral feeding independent of parenteral nutrition. Systematic review of nine

trials did not provide any evidence that early trophic feeding affected feed

tolerance or growth rates in VLBW infants (Bombell and McGuire 2009). Trials

of supplementation of breast milk or formula milk with enzyme or hormone to

improve short and long term effects of feed intolerance were also conducted. A

randomized trial did not provide evidence of significant benefit to preterm infants

68


from adding lactase to their feeds (Tan-Dy and Ohlsson 2005). A study of eight

preterm infants who were given 4 U/kg/day insulin enterally from 4 to 28 days of

age showed higher lactase activity and less feed intolerance (30% shorter time

to full enteral feeds; fewer gastric residuals per infant) than the matched

historical controls (Shulman 2002). However further large controlled studies are

warranted before its use in preterm population.

Recent literature abounds in the study of probiotics, prebiotics, or synbiotics in

preterm infants primarily looking to improve growth and reduce mortality due to

NEC and infections. Some of these trials reported on their effect on feed

tolerance and/or days to reach full enteral feeds as a surrogate marker of feed

tolerance. L. sporogenes supplementation at the dose of 350,000,000 c.f.u/day

was not effective in reducing the incidence of death or NEC in VLBW infants;

however, it could improve the feeding tolerance (Sari, Dizdar et al. 2011).

Although the time to reach full enteral feeds was significantly shorter in the early

enteral probiotic group than in controls (Deshpande, Rao et al. 2007) an

updated meta-analysis did not show this effect which was attributed to the

significant heterogeneity among the included trials (Deshpande, Rao et al.

2010). The results of ProPrems trial (Garland, Tobin et al. 2011)- a multi-centre,

randomised, double blinded, placebo controlled trial investigating

supplementing preterm infants born at < 32 weeks' gestation weighing < 1500

g, with a probiotic combination (Bifidobacterium infantis, Streptococcus

thermophilus and Bifidobacterium lactis) is awaited which in addition is

investigating time to reach full enteral feeds. Supplementation with

Bifidobacterium longum and Lactobacillus rhamnosus may improve the

gastrointestinal tolerance to enteral feeding in infants weighing >1000 g (Rouge,

Piloquet et al. 2009). Enteral supplementation of a prebiotic mixture consisting

of neutral and acidic oligosaccharides decreased stool viscosity and stool pH

with a trend towards increased stool frequency without demonstrating benefit on

feed tolerance (Westerbeek, Hensgens et al. 2011). Evidence is still emerging

with regards to the strain type, supplementation to breast or formula and dose,

frequency and duration of prebiotics and probiotics in relation to its effects on

feed intolerance and other important clinical outcomes including safety data.

The ESPGHAN Committee on nutrition considers that the supplementation of

formula with probiotics and/or prebiotics does not raise safety concerns but

69


there is insufficient data to recommend routine supplementation and therefore is

an important field of research (Braegger, Chmielewska et al. 2011).

In clinical practice, failure of pharmacological intervention may necessitate trial

of transpyloric feeding as a last resort. However transpyloric route cannot be

recommended for preterm infants (McGuire and McEwan 2007) due to adverse

effects such as greater incidence of gastrointestinal disturbances and some

evidence of increased mortality. The fallback position when all intervention fails

is to continue total parenteral nutrition until the feed intolerance settles.

In conclusion management of feed intolerance in preterm infants is varied

reflecting the dilemma surrounding the definition of feed intolerance. They

include pharmacological and biological interventions the efficacy of which is

variable. Further trials are awaited for evidence based management of this

common condition.

70


What was wrong with this section?

Chapter 5 – Literature Review: Gestational Diabetes Mellitus

Chapter 6- Literature Review: Gestational Diabetes Mellitus

6.1 Definition of Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) is defined as any degree of glucose

intolerance with onset or first recognition during pregnancy (Metzger and

Coustan 1998)


3-10% of all pregnancies (Hoffman, Nolan et al. 1998) experience GDM. This

number may rise significantly in the next decade as the current significantly

overweight paediatric population heads into their child-bearing years. GDM is

not only associated with substantial rates of maternal adverse effects but also

periconceptional, fetal, neonatal, and long term morbidities (Nold and Georgieff

2004). Long-term adverse health outcomes reported among infants born to

mothers with gestational diabetes (IMDM) include sustained impairment of

glucose tolerance (Silverman, Metzger et al. 1995), subsequent obesity (Petitt,

Bennett et al. 1985), and impaired intellectual achievement (Rizzo, Metzger et

al. 1997; Rizzo, Silverman et al. 1997).

6.3 Physiology of Insulin Secretion

Glucose and several amino acids stimulate insulin secretion under physiologic

conditions. The rate of insulin secretion is dependent on the ratio of ATP to ADP

within the pancreatic beta cell. An increase in the intracellular ATP/ADP ratio

activates membrane ATP-sensitive potassium-dependent channels (KATPs)

leading to closure of the potassium channel and depolarization of the cell

membrane allowing influx of calcium through voltage-gated calcium channel

resulting in insulin secretion. The rate of glucose entry into the beta cell

exceeds its oxidation rate which is facilitated by a glucose transporter. The first

rate-limiting step in glycolysis which is conversion of glucose to glucose-6-

phosphate by the enzyme glucokinase regulates the rate of glucose oxidation

and subsequent insulin secretion.

71


6.4 Physiology of Gestational Diabetes Mellitus

Pregnancy is a physiologic condition in which maternal glucose is the main

energy substrate for intrauterine growth (Combs, Gunderson et al. 1992). Early

in pregnancy, increased levels of both oestrogen and progesterone act as

counter-regulatory hormones affecting glucose homeostasis. As pregnancy

progresses, human placental lactogen released by the syncytiotrophoblast

leads to lipolysis and the subsequent release of glycerol and fatty acids which

reduces maternal use of glucose and amino acid, thus preserving these

substrates for the fetus. As placental growth and pregnancy continue, maternal

insulin needs increase by 30% due to the release of increasing amounts of

counter-insulin factors. Glucose and amino acids readily traverse the placental

membrane while insulin is unable to cross materno-fetal barrier in physiological

concentrations. In pregnancies complicated by diabetes, high concentrations of

glucose, particularly after a meal, give rise to an increased nutrient transfer to

the fetus. This results in beta-cell hyperplasia, stimulating an increased release

of insulin which adversely influences birth weight with its pregnancy

complications (Jovanovic-Peterson, Peterson et al. 1991; Combs, Gunderson et

al. 1992).

6.5 Strategies to Prevent Fetal and Neonatal Hyperinsulinaemia

The Diabetes Prevention Program randomised clinical trial showed reduction of

diabetes incidence in high-risk adults by 58% with intensive lifestyle intervention

and by 31% with metformin (Knowler, Barrett-Connor et al. 2002) the benefits of

which persisted for at least 10 years (Knowler, Fowler et al. 2009). A

randomised trial of dietary advice, blood glucose monitoring, and insulin therapy

or routine care in women between 24 and 34 weeks' gestation who had

gestational diabetes showed significantly lower serious perinatal complications

such as death, shoulder dystocia, bone fracture, and nerve palsy (Crowther,

Hiller et al. 2005). Continuous glucose monitoring during pregnancy is

associated with improved glycaemic control in the third trimester, lower birth

weight, and reduced risk of macrosomia (Murphy, Rayman et al. 2008).

Compared to normal pregnant women in the low-glycaemic index group, women

in the moderate-to-high glycaemic index group gave birth to infants who were

72


heavier and had a higher birth centile, a higher ponderal index, and a higher

prevalence of large-for-gestational age (Moses, Luebcke et al. 2006). Low–

glycaemic index diet for women with gestational diabetes also halved the

number needing to use insulin, with no compromise of obstetric or fetal

outcomes (Moses, Barker et al. 2009). Both intensive lifestyle and metformin

were highly effective in delaying or preventing diabetes in women with impaired

glucose tolerance test and a history of gestational diabetes (Ratner, Christophi

et al. 2008). On the other hand, a multicentre randomised control trial of

treatment of mild gestational diabetes did not significantly reduce the frequency

of a composite outcome that included stillbirth or perinatal death and several

neonatal complications; however it did reduce the risks of fetal overgrowth,

shoulder dystocia, caesarean delivery, and hypertensive disorders (Landon,

Spong et al. 2009). No substantial maternal or neonatal outcome differences

were found with the use of glyburide or metformin compared with use of insulin

in women with GDM (Nicholson, Bolen et al. 2009). Fat mass, body mass index,

ponderal index, skinfold sum, arm fat area, and cord concentrations of

biomarkers were not different in infants of women treated for gestational

diabetes with glyburide or insulin (Lain, Garabedian et al. 2009). In women with

gestational diabetes at 20-33 weeks of gestation, metformin (alone or with

supplemental insulin) as compared with insulin was not associated with

increased perinatal complications (Rowan, Hague et al. 2008). Glucose control

in women with gestational diabetes mellitus treated with metformin and/or

insulin was strongly related to outcomes. The lowest risk of complications was

seen when fasting capillary glucose was <4.9 mmol/l (mean ± SD 4.6 ± 0.3

mmol/l) compared with 4.9–5.3 mmol/l or higher and when 2-h postprandial

glucose was 5.9–6.4 mmol/l (6.2 ± 0.2 mmol/l) or lower (Rowan, Gao et al.

2010). Although treatment of GDM substantially reduced macrosomia at birth, it

did not result in a change in BMI at age 4-5 years (Gillman, Oakey et al. 2010).

Thus strict control of blood sugar during pregnancy in GDM prevents fetal and

neonatal hyperinsulinaemia and related complications but may not help in

reversing intrauterine programming resulting in future adverse outcomes.

73

Chapter 6 – Literature Review: IMDM

Chapter 7- Literature Review: IMDM


Currently, 3-10% of pregnancies are affected by abnormal glucose regulation

and control. Of these cases, 80-88% are related to gestational diabetes

mellitus. Of mothers with preexisting diabetes, 35% have been found to have

type 1 diabetes mellitus, and 65% have been found to have type 2 diabetes

mellitus.

Notably infants born to mothers whose pregnancy was complicated by diabetes

(gestational and types 1 and 2 diabetes) have experienced nearly 30-fold

decrease in morbidity and mortality rates since the development of specialized

maternal, fetal, and neonatal care for women with diabetes and their offspring.

However adverse neonatal outcomes are still 3-9 times greater in infants born

to mothers with preexisting types 1 and 2 diabetes compared to those of

nondiabetic mothers (Yang, Cummings et al. 2006). In mothers with insulin-

dependent diabetes, the stillbirth and perinatal mortality rate is 5 times the rate

in the general population, and neonatal and infant mortality rates are 15

times and 3 times the rate in the general population, respectively. Major

congenital malformations are found in 5-9% of affected infants and account for

30-50% of perinatal deaths of infants of mothers with gestational diabetes.

IMDM (gestational, types 1 and 2 diabetes) are 3 times more likely to be born

by caesarean delivery, twice as likely to suffer serious birth injury, and 4 times

as likely to be admitted to a neonatal intensive care unit. In addition they are at

increased risk of morbidity and mortality related to the abnormalities in fetal

growth (either overgrowth or undergrowth), birth injuries, hypoglycaemia,

hypocalcaemia, feed intolerance, polycythaemia and thrombocytopenia,

jaundice, prematurity, respiratory distress syndrome, asymmetric septal

hypertrophy, congenital malformations and intrapartum asphyxia.

7.2 Physiology of Fetal Hyperinsulaemia

The physiology of fetal hyperinsulinaemia in mothers with gestational diabetes

is explained by Pedersen hypothesis which states that maternal hyperglycemia

results in fetal hyperglycemia as glucose readily traverses the placenta

74


(Pedersen 1977). Before 20 weeks' gestation, fetal islet cells are not capable of

responsive insulin secretion. After 20 weeks’ gestation, the fetus has a

functioning pancreas and is responsible for its own glucose homeostasis.

Therefore fetal hyperglycemia induces fetal pancreatic beta-cell hyperplasia

with resultant hyperinsulinaemia. In addition, proinsulin hormones such as

insulin like growth factor-1 [IGF-1] and insulin like growth factor–binding protein-

3 [IGFBP-3] act as growth factors that, in the presence of increased fetal amino

acids result in fetal macrosomia noted as early as 24 weeks' gestation. The

combination of hyperglycaemia and insulin increases fetal fat and glycogen

stores, resulting in weight increases marked by hepatosplenomegaly and

cardiomegaly. Chronic fetal hyperglycaemia and hyperinsulinaemia increase the

fetal basal metabolic rate and oxygen consumption, leading to a relative hypoxic

state. The fetus responds by increasing oxygen-carrying capacity through

increased erythropoietin production, potentially leading to polycythaemia. Prior

to birth, elevated insulin levels may inhibit the maturational effect of cortisol on

the lung, including the production of surfactant from type 2 pneumocytes. This

puts the fetus at risk for developing respiratory distress syndrome after birth at a

gestational age when normal lung function is expected. Withdrawal of the

transplacental supply of glucose after birth leads to a precipitous drop in the

concentration of glucose; many require infusion of large quantities of glucose to

maintain normal blood glucose levels.

7.3 Feed Intolerance in IMDM

Feed intolerance occurs in up to 37% of IMDM (Kitzmiller, Cloherty et al. 1978)

and a key reason for prolongation of hospital stay and parent-infant separation

(Lee-Parritz 2004). It is often present in the absence of prematurity, respiratory

distress, and polyhydramnios, and the incidence is similar among large-for-

gestational-age and appropriate-for-gestational-age infants (Lee-Parritz 2004).

This frequently observed problem of feed intolerance in IMDM is overshadowed

by previously described morbidities (section 5.1) and therefore not widely

reported. The management of feed intolerance in IMDM relies on tube feeding

with or without intravenous dextrose solution until the infant establishes to

demand oral feeds. The conservative approach to management may be due to

75


the fact that the aetiology and pathophysiology of feed intolerance in IMDM is

yet to be determined.

7.4 Proposed Patho-Physiology of Feed intolerance in IMDM

Elevation of plasma amylin levels has been described in pregnant women with

gestational diabetes mellitus (Ludvik, Kautzky-Willer et al. 1997). Insulin

resistance and hyperinsulinaemia probably due to hyperleptinemia has been

demonstrated in IMDMs (Vela-Huerta, San Vicente-Santoscoy et al. 2008). It is

plausible that this hyperinsulinaemia and insulin resistance may also predispose

to hyperamylinaemia which may have a role to play in the pathogenesis of feed

intolerance in IMDMs. This plausible mechanism and hypothesis is yet to be

proven and a subject of investigation of this dissertation.

76

Chapter 7 – Overview of Project Plan

Chapter 8- Overview of Project Plan

8.1 General Design

The overall project plan (Figure 16) was to test the hypothesis that amylin would

be raised in neonates with clinical signs of feed intolerance.

Figure 16. Project plan Gantt chart

77


As there was no published information regarding amylin and its concentrations

in the newborn, the first part of the study was to establish normative values of

amylin at birth and postnatal period in term and well preterm infants.

Measurement of amylin levels at more than one time point during the first weeks

of life following birth would help in the assessment of its trend and decay with

time. It was also essential to identify two or more time points after birth that

would allow for blood collection to be undertaken without causing discomfort

and pain to the study participants. Umbilical cord blood at birth and Guthrie

blood on postnatal day 5 were identified as practically feasible time points

requiring minimal or no intervention in the mother and the baby. This approach

required collection of blood both in the hospital (from umbilical cords and at the

time of Guthrie of in-house babies) and community setting (at the time of

Guthrie of those babies discharged home from the hospital before day 5). A

pilot study of community blood collection identified unacceptable compromises

to the sampling process and maintenance of cold chain. Therefore the study

plan was amended to abandon community blood collections. Umbilical cord

blood could be collected from discarded cords immediately after birth without

causing pain or discomfort to the mother and the baby. The blood for Guthrie

test is collected from a heel-prick sample and is offered to all infants in the UK

with the aim of screening for up to five disorders typically on day 5 after birth.

Thus it was feasible to collect blood for amylin during routine Guthrie test

minimizing pain and discomfort. A third time point in the 2nd week of postnatal

life although necessary was deemed impractical due to logistical reasons

around blood sampling and ethical concerns of blood collection purely for study

purposes.

Originally, the second part of the project was to determine amylin levels in feed

intolerant infants of mothers whose pregnancy was complicated by diabetes

mellitus. To address the hypothesis that amylin levels are raised in IMDM with

feed intolerance, a case-control study of feed intolerant and feed tolerant IMDM

was considered. However an audit showed that not all IMDM were admitted to

the neonatal intensive care or special care baby unit. Additionally, not all

admitted IMDM required gastric tube placement as part of their routine care.

Insertion of gastric tube to measure prefeed gastric residual volumes was

essential to define feed intolerance. This meant designing a new guideline for 78


the purpose of the study with inherent ethical issues. Therefore case-control

study design was abandoned in favor of a cohort or prospective observational

study of IMDM as an appropriate alternative methodology to address the

hypothesis. Thus if amylin levels were observed to be raised in IMDM then it

was not necessary to provide proof of concept as amylin was shown in previous

studies to be a potent inhibitor of gastric emptying. Measurement of amylin

levels at more than one time point during the first weeks following birth would

help analyse its trend and decay with time. It was therefore necessary to identify

at least two points in time following birth of the IMDM for the bloods to be

collected causing minimal discomfort and pain. Umbilical cord blood at birth and

Guthrie blood on postnatal day 5 were identified as practically feasible time

points requiring minimal or no intervention in the mother and the baby. A third

time point for opportunistic blood collection was also agreed when IMDM were

admitted to the neonatal intensive care unit or transitional care unit for IMDM

related problems and needing routine blood sampling.

The third part of the study was to test the hypothesis that amylin was raised in

preterm infants with feed intolerance. A cohort study of preterm infants with feed

intolerance alone may not conclusively address the hypothesis as measurement

of gastric emptying times in addition to gastric residual volumes would have to

be incorporated into the study design. Review of currently available tests to

measure gastric emptying times at both the study centres were not considered

to be practical, feasible and for some tests to be ethical: The expertise to

measure gastric emptying time using currently available tests such as

scintigraphy, ultrasonography, bio impedence, and 13C-octanoate breath test

was not available at Doncaster General Hospital. Similarly bioimpedence

technology and 13C-octanoate breath test were not available at Jessop Wing.

Additionally the research team felt that serial ultrasonographic assessment of

ACSA at Jessop Wing was not practical and feasible due to non-availability of

radiology expertise out of hours and on weekends. Although gamma camera

was available within the hospital building, the need to transfer the infants to the

radiology department for this to be undertaken would put strain on existing

resources in addition to a small risk of exposure to radiation. The question

regarding safety of paracetomol in preterm infants and need for serial blood

sampling for paracetomol absorption test would raise ethical concerns. A case-

79


control study was therefore adopted to answer the hypothesis. Preterm infants

less than 34 weeks are routinely admitted to the neonatal unit and most of these

infants require naso/orogastric tube placement and routine blood sampling as

part of their care.

8.2 General Study Configuration and Methodology

The study project involved observation of blood concentration of amylin in

neonatal population and therefore did not require randomisation or blinding. The

project was conducted simultaneously as three interrelated studies (Figure 17 &

Figure 18) for operational reasons and initially required a single-centre research

ethics application.

80


Figure 17. Study plan Gantt chart

81

PROJECT TITLE: AMYLIN PEPTIDE: AN ASSOCIATION WITH FEED INTOLERANCE IN PRETERM NEONATES AND IMDM

Study AAmylin levels at birth and postnatal day 5 in healthy preterm and term infants

Study BAmylin levels at birth and

postnatal day 5 in preterm and term IMDM

Study CAmylin levels in preterm

infants with feed intolerance

Single Centre Cohort Study Two Centre Case-Control Study

Labour ward, postnatal wards, Neonatal intensive care unit

Jessop wing, Royal Hallamshire Hospital

Neonatal intensive care unit Jessop wing, Royal Hallamshire Hospital

& Doncaster General Hospital

Prefeed GRV > 50% on 2 consecutive occasions

Inclusion24-36 weeks gestation admitted

to intensive care unit and requiring gastric tube feeding

ELISAAmylin levels

Insulin levels (where possible)

Yes=nTOL No=TOL

0.5-1.0 ml blood collected from discarded umbilical cord, during routine collection and Guthrie

Plasma stored at -70 degrees

Parental consent

0.5-1.0 mlblood collected within an

hour

0.5-1.0 mlblood collected at the next

routine collection

Inclusion≥ 24 week gestation infants born without major antenatal

or labour complications

Inclusion≥ 24 weeks gestation born to

mothers with diabetes mellitus (gestational, insulin or non-insulin dependent)

Matched for birthweight, gestation, postnatal age


Figure 18. General study methodology

82


These three studies were planned to complete in 12 months; 10 months for

recruitment and sample collection; and 2 months for completion of laboratory

and data analysis.

Study A was a single centre prospective observational study in well preterm and

term infants to determine normal ranges of amylin in umbilical cord (birth) and

Guthrie (postnatal day 5) blood.

Study B was a single centre prospective observational study of IMDM to

determine amylin levels in umbilical cord (birth), during routine blood sampling

(neonatal intensive care stay) of admitted babies and Guthrie (postnatal day 5).

Study C was a two centre case-control study to determine amylin levels in

preterm infants admitted to the neonatal intensive care unit who subsequently

developed feed intolerance and compared to amylin levels in birth weight,

gestation and postnatal day of life matched preterm infants without feed

intolerance. Some of these preterm infants were also recruited to study A. A

second centre was enrolled to ensure the study was conducted to time lines.

There were no modifications to the treatment of care received by the study

infants. Blood samples for the study were obtained only when blood was being

collected for routine clinical monitoring as this approach minimised discomfort to

the infant. Doctors, nurses and midwifes working in the neonatal unit and labour

ward performed routine blood sampling. Further training was imparted by the

research team to nurses, midwives and doctors in blood collection, processing

and storage. The transportation of the stored plasma to the laboratory was

performed under controlled conditions using liquid nitrogen to maintain the cold

chain.

8.3 Ethics Consideration

The study was conducted according to the standards of International

Conference on Harmonisation Good Clinical Practice Guideline, Research

Ethics Committee regulations, Royal Hallamshire Hospital’s policies and

procedures, and any applicable government regulations.

83


The study protocol and any amendments were submitted to a properly

constituted Ethics Committee (REC) for approval of the study conduct. The

REC requested minor amendments to the protocol (Appendix 4 & Appendix 5),

information leaflets (Appendix 6, Appendix 7 & Appendix 8), and adverts

(Appendix 11, Appendix 12 & Appendix 13) which were incorporated before

final approval in writing.

All parents/guardians were provided with an information sheet (Appendix 6,

Appendix 7, Appendix 8) describing the elements of this study, with sufficient

information for them to make an informed decision about their infant

participating in this study. The parents/guardians completed and signed a

consent form to indicate that they were giving consent for their infant to

participate (Appendix 9 & Appendix 10). It was made clear to the

parents/guardians that the study will offer no direct benefit to their own child.

Additionally they were free to withdraw their infant from the study at any time.

Participants were discontinued from study at any time for safety reasons as

judged by the investigator or clinician responsible for the child and when

parents withdrew consent.

The possible ethical considerations arising from the conduct of the study were:

(1) Blood collection from umbilical cord attached to the baby. An amendment

was accepted to collect the blood samples from the discarded umbilical cord to

prevent discomfort, bleeding and possible parent-baby separation.

(2) Consenting pregnant mothers in active labour. The ethics committee agreed

for the blood to be collected from umbilical cords immediately after birth of the

baby without prior consent. The study investigators may approach the mothers

after delivery for consent and collected blood sample discarded if consent is

refused. To circumvent this problem and to fulfil research governance

obligations, the research team decided to amend the study plan to recruit

mothers that were admitted for elective induction of labour or caesarean section

and umbilical cord blood collected after informed and written consent.

(3) The collection and possession of human tissue. The umbilical cords were

disposed immediately after blood collection according to hospital policy. The left

84


over plasma was disposed immediately after analysis.

(4) Blood collection from preterm infants and volume of blood. Blood samples

were obtained during times when blood was collected for routine clinical

monitoring. This approach minimised any extra discomfort to the infant. All

blood handling was performed as per guidance for the minimization of infection

risk to both participants and investigators. Not more than 2 ml of blood was

collected from any infant during the course of the study which was within the

safe limit.

8.4 Data Handling and Record Keeping

Confidentiality: Information about study subjects was kept confidential and

managed according to the requirements of the Data Protection Act, NHS

Caldicott Guardian, The Research Governance Framework for Health and

Social Care and Ethics Committee Approval. The chief investigator acted as

custodian of the data.

Each participant was assigned a unique study number, allocated for use on

data collection forms, other documentation and the electronic database. The

data collection form was the only form to contain identifying data and were

treated as confidential documents and held securely in accordance with the

relevant regulations. Access was restricted to those personnel approved by the

chief investigator. Blood samples were sent to the laboratory for analysis

labelled only with the unique identification number. Data were anonymised on a

computer database for the study by using the subject identification number only

and this anonymised data was used for the statistical analyses specific to this

study.

Data Collection Forms or Case Report Forms (Appendix 14, Appendix 15 &

Appendix 16) recorded participants’ biochemistry and demographics. Relevant

information was retrieved from an electronic patient database and patient notes.

Infant data included demographic variables such as birth weight, gestation, and

gender, and postnatal age, date of blood collection which were recorded for all

neonates. Maternal data included medical, antenatal, and labour history. For

Study C the neonatal data also included ventilation days, % GRV just prior to

amylin sampling, time from initiation to full enteral feeds (defined as 150 ml/kg 85


per day) and time to discharge. Additionally, inflammatory markers such as C-

reactive protein, white cell count, and neutrophil cell count were also recorded

nearest to the date of blood sampling. Incomplete datasets from subjects

discontinued from the study were not used.

Records Retention: Documents were stored in a locked cabinet with limited

access. Good Clinical Practice Guidelines stipulated that for patients involved in

clinical trials documentation should be retained for 25 years after conclusion of

treatment.

8.5 Benefits and Risks

There would be no health benefit to the participants themselves. The results

obtained from this study will help to establish normative amylin data in healthy

preterm and term infants. The study may also help unravel the pathophysiology

of feed intolerance which may lead to intervention studies in IMDM and preterm

infants. Blood was collected from discarded umbilical cords for study A and B.

No more than a total of 2.0 ml of blood was collected during the whole study

from each baby in study A, B & C. This is also an acceptable amount to take

from a pre-term baby (Directive 2008).

8.6 Funding

The investigators received £5000 from Jessop Wing Baby Fund to complete the

three studies. Three thousand and five hundred pounds was returned as only

£1500 was spent on the entire project.

The money was utilized for the following:

Amylin Kits £1440

Serum tubes £50

Technical staff time £0

Trasylol £10

86

Chapter 8 – Clinical Study A

Chapter 9- Clinical Study A: Umbilical & Guthrie Amylin in Preterm and Term Infants

9.1 Introduction

The aim of this study was to determine the normal range of serum amylin levels

in healthy preterm and term neonates at birth and postnatal day 5. This

approach required collection of blood both in the hospital (from umbilical cords

and at the time of Guthrie of in-house babies) and community setting (at the

time of Guthrie of those babies discharged home from the hospital before day

5). A pilot study of community blood collection however identified unacceptable

compromises to the sampling process and maintenance of cold chain.

Therefore the study aim was amended in favor of blood collection of those

infants who remain in house on postnatal day 5.

9.2 Methodology

9.2.1 Study Design & Setting

This single centre prospective observation study was conducted at the

University Hospitals of Sheffield NHS Trust, Jessop Wing neonatal and obstetric

departments, over a one year period. Analysis of the blood samples were

conducted in the Academic Unit of Reproductive and Developmental medicine

located at Jessop Wing, Royal Hallamshire Hospital.

9.2.2 Consent

The Infants were recruited once informed consent was obtained from their

parents/guardians. Infants whose parents refused consent were excluded from

the study.

9.2.3 Inclusion Criteria

All infants born at a gestational age greater than 24 weeks were eligible for the

study. We chose to use the term ‘healthy’ to identify well from unwell preterm

neonates who were deemed clinically stable by the attending clinical team.

Preterm infants between 35-37 weeks admitted to the neonatal unit, transitional

care ward or postnatal ward were establishing feeds or required prophylactic

87


antibiotics pending blood cultures. Preterm infants less than 35 weeks were

admitted to the neonatal unit primarily because of prematurity and low birth

weight were generally well and establishing feeds. Extremely premature

neonates (e.g. 24 weeks) who were ventilated or required continuous positive

airway pressure (CPAP) for poor respiratory effort were clinically stable, and

therefore considered to be ‘healthy’ for inclusion into the study.

9.2.4 Exclusion Criteria

1. The Infant born to mother whose pregnancy was complicated by

diabetes (including gestational, insulin dependent and non-insulin

dependent diabetes mellitus).

2. Deliveries associated with chorioamnionitis, or other significant antenatal

or labour complications.

3. Infants with congenital and chromosomal abnormality.

Rationale for exclusion criteria: Amylin has been shown to have both vasodilator

(Hall and Brain 1999) and anti-inflammatory (Clementi, Caruso et al. 1995)

properties. Since amylin shares 50% homology with calcitonin gene related

peptide, which is recognised to be raised in acute inflammatory states such as

trauma (Onuoha and Alpar 1999), sepsis (neonatal and adult) (Parida,

Schneider et al. 1998), and hypotensive shock (Joyce, Fiscus et al. 1990), it

may also share these properties. We therefore excluded infants born to mothers

with chorioamnionitis and those with significant antenatal and labour

complications. Similarly, amylin levels are influenced by diabetes and therefore

mothers whose pregnancy was complicated by diabetes were also excluded

from the study.

9.2.5 Study Procedure

The research team approached the pregnant mothers for consent in the

antenatal clinic and on admission to the labour ward prior to delivery. Mothers

admitted to the labour ward for elective induction of labour and elective

caesarean section were also approached. A pilot study highlighted concerns of

consenting pregnant women during busy antenatal clinics and in active labour.

Moreover it was acknowledged that pregnant mothers attending the antenatal 88


?sense


clinics may not remember the information provided in the information leaflets at

the time of admission to the labour ward. Therefore following feedback from

mothers and midwives, pregnant women admitted for elective induction of

labour and elective cesarean section were chosen as the target population.

These pregnant mothers were identified by the research team from admission

book and theatre lists in the antenatal ward and labour ward prior to elective

induction of labour and elective caesarean section. The study was discussed

with the prospective mothers and information leaflet provided. Written consent

was obtained from mothers for collection of blood from umbilical cord and

Guthrie bloods. Additionally, researchers also identified infants who were

staying in the hospital for maternal reasons and who required Guthrie blood on

day 5 and parents approached for consent.

Blood was collected immediately after birth from umbilical cord stump that was

clamped and separated from the baby. One to two ml of free flowing blood was

collected from the umbilical vein and/or artery, were possible. The postnatal day

5 Guthrie blood was collected from in-house infants on the neonatal unit and

postnatal wards and at home as a pilot for discharged babies. For in-house

infants in the postnatal wards, special care baby unit and transitional care

wards, 0.5-1.0 ml blood sample was collected from heel prick at the time of

routine day 5 Guthrie. In infants admitted for neonatal intensive care, blood from

capillary, vein or indwelling arterial catheters were obtained at the time of

routine collection. Guthrie blood collection of term infants discharged home

before their 5th day was conducted by their health visitors in the community.

However community blood collection was abandoned due to the technical

difficulty of maintaining cold chain and transporting the blood samples on ice.

The study plan was therefore amended to recruit in-house infants alone who

required Guthrie bloods during their stay in the neonatal intensive care, special

care baby unit, transitional care and those staying in the hospital for maternal

reasons.

9.2.6 Study End Point

The study end point was reached once blood was collected at birth from the

umbilical cord and postnatal period at the time of Guthrie test respectively.

89


9.2.7 Sample Collection & Storage

Whole blood was drawn into refrigerated EDTA tube containing 500 IU of

aprotinin, a serine protease inhibitor, known to inhibit trypsin, chymotrypsin,

plasmin, kallikrein, and thrombin. Addition of aprotinin helps to inhibit protein

degradation prior to separation of plasma (Mintz 1993) which was obtained by

centrifugation immediately at 1000 xg for 10 minutes in refrigerated centrifuge.

The plasma specimens were stored at ≤ -70°C. Blood samples with gross

haemolysis or lipaemia were discarded as these were known to affect amylin

levels.

9.2.8 Sample Analysis

Linco research laboratories supplied two amylin assay kits namely, “Human

amylin kit” and “Total amylin kit”. These amylin kits had been validated for use

with human plasma and were sensitive to 1 pmol. The “Human amylin kit”

reflected biologically active amylin more faithfully than “Total amylin kit”. In

some cell types the reduced form (disulphide bond broken) is not biologically

active although it may act as a competitive inhibitor (receptor antagonist) of the

unreduced form. Since the disulphide bond is sometimes reduced in

physiological conditions, the “Total amylin kit” tended to be higher than the

“Human amylin kit”. The “Total amylin kit” was however considered to be more

robust when reassurance about the controls with regards to the sampling

variables, time to separation, and storage was not absolutely certain. This is

because the rearrangements of the thiols in blood sample were common when

there were unavoidable delays in processing due to strict clinical protocol. The

research team concluded that it was impossible to strictly control sampling

variables, time to separation and storage. Furthermore amylin assay developer

and biochemical scientist opined that the marginal higher estimation of plasma

amylin by “Total amylin kit” was very small to affect study results and

conclusions. Therefore on balance, the “Total Human amylin kit” was chosen for

the project.

All plasma samples were analysed for amylin, and where volume of plasma was

sufficient, insulin assay was also performed. Plasma total amylin

immunoreactivity was quantified using Human Amylin (Total) ELISA KIT, a

90


monoclonal antibody based sandwich immunoassay system developed by

LINCO Research, Inc. (Missouri, USA). After the receipt of the ELISA kit, all

components were stored at 2-8°C. This ELISA has sensitivity of 1 pmol (in 50 µl

plasma) with an inter-assay coefficient of variation of 13-18% and intra-assay

coefficient of variation of 2.5-5% at 50 pmol spiked concentration of amylin

(Appendix 3). The basic steps of the assay are detailed in Appendix 2. Plasma

immunoreactive insulin was measured by ELISA that has inter- and intra-assay

coefficients of variation of 5.6% and 5.3% (BioSource Labs, Belgium)

respectively.

The principle of the sandwich ELISA (Appendix 1) is that the capture antibody

and detection antibody binds to reduced or unreduced human amylin which is

complexed with Streptavidin-Alkaline Phosphatase to form a sandwich.

Substrate 4-Methylumbelliferyl Phosphate (MUP) is applied to the completed

sandwich and fluorescent signal monitored at 355 nm/460 nm which is

proportional to the amount of amylin present in the sample.

9.2.9 Sample Size

A Priori statistical advice suggested a sample size of at least 200 observations

from cord blood samples for the calculation of a reference interval (Altman DG

1991).

9.2.10 Statistical Analysis

Statistical analysis was performed using Medcalc version 11.4.4.0. 1993-2010.

Data were assessed for normality using coefficient of skewness, coefficient of

kurtosis, D’Agostino-Pearson and Kolmogorov-Smirnov tests. Non-parametric

tests (such as Wilcoxon rank sum test & Mann-Whitney U test) were employed

where data were non-normal and presented as median values with interquartile

range (25th-75th centile). The data were analysed for group differences with chi-

square or Fisher’s exact tests for the categorical variables and with t-tests for

the continuous variables. Pearson’s and Spearman’s correlation coefficient was

used to assess correlation between normally and non-normally distributed

amylin and insulin levels respectively. A p value of less than 0.05 was

considered significant. A Bland-Altman plot of difference between the methods

(y axis) against their average (x axis) was used to measure agreement between

91


the two sampling methods (cord arterial and cord venous). This method aims to

investigate a possible relation between measurement error and the true value

which is unknown. Therefore the mean, average discrepancy (bias), trend, and

scatter of the two measurements are used as a guide to the comparability of the

two methods.

9.2.11 Data Collection & Case Report Form

Case report forms (Appendix 14) of infants recruited into the study consisted of

demographic details, clinical details, and time of blood sampling. Maternal

details and clinical history were also entered on a CRF.

9.2.12 Study Summary

Table 2 highlights the key features of the study.

92


Table 2. Study A Summary

TitleUmbilical and Guthrie amylin levels in

healthy preterm and term infants

Study DesignSingle centre, Prospective

Observational Study

Study Duration 12 months

ObjectivesTo determine plasma amylin levels in

healthy preterm and term infants

Number of blood samples

/ Participants

200 umbilical cord samples

Main Inclusion Criteria Infants ≥ 24 weeks gestation

Main Exclusion Criteria

Infants born to mothers whose

pregnancy was complicated by

diabetes

Statistical Methodology

Median and interquartile ranges for

summary statistics. Corelation

Coefficient to evaluate the

relationship between variables. Bland

Altman test for method comparison

between umbilical arterial and

umbilical venous blood sampling

method

Outcome variable Plasma amylin levels

Measurement Assay ELISA

Figure 19 below shows the overall study plan in a Gantt chart.

93


Figure 19. Study A Gantt chart

9.3 Results

Figure 20 below shows participant flow and collection of blood samples.

94

Paired umbilical cord arterial blood where

possible (n=26)

14 mothers excluded due to significant perinatal complications

196 mothers eligible(194 singleton & 2 twin)

Paired umbilical cord arterial blood

where possible (n=8)

14 infants were inpatients for maternal reasons

(Post caesarean section)

25 infants were inpatient due to problems relating to

prematurity

Healthy Term infants (n=138)

Cord Venous Amylin levels (181)Paired Cord Arterial Amylin levels (34)

Paired Cord Insulin levels (33) Postnatal Day 5 Amylin levels (39)

Umbilical cord venous blood

(n=138)

Healthy Preterm infants (n=43)

Guthrie Blood D5 (n=14)

Guthrie Blood D5 (n=25)

Umbilical cord venous blood

(n=43)

3 infants excluded(2 Downs syndrome, 1 CDH)

182 mothers consented(180 singleton & 2 twin)

124 infants discharged on D1 18 infants

discharged on D1

184 infants eligible

ELISA


Figure 20. Study A flow diagram of participants and blood samples

95


During the course of the study, 196 pregnant mothers (194 singleton and 2 twin

gestation) were eligible for the study of whom 19 consented antenatally, 10

during early labour, and 169 prior to elective caesarean section and induction of

labour. Fourteen mothers with singleton pregnancies subsequently developed

severe perinatal complications (7 preeclampsia, 4 sepsis/chorioamnionitis, and

3 gestational diabetes requiring insulin therapy). Thus 182 mothers (180

singleton and 2 twin gestation) who consented gave birth to 184 infants. Three

singleton mothers gave birth to infants who did not meet the inclusion criteria (2

Downs syndrome and 1 Congenital Diaphragmatic Hernia) and were therefore

excluded from the study.

Of the 181 infants who were eligible for blood collection, 138 were healthy term

infants and 43 healthy preterm infants. Umbilical venous blood was collected

from 138 term and 43 preterm infants. In addition, paired umbilical arterial blood

was also collected from 26 term and 8 preterm infants. A total of 124 term and

18 preterm (> 35 weeks gestation) infants were discharged between D1-D4.

Guthrie blood was collected from 14 term infants who were inpatient due to

maternal reasons (post caesarean section) and 25 preterm infants who were

inpatients due to problems associated with prematurity (9 feeding

establishment, 5 temperature control, 7 mild respiratory distress, 4 monitoring

for presumed sepsis).

A total of 254 blood samples (181 umbilical cord venous, 34 paired (meaning

when arterial blood was collected along with venous blood from the same

umbilical cord where possible) umbilical cord arterial, and 39 Guthrie) were

analysed for amylin levels. In addition, 33 paired umbilical cord blood samples

were also analysed for insulin levels.

The median (interquartile range) amylin concentration in 138 healthy term

infants at birth (umbilical cord) was 6.10 (3.30-9.70) pmol/L. Table 3 shows the

summary statistics of amylin concentration, birth weight and gestation at birth

(umbilical cord group) in healthy term infants. Figure 21 provides the box and

whisker plot showing distribution of individual amylin level at birth in 138 healthy

term infants.

96



characteristics at birth in healthy term infants

Healthy Term Infants(Umbilical Cord Group)

Birth weight(Kg)

Gestation(Weeks)

Amylin(pmol/L)

Median 3.28 39.00 6.10

95% CI 3.15 – 3.42 38.00 - 39.00 5.20 - 7.49

25-75 percentile (IQR) 2.98 – 3.67 38.00 - 40.00 3.30 - 9.70

Min-Max 1.86-4.64 37-42 0.50-27.40

Coefficient of Skewness -0.06

(p=0.74)

0.60

(p=0.00)

1.14

(p<0.00)

Coefficient of Kurtosis 0.21

(p=0.49)

-0.43

(p=0.26)

1.49

(p=0.01)

*D-P test for Normal

distribution

0.75

Accept

0.00

Reject

<0.00

Reject

* D’Agostino-Pearson test

97


0

5

10

15

20

25

30

Healthy Term Infant Group (n=138)

Um

bilic

al C

ord

Ven

ous

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 21. Box-and-whisker plot of umbilical cord amylin levels in healthy term

infants

The central box represents the values from 25th to 75th percentile, middle line

represents the median, and the vertical line joining the horizontal line extends

from the minimum to the maximum value (range), excluding "outside" values

which are displayed as separate points. An outside value is defined as a value

that is smaller than the lower quartile minus 1.5 times the interquartile range, or

larger than the upper quartile plus 1.5 times the interquartile range. D’Agostino

test for normal distribution was rejected (p<0.0001).

The amylin concentration in 14 healthy term infants on postnatal day 5 (Guthrie)

was 5.65 (3.10-8.20) pmol/L (Table 4 & Figure 22).

98



characteristics at postnatal day 5 in healthy term infants

Healthy Term Infants(Guthrie Group)

Birth weight(Kg)

Gestation(Weeks)

Amylin(pmol/L)

Median 3.40 38.5 5.65

95% CI 3.11-3.58 37.8-41.1 3.05-8.23

25-75 percentile (IQR) 3.12-3.58 38-41 3.10-8.20

Min-Max 2.02-4.42 37-42 1.4-21.0


(p=0.24)

0.52

(p=0.35)

1.97

(p=0.00)


(p=0.05)

-1.27

(p=0.23)

5.26

(p=0.00)

*DP test for Normal

distribution

p=0.05

Reject

p=0.32

Accept

p=0.00

Reject


99


0

5

10

15

20

25

Healthy Term InfantGuthrie Group (n=14)

Gut

hrie

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 22. Box-and-whisker plot of Guthrie amylin levels in healthy term infants

The central box represents 25th to 75th percentiles; middle line, median, and

the vertical lines are limit lines (range) excluding "outside" value which is

displayed as separate point. D’Agostino test for normal distribution was rejected

(p value of p<0.003).

There was no difference in the demographic characteristics and serum amylin

levels at birth (umbilical cord) and postnatal day 5 (Guthrie) in healthy term

infants (Table 5 & Figure 23).

100


Table 5. Demographic characteristics of healthy term infants at birth and

postnatal day 5

Healthy Term infants Birth (Umbilical Cord venous)

Postnatal Day 5 (Guthrie)

p value

n 138 14

Amylin (pmol/L) 6.10

(3.30-9.70)

5.65

(3.10-8.20)

*NS

Gestation (wks) 39

(38.0-40.0)

38.5

(38.0-41.0)

*NS

Birth Weight (kg) 3.28

(2.98-3.67)

3.40

(3.12-3.58)

*NS

Gender

Male 71 7

Female 67 7

* NS- not significant

Median and interquartile range, Mann Whitney U test

101


Am

ylin

con

cent

ratio

n (p

mol

/L)

0

5

10

15

20

25

30

Umbilical Cord GroupHealthy Term Infants

(n=138)

Guthrie GroupHealthy Term Infants

(n=14)

Figure 23. Notched box-and-whisker plot of amylin levels at birth and postnatal

day 5 in healthy term infants

The central box represents 25th to 75th percentiles; the middle line, median and

a vertical line, limits (range), excluding "outside" values which are displayed as

separate points. The confidence intervals for the medians are provided by

notches surrounding the medians which overlap; therefore the medians are not

significantly different at a ± 95% confidence level. To test the difference

between two groups, Mann- Whitney U test for independent samples was used,

p=0.58.

Table 6 shows the demographic characteristics in healthy preterm infants at

birth. Although the group included preterm infants of 26 weeks gestation, the

median gestational age at birth of 34 weeks suggests that the population

studied were dominated by late preterm infants. The amylin level at birth

(umbilical cord) in 43 healthy preterm infants was 4.60 (1.90-8.30) pmol/L

(Table 6 & Figure 24).

102


All these figures need legends to describe statistical methods/distribution etc



characteristics at birth in healthy preterm infants

Healthy Preterm Infants(Umbilical cord Group)

Birth weight(Kg)

Gestation(Weeks)

Amylin(pmol/L)

Median 2.05 34.00 4.60

95% CI 1.65-2.30 33.00-34.39 3.16-6.19

25-75 percentile (IQR) 1.53-2.39 30.50-35.00 1.90-8.30

Min-Max 0.83-3.92 26-36 1.0-11.5

Coefficient of Skewness 0.52

(p=0.13)

-0.87

(p=0.02)

0.35

(p=0.31)


(p=0.60)

-0.36

(p=0.47)

-1.16

(p=0.12)

*DP test for Normal

distribution

p=0.29

Accepted

p=0.05

Rejected

p=0.18

Accepted


103


0

2

4

6

8

10

12

Healthy Preterm Infant Group (n=43)

Um

bilic

al C

ord

Ven

ous

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 24. Box-and-whisker plot of umbilical cord amylin levels at birth in

healthy preterm infants

The central box represents 25th to 75th percentiles; middle line, median, and

the vertical lines, limit lines (range). D’Agostino test for normal distribution was

accepted (p value of p=0.18).

The amylin level on postnatal day 5 (Guthrie) was 6.9 (2.75-9.50) pmol/L (Table

7 & Figure 25) in 25 healthy preterm infants.

104



characteristics at postnatal day 5 in healthy preterm infants

Healthy Preterm Infants(Guthrie Group)

Birth weight(Kg)

Gestation(Weeks)

Amylin(pmol/L)

Median 1.72 33.0 6.9

95% CI 1.56-2.14 32.14-34.00 2.90-8.55

25-75 percentile (IQR) 1.50-2.24 30-35 2.75-9.50

Min-Max 0.83-2.75 26-36 1.0-19.7


(p=0.99)

-0.86

(p=0.06)

0.93

(p=0.04)

Coefficient of Kurtosis -0.62

(p=0.37)

-0.32

(p=0.55)

-0.08

(p=0.73)

*DP test for Normal

distribution

p=0.67

Accepted

p=0.15

Accepted

p=0.13

Accepted


105


0

5

10

15

20

Healthy Preterm Infant Group(n=25)

Gut

hrie

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 25. Box-and-whisker plot of amylin levels at postnatal day 5 in healthy

preterm infants


represents the median, and the horizontal line extends from the minimum to the

maximum value, excluding "outside" value which is displayed as separate point.

D’Agostino test for normal distribution was accepted (p value=0.13).

There was no difference in the demographic characteristics and serum amylin

levels of healthy preterm infants at birth (umbilical cord) and postnatal day 5

(Guthrie) (Table 8 & Figure 26).

106


Table 8. Demographic charateristics of healthy preterm infants at birth and

postnatal day 5.

Healthy Preterm infants

Birth (Umbilical Cord venous)


P value

n 43 25

Amylin (pmol/L) 4.60 (1.90-8.30) 6.9 (2.75-9.50) NS

Gestation (wks) 34 (30.5 - 35.0) 33.0 (30.0 - 35.0) NS

Birth Weight (kg) 2.05 (1.53 – 2.39) 1.72 (1.50 - 2.24) NS

Gender

Male 25 13

Female 18 12

NS- not significant


107


Am

ylin

Con

cent

ratio

n (p

mol

/L)

0

5

10

15

20

Umbilical Cord GroupHealthy Preterm Infants

(n=43)

Guthrie GroupHealthy Preterm Infants

(n=25)

Figure 26. Notched box-and-Whisker plot of amylin levels at birth and postnatal

day 5 in healthy preterm infants

The central box represents 25th to 75th percentiles; the middle line, median;

and vertical line the limits (range) excluding “outside" value which is displayed

as a separate point. The confidence intervals for the medians are provided by

notches surrounding the medians which overlap and therefore the medians are

not significantly different at a ± 95% confidence level. Mann-Whitney U test for

difference between the groups was not significant (p value=0.279). Independent

sample t test was also not significant (p=0.07)

The gestation and birth weight were significantly lower in preterm infants at birth

(umbilical cord) and postnatal day 5 (Guthrie) compared to term infants. Amylin

levels were significantly lower at birth (umbilical cord) but not on postnatal day 5

(Guthrie) in preterm infants when compared to term infants (Table 9 & Figure 27

& Figure 28).

108


Table 9. Comparison of demography and amylin levels in healthy term and

preterm infants at birth and postnatal day 5

Healthy TermBirth

Healthy Preterm

Birth

Healthy Term

Postnatal Day 5

Heathy Preterm

Postnatal Day 5

p value

n 138 43 14 25

Amylin (pmol/L)

6.10(3.30 -9.70)

*4.60(1.90-8.30)

5.65(3.10-8.20)

**6.90 (2.75-9.50)

*p=0.04**p=0.63

Gestation (wks)

39(38.0 - 40.0)

*34(30.5 - 35.0)

38.5(38.0-41.0)

**33.0(30.0 - 35.0)

*p<0.00**p<0.00

Weight(kg)

3.28(2.98-3.67)

*2.05(1.53- 2.39)

3.40(3.12-3.58)

**1.72 (1.50 - 2.24)

*p<0.00**p<0.00

GenderMale 71 25 7 13Female 67 18 7 12


109


Am

ylin

Con

cent

ratio

n (p

mol

/L)

0

5

10

15

20

25

30

Umbilical Cord Group Healthy Preterm Infants

(n=43)

Umbilical Cord Group Healthy Term Infants

(n=138)

Figure 27. Comparison Box-and-Whisker plot of amylin level in term and

preterm infants at birth

The central box represents 25th to 75th percentiles; the middle line, median;

and vertical line, limits (range), excluding "outside" values. The confidence

intervals for the medians are provided by means of notches surrounding the

medians which do not overlap and therefore the medians are significantly

different at a ± 95% confidence level, Mann-Whitney U test for independent

samples was significant (p=0.04).

110


Am

ylin

Con

cent

ratio

n (p

mol

/L)

0

5

10

15

20

25

Guthrie GroupHealthy Preterm Infants

(n=25)

Guthrie GroupHealthy Term Infants

(n=14)

Figure 28. Comparison Box-and-Whisker plot of serum amylin level in term and

preterm infants on postnatal day 5

The central box represents 25th to 75th percentiles; middle line, median; and

vertical line, limits (range), excluding "outside" values. The notches about two

medians overlap and therefore the medians are not significantly different at a ±

95% confidence level, Mann-Whitney U test for independent samples was not

significant (p=0.93).

Thus the study established reference interval for amylin concentrations at birth

and postnatal day 5 in healthy term infants (6.10 (3.30-9.70) and 5.65 (3.10-

8.20) pmol/L) and healthy preterm infants (4.60 (1.90-8.30) and 6.90 (2.75-

9.50)) pmol/L respectively.

111


Mean ranges here


Paired umbilical cord arterial and umbilical cord venous amylin levels were

available from 34 infants. The median amylin level in 34 paired cord venous and

arterial blood samples were 5.3 (2.97-10.17) and 5.8 (2.5-9.7) pmol/L

respectively. The amylin levels in paired samples correlated positively

(Spearman’s coefficient of rank correlation rho=0.94, 95% CI 0.89-0.97,

p<0.0001) (Figure 29).

0 5 10 15 20 250

5

10

15

20

25

Umbilical Cord Venous Amylin Concentration (pmol/L)

Um

bilic

al C

ord

Art

eria

l Am

ylin

Con

cent

ratio

n (p

mol

/L)


association between paired umbilical cord venous amylin (x axis) and umbilical

cord arterial amylin (Y axis)

Since the distribution of cord venous amylin levels were non-normal, rank

correlation was used. The data were ranked in order of size, and calculations

were based on the ranks of corresponding values X and Y to provide Spearman

correlation coefficient rho with p-value, and a 95% CI for rho, i.e. the range of

values which contains the true correlation coefficient with 95% probability

(n=34, r=0.98, 95% CI 0.96-0.99, p<0.0001).

112


A Bland-Altman plot (Figure 30 & Figure 31) or difference plot is a graphical

method of the differences between cord venous and cord arterial amylin (y axis)

against their average (x axis) to demonstrate agreement between the two

sampling methods (cord venous versus cord arterial).

0 5 10 15 20 25-10

-8

-6

-4

-2

0

2

4

AVERAGE of Cord Venous Amylin and Cord Arterial Amylin (pmol/L)

Cor

d V

enou

s A

myl

in -

Cor

d A

rter

ial A

myl

in (

pmol

/L)

Mean-0.8

-1.96 SD-4.4

+1.96 SD2.8

Figure 30. Bland Altman method comparison plot of paired venous and arterial

levels

The plot is useful to reveal a relationship between the differences and the

averages, to look for any systematic biases and to identify possible outliers. In

this graphical method the differences between the two sampling techniques (Y

axis) are plotted against the averages of the two techniques (X axis). Horizontal

lines are drawn at the mean difference, and at the limits of agreement, which

are defined as the mean difference plus and minus 1.96 times the standard

deviation of the differences. Thus the average discrepancy between the two

levels is -0.8 which is clinically small. The difference between the levels and

scatter around the mean (bias) generally remain the same as the average

increases (n=34, mean bias -0.8, and limits of agreement +2.8 to -4.4).

113


0 5 10 15 20 25-140

-120

-100

-80

-60

-40

-20

0

20

40

60

AVERAGE of Cord Venous Amylin and Cord Arterial Amylin

(Cor

d V

enou

s A

myl

in -

Cor

d A

rter

ial A

myl

in)

/ Ave

rage

%

Mean

-13.9

-1.96 SD-69.4

+1.96 SD41.5

Figure 31. Bland Altman method comparison, percentage difference plot, of

paired venous and arterial levels

In this graphical method the differences are expressed as percentages of the

values on the axis (i.e. proportionally to the magnitude of measurements). This

option is useful when there is an increase in variability of the differences as the

magnitude of the measurement increases.

The Bland-Altman method aims to investigate a possible relation between

measurement error and the true value which is unknown. Therefore the mean,

average discrepancy (bias), trend, and scatter of the two measurements are

used as a guide to the comparability of the two methods. The Bland-Altman plot

is interpreted informally by investigating the size of the average discrepancy

between the methods (bias), the trend of the the difference between methods

(tend to get larger (or smaller) as the average increases), and the consistency

of variability across the graph (Does the scatter around the bias line get larger

as the average gets higher?). Horizontal lines are drawn at the mean difference,

and at the limits of agreement, which are defined as the mean difference plus

114


and minus 1.96 times the standard deviation of the differences. If the

differences within mean ± 1.96 SD are not clinically important, the two methods

may be used interchangeably. The mean bias was -0.8 and the limits of

agreement +2.8 to -4.4 which is clinically acceptable.

Thus umbilical venous or arterial blood may be used to analyse amylin levels in

both term and preterm infants.

Paired amylin and insulin levels were analysed from 33 umbilical cord blood

samples from healthy preterm and term infants. The median insulin level at 38.5

(37-40) week gestation and birth weight 3.14 kg (2.61-3.76) was 5.0 (3.00-8.25)

mu/L. The amylin levels showed statistical correlation with insulin levels (rho=

0.46, 95% CI=0.14-0.69, p<0.006) (Figure 32). However analysis of Figure 32

shows wide scatter suggesting this correlation is not strong.

115


0 5 10 15 200

5

10

15

20

25

Umbilical cord blood insulin concentration (mu/L)

Um

bilic

al c

ord

bloo

d am

ylin

con

cent

ratio

n (p

mol

/L)


association between paired umbilical cord insulin (x axis) and umbilical cord

amylin (Y axis) in healthy preterm and term infants

Since the distribution of cord amylin (DP test, p=0.002) and insulin (DP test,

p=0.0004) levels were non-normal, rank correlation was used. The data are

ranked in order of size, and calculations were based on the ranks of

corresponding values X and Y to provide Spearman correlation coefficient (rho)

with p-value, and a 95% CI for rho, i.e. the range of values which contains the

true correlation coefficient with 95% probability (n=33, spearman’s rho= 0.46,

95% CI=0.14-0.69, p<0.006).

The umbilical cord venous amylin levels correlated both with gestation (Figure

33, r=0.20, 95% CI= 0.04-0.35, p=0.012) and birth weight (Figure 34, r=0.21,

95% CI=0.05-0.36, p=0.002).

116


Draw the correlation curve in this figure please


25 30 35 40 450

5

10

15

20

25

30

Gestation at Birth (weeks)

Um

bilic

al C

ord

Am

ylin

Con

cent

ratio

n (p

mol

/L)


association between gestational age at birth (x axis) and umbilical cord amylin

(Y axis)

Since the distribution of cord venous amylin levels were non-normal, rank

correlation was used to calculate Spearman correlation coefficient rho with p-

value, and a 95% CI for rho (n=181, Spearman’s rho=0.20, 95% CI= 0.04-0.35,

p=0.012).

117


Draw curves


0 1000 2000 3000 4000 50000

5

10

15

20

25

30

Birth Weight (Grams)

Um

bilic

al C

ord

Am

ylin

con

cent

ratio

n (p

mol

/L)


association between birth weight (x axis) and umbilical cord amylin (Y axis)

Since the distribution of cord amylin levels were non-normal, rank correlation

was used to calculate Spearman correlation coefficient rho with p-value, and a

95% CI for rho (n=181, Spearman’s rho= 0.21, 95% CI=0.05-0.36, p=0.002).

9.4 Discussion

This single centre prospective observational study was the first to establish

normal range of plasma amylin concentration in healthy term and preterm

neonates at birth and at the fifth postnatal day.

We chose to use the term ‘healthy’ to identify well from unwell preterm neonates

who were deemed clinically stable by the attending clinical team. Term infants

who stayed in the hospital for maternal reasons were well and did not need any

medical interventions. Preterm infants between 35-37 weeks admitted to the

neonatal unit, transitional care ward or postnatal ward were establishing feeds

or required prophylactic antibiotics pending blood cultures. Preterm infants less

118


Draw curves


than 35 weeks were admitted to the neonatal unit primarily because of

prematurity and low birth weight were generally well and establishing feeds.

Premature neonates who were ventilated or required continuous positive airway

pressure (CPAP) for poor respiratory effort were clinically stable, and therefore

included in the study.

The plasma amylin levels at birth and postnatal day 5 in healthy term (6.10 and

5.65 pmol/L) and preterm infants (4.60 and 6.90 pmol/L) were similar to the

levels observed (mean (SD)) in the paediatric (5.0 (1.94) pmol/l) and adult (5.0

(0.4) pmol/l) populations (Mitsukawa, Takemura et al. 1990; Akimoto, Nakazato

et al. 1993). There was no difference in amylin concentration and birth weight,

gestation, and gender between the umbilical cord and Guthrie group in both

term and preterm infants. The amylin concentration in umbilical cord of preterm

infants however was lower than terms infants (6.10 versus 4.60) most probably

due to small sample size and differences in birth weight.

In adults, like insulin, amylin levels show variation depending on fasting or

postprandial state. It is not known if a similar pattern also exists in neonatal

population. It is plausible that the amylin levels observed on postnatal day 5 in

this study may have been influenced by the timing of blood sampling in relation

to the infants’ pre-prandial or post-prandial state. This is less likely as the amylin

levels were similar between the cord and Guthrie groups. We were unable to

study diurnal variation of amylin and the influence of feeding due to the

limitation of the study protocol to opportunistic blood collection.

We propose that plasma amylin levels in the neonatal period are due to

endogenous production as explained previously in section 3.4 on page 34, and

are in the range reported in the paediatric and adult populations.

Agreement was shown between paired cord arterial and venous amylin levels;

therefore either of the two sampling methods could be used to measure amylin.

This observation will help future studies investigating amylin levels in umbilical

cord blood.

Although umbilical cord amylin levels showed statistically significant correlation

with insulin levels, due to wide scatter of the data, we suggest that amylin

secretion may be in some way associated with insulin. In our study, umbilical

119


?sense


vein insulin level from 34 neonates was 36.0 pmol/L (22.0-63.5) which was

higher than 21.0 pmol/L (14.0-33.0) (Ong, Kratzsch et al. 2000) and in the range

reported in previous studies (39.0±5 pmol/L and 33.0 pmol/L (24.0-41.0)

(Thomas, de Gasparo et al. 1967; Gesteiro, Bastida et al. 2009).

Fetal growth and development is dependent on various growth factors such as

insulin, growth hormone and insulin like growth factor. The umbilical venous

plasma insulin concentration and fetal insulin/glucose ratio increase

exponentially with gestation, reflecting maturation of the endocrine pancreas

(Economides, Nicolaides et al. 1990). Body weight at birth is related to the

amount of functioning pancreatic tissue, with hyperinsulinemic babies being

macrosomic and SGA babies having reduced amounts of pancreatic tissue

(Fowden 1989). In this study, a positive correlation was observed between birth

weight and umbilical vein insulin levels (Delmis, Drazancic et al. 1992). We also

observed a direct correlation between amylin levels with ascending gestation

and birth weight, finding shared with its cousin calcitonin gene related peptide

(Parida, Schneider et al. 1998). It is possible that amylin may play a similar role

to insulin in the maturation of pancreas. Although amylin levels showed an

association with birth weight and gestation, the precise clinical significance of

this observation is unclear. Since amylin is co-secreted with insulin from

pancreas, we assessed cord amylin levels in a cohort of small for gestational

age infants to assess the possible influence of faltering intrauterine growth. The

median amylin level in the umbilical cord blood of 23 small for gestational age

infants with median birth weight 1.80 kg (1.50-2.12) and gestation 36 weeks

(33-37) was 4.0 (1.20-7.60) pmol/L. Although the amylin levels were lower that

the levels observed in term and preterm infants, this did not reach statistical

significance.

Amylin has been shown to be raised in rat intestinal ischaemic injury (Phillips,

Abu-Zidan et al. 2001), and may have a biologic role in sepsis (Parida,

Schneider et al. 1998). Future research investigating the role of amylin in feed

intolerance, necrotising enterocolitis and sepsis in the neonatal population may

benefit from normal values established by this study.

The main strength of the study was adherence to study methodology with

regards to sample processing, storage and analysis. In addition, an experienced

120


technician who was blinded to the patient details performed all laboratory

analyses. To be absolutely certain that protein degradation does not take place

in the blood collected prior to centrifugation, separation and storage, EDTA

tubes were primed with aprotinin which is a protease inhibitor. The major

limitations of the study were selection bias towards inpatient infants especially

of term infants in the Guthrie group which also contributed to the small sample

size; and blood sampling covering first postnatal week alone. Serial amylin

measurements beyond first week of life in preterm and term infants may have

helped understand the influence of transplacental passage, diurnal variations,

influence of feeds and decay time of amylin. In hindsight, maternal amylin may

have also helped determine transplacental passage of amylin. However we

could not accommodate this into the study design due to logistical reasons.

9.5 Conclusions

This single centre prospective observational study provides normative data of

amylin levels in neonatal population. Further data from similar studies would be

helpful in establishing a normal range. In healthy term infants, the median

amylin level is 6.10 pmol/L at birth and 5.65 pmol/L at postnatal day 5. In

healthy preterm infants, the median amylin level is 4.60 pmol/L at birth and 6.90

pmol/L at postnatal day 5.

Umbilical cord venous amylin levels showed agreement with paired umbilical

cord arterial amylin levels. Thus umbilical venous or arterial blood may be used

to analyse amylin levels in term and preterm infants.

The median insulin concentration in healthy preterm and term infants is 5.0

(3.00-8.25) mu/L and its correlation with amylin levels suggests an association

and possibly co-secretion.

Amylin levels demonstrate an association with gestation and birth weight, the

exact significance of which is not clear.

121


Shy not?

Chapter 9 – Clinical Study B

Chapter 10- Clinical Study B: Umbilical and Guthrie Amylin in Preterm and Term IMDM

10.1 Introduction

In view of the physiological changes known to take place in fetus and neonate

exposed to maternal diabetes, we hypothesised that amylin levels may be

raised in new-born IMDM. The aim of this study was to determine serum amylin

levels at birth (umbilical cord) and postnatal day 5 (Guthrie) in preterm and term

IMDM.

10.2Methodology


This single-centre prospective observational study was conducted at the

University Hospitals of Sheffield NHS Trust neonatal and obstetric department,

over a one year period. Analysis of the blood samples was conducted in the

Academic Unit of Reproductive and Developmental medicine located at Jessop

Wing, Royal Hallamshire Hospital.

10.2.2 Consent

Infants were recruited once informed consent was obtained from their


the study.


Infant’s ≥ 24 weeks gestation at birth born to mothers whose pregnancies were

complicated by diabetes mellitus (including gestational, insulin dependent and

non-insulin dependent diabetes mellitus) were eligible for the study.


1. Deliveries associated with chorioamnionitis, or other significant antenatal

or labour complications.

2. Infants with congenital and chromosomal abnormality

122


This is all repeated from p 77



Mothers whose pregnancies were complicated by diabetes were identified by

the research team in the antenatal ward and labour ward before elective

induction of labour and elective caesarean section. Information leaflet and

consent were discussed with parents. Written consent was obtained for

collection of blood from umbilical cord and during routine Guthrie. IMDM who

stayed in the hospital for maternal reasons and requiring Guthrie bloods on day

5 were also identified. Additionally, IMDM admitted to the neonatal unit for

monitoring of blood sugars were identified and parents approached for consent.

Blood was collected immediately after birth from umbilical cord stump that was

clamped and separated from the baby. One to two ml of free flowing blood was

obtained from the umbilical vein and artery, where possible. Postnatal blood

samples were collected at the time of routine day 5 Guthrie or during routine

blood sampling (such as bedside glucose monitoring) from capillary, vein or

indwelling arterial catheters as deemed necessary for routine collection.


The infants were no longer followed up after collection of cord and postnatal

bloods.


A 0.5 to 1 ml whole blood was collected and processed as described in Study A,

section 9.2.7 on page 90.


All samples were analysed for amylin, and where volume of plasma was

sufficient, insulin assay was also carried out as described in study A, section

9.2.8 on page 90.

10.2.9 Sample Size

At least 21 IMDM were required, to detect a difference in means of 15,

assuming a standard deviation of 13 with 5% two sided significance level and

123


repetition


90% power. The mean and standard deviation were derived from the data

published by (Mayer, Durward et al. 2002)



as described in study A, section 9.2.10 on page 91.


Case report form (Appendix 15) of infants recruited into the study consisted of

demography, clinical details, and time of blood sampling. Maternal details and

clinical history were also entered on a CRF.


Table 10 below highlights the key features of the study methodology.

124


Table 10. Study B summary

Title Umbilical and Guthrie amylin levels in

preterm and term IMDM

Study Design Prospective Observational Study


Study Centres Single Centre

Objectives To determine plasma amylin levels in

cord and Guthrie blood of IMDM

infants

Number of Samples/Participants 21 IMDM infants

Main Inclusion Criteria IMDM ≥ 24 weeks gestation

Statistical Methodology Median and interquartile ranges and

non-parametric tests. Correlation

Coefficient was performed to evaluate

the relationship between variables.



Figure 35 below provides details of the study plan in a Gantt chart.

125


Figure 35. Study B Gantt chart

126

Paired umbilical cord arterial blood (n=3)

Term infants (n=17)Umbilical cord venous blood

31 singleton mothers were eligible who consented

Paired umbilical cord arterial blood (n=3)

Preterm infants (n=14)Umbilical cord venous blood

Guthrie blood (n=9)

Guthrie blood (n=5)

Cord Amylin (n=31)Guthrie Amylin (n=14)Paired insulin (n=6)

Discharged on D1-4 (n=17)

Inpatients

Feed intolerance

(n=4)

Feed intolerance

(n=8)


10.3Result

The Figure 36 below shows the participant flow and blood samples.

Figure 36. Study B-flow diagram of participants and blood samples

Thirty-one singleton mothers whose pregnancy was complicated by diabetes

consented for the study. 20 mothers had gestational diabetes and 11 mothers

had insulin dependent diabetes mellitus. Umbilical cord blood was collected

from 17 term infants and 14 preterm infants. Of these seventeen infants were

discharged between days 1-4. Guthrie blood was collected from 5 term and 9

preterm inpatient infants who were admitted to the neonatal unit for respiratory

distress, hypoglycemia, prematurity, or feeding problems.

The null hypothesis of the study was that there was no difference in amylin

concentration at birth and postnatal day 5 between preterm and term IMDM and

preterm and term healthy infants (controls).

The median (interquartile range) amylin concentration at birth (umbilical cord) in

term IMDM was 34.30 (28.35-50.00) pmol/L (Table 11 & Figure 37).

127


Which healthy controls?



characteristics at birth in term IMDM infants

Term IMDM(umbilical cord Group)

Birth weight(kg)

Gestation(weeks)

Amylin(pmol/L)

Median 3.74 39.00 34.30

95% CI 3.44-3.98 38.00 - 39.98 32.79-47.39

25-75 percentile (IQR) 3.41 – 4.07 37.75- 40.00 28.35-50.00

Min-Max 2.51-4.85 37-42 22.10-70.10


(p=0.62)

0.58

(p=0.26)

0.59

(p=0.25)


(p=0.71)

-0.21

(p=0.66)

-0.65

(p=0.417)


distribution

0.82

Accept

0.49

Accept

0.38

Accept


128


20

30

40

50

60

70

80

IMDM Term InfantUmbilical Cord Group (n=17)

Um

bilic

al C

ord

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 37. Box-and-whisker plot of umbilical cord amylin levels in term IMDM

infants

The central box represents 25th to 75th percentile, middle line represents the

median, and the vertical line represents range. D’Agostino-Pearson test for

normal distribution of amylin levels was accepted (p=0.38).

The plasma amylin level in term IMDM on postnatal day 5 (Guthrie) was 25.20

(22.20 – 48.75) pmol/L (Table 12 & Figure 38).

129



characteristics on postnatal day 5 in term IMDM infants

Term IMDM(Guthrie Group)

Birth weight(kg)

Gestation(weeks)

Amylin(pmol/L)

Median 3.53 37 25.20

95% CI 2.67 – 4.67 35.22 – 40.77 12.18 – 56.29

25-75 percentile (IQR) 3.21 – 4.43 37 – 38.25 22.20 – 48.75

Min-Max 2.51-4.46 37-42 17.10-60.00


(p=0.74)

2.23 0.85


(p=0.75)

5.00

(p=0.02)

-0.988

(p=0.59)


distribution

Sample size too

small

Sample size

too small

Sample size

too small


Four of five infants in the Guthrie group also had feed intolerance at the time of

blood collection.

130


15

20

25

30

35

40

45

50

55

60

IMDM Term InfantGuthrie group (n=5)

Gut

hrie

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 38. Box-and-whisker plot of Guthrie amylin levels in term IMDM infants


represents the median, and the vertical line joining the horizontal line extends

from the minimum to the maximum value (range). The sample size was too

small to test normal distribution.

131


There was no difference in the demographic characteristics and plasma amylin

levels of term IMDM at birth (umbilical cord) and postnatal day 5 (Guthrie) as

shown in Table 13 & Figure 39.

Table 13. Demographic characteristic of term IMDM at birth and postnatal day 5

Term IMDMGroup

Birth(Umbilical Cord)


p value

n 17 5


(28.35-50.00)

25.20

(22.20-48.75)

*NS

Gestation (wks) 39

(37.75-40.00)

37

(37-38.25)

*NS

Weight

(kg)

3.74

(3.41-4.07)

3.53

(3.21-4.43)

*NS

Gender

Male 8 2 *NS

Female 9 3 *NS

* NS-not significant


132


Am

ylin

Con

cent

ratio

n (p

mol

/L)

10

20

30

40

50

60

70

80

Term IMDMUmbilical Cord Group

(n=17)

Term IMDMGuthrie Group

(n=5)

Figure 39. Comparison Box-and-Whisker plot of amylin levels at birth and

postnatal day 5 in term IMDMs

Mann-Whitney test for independent samples was not significant, p=0.25.

133


Add legend


Sort out IDM vs IMDM


In preterm IMDM, amylin level at birth (umbilical cord) was 32.0 (18.65-44.27)

pmol/L (Table 14 & Figure 40).


characteristics at birth in preterm IMDM infants

Preterm IMDM(umbilical cord group)

Birth weight(kg)

Gestation(weeks)

Amylin(pmol/L)

Median 2.88 34 32.00

95% CI 1.97-3.51 33 – 35.46 18.51 –45.38

25-75 percentile (IQR) 1.97-3.35 33 – 35.25 18.65-44.27

Min-Max 0.82-4.21 26-36 13.90-90.00


(p=0.73)

-1.69

(p=0.00)

1.25

(p<0.04)


(p=0.49)

2.91

(p=0.05)

1.28

(p=0.24)


distribution

0.74

Accept

0.00

Reject

<0.06

Accept


134


10

20

30

40

50

60

70

80

90

IMDM Preterm InfantsUmbilical Cord Group (n=13)

Um

bilic

al C

ord

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 40. Box-and-whisker plot of umbilical cord amylin levels in preterm IMDM

infants

The central box represents 25th to 75th percentile, middle line represents the



135


In preterm IMDM, amylin level on postnatal day 5 (Guthrie) was 23.4 (15.37-

46.57) pmol/L (Table 15 & Figure 41).


characteristics at postnatal day 5 in preterm IMDM infants

Preterm IMDM(Guthrie group)

Amylin(pmol/L)

Birth weight(kg)

Gestation(weeks)

Median 23.4 2.61 34

95% CI 13.84 - 82.01 1.53 - 4.02 33.00 - 36.00

25-75 percentile (IQR) 15.37 - 46.57 1.96 - 3.92 38.0 - 40.0

Min-Max 4.90-92.00 0.82-4.21 26-36


(p<0.05)

-0.05

(p=0.92)

-1.45

(p=0.03)


(p=0.64)

-1.22

(p=0.30)

1.58

(p=0.22)


distribution

0.14

Accept

0.59

Accept

0.05

Accept


136


0

10

20

30

40

50

60

70

80

90

100

IMDM Preterm InfantsGuthrie Group (n=9)

Gut

hrie

Am

ylin

Con

cent

ratio

n (p

mol

/L)

Figure 41. Box-and-whisker plot of Guthrie amylin levels in preterm IMDM

infants

The central box represents 25th to 75 percentile, middle line represents the



At the time of Guthrie blood collection, eight of nine preterm IMDM were

experiencing feed intolerance.

137


There was no difference in the demographic characteristics and amylin levels of

preterm IMDM at birth (umbilical cord) and postnatal day 5 (Guthrie) as shown

in Table 16 & Figure 42.

Table 16. Demographic characteristics of preterm IMDM at birth and postnatal

day 5

Preterm IMDM Birth(Umbilical cord)

Postnatal day 5(Guthrie)

P value

n 14 9


(18.65-44.27)

23.4

(15.37-46.57)

*NS

Gestation (wks) 34

(33-35.25)

34

(38-40)

*NS

Weight (kg) 2.88

(1.93-3.35)

2.61

(1.96-3.92)

*NS

Gender *NS

Male 9 5

Female 5 4

* NS-not significant


138


Am

ylin

Con

cent

ratio

n (p

mol

/L)

0

10

20

30

40

50

60

70

80

90

100

Umbilical Cord GroupPreterm IMDM

(n=13)

Guthrie GroupPreterm IMDM

(n=9)

Figure 42. Comparison box-and-whisker plot of amylin concentration at birth

and postnatal day 5 in preterm IMDM

Mann-Whitney test for independent samples did not reach statistical

significance, p=0.48.

Figure below summarises the serum amylin levels at birth and postnatal day 5

in term and preterm IMDM. There was no statistical difference in plasma amylin

levels between term and preterm IMDM at birth (p=0.25) and postnatal day 5

(p=0.64).

139


0

10

20

30

40

50

60

70

80

90

100

p=0.25 p=0.64

Am

ylin

Con

cent

ratio

n (p

mol

/L)

TermIMDMCord PretermIMDMCord TermIMDMGuthrie PretermIMDMGuthrie

Figure 43. Comparison box-and-whisker plot of amylin concentration in

umbilical cord and Guthrie blood in term and preterm IMDM

Mann-Whitney test for independent samples was not significant, cord group

p=0.25 and Guthrie group p=0.64.

140


Still not clear


What are the shaded boxes?


There was no baseline difference in the demographic characteristics at birth

(umbilical cord) and postnatal day 5 (Guthrie) between term IMDM and term

healthy controls (Table 17). However, the median amylin level at birth in term

IMDM was significantly increased at 34.30 pmol/L compared to 6.10 pmol/L in

term healthy controls (p <0.0001). Similarly, the median amylin level at

postnatal day 5 in term IMDM was also significantly increased at 25.20 pmol/L

compared to 5.65 pmol/L in term healthy controls (p < 0.0001) (Figure 44).

Table 17. Demographic characteristics and serum amylin levels of term healthy

controls and term IMDM at birth and postnatal day 5

Term Controls

Birth

Term IMDMBirth

Term Controls Postnatal

Day 5

Term IMDMPostnatal

Day 5

p value

n 138 17 14 5

Amylin (pmol/L)

6.10(3.30-9.70)

*34.30(28.35-50.00)

5.65(3.10-8.20)

**25.20(22.20-48.75)

*p<0.00**p<0.00

Gestation (wks)

39.00(38.00-40.00)

39(37.75-40.00)

38.5(38.00-41.00)

37.0(37-38.25)

NS

Weight(kg)

3.28(2.98-3.67)

3.74(3.41-4.07)

3.40(3.12-3.58)

3.53(3.21-4.43)

NS

GenderMale 71 8 7 2Female 67 9 7 3

NS-not significantMedian and interquartile range, Mann Whitney U test

141


Use 2 places of decimals throughout


0

10

20

30

40

50

60

70

80

p<0.0001 P<0.001

Am

ylin

Con

cent

ratio

n (p

mol

/L)

HealthyTermCord TermIMDMCord HealthyTermGuthrie TermIMDMGuthrie


postnatal day 5 between term IMDM and healthy term infants (controls)

Mann-Whitney test for independent samples was significant, cord group

p<0.0001 and Guthrie group p <0.001.

The null hypothesis was therefore rejected. Thus amylin levels are significantly

raised in term IMDM both at birth and postnatal day 5 when compared to term

healthy control infants.

142


There was no difference in the demographic characteristics at birth (umbilical

cord) and postnatal day 5 (Guthrie) in preterm IMDM and preterm healthy

controls (Table 18). However, the median amylin level at birth in preterm IMDM

was significantly increased at 32.00 pmol/L compared to 4.60 pmol/L in preterm

healthy controls (p<0.0001). Similarly, the median amylin level at postnatal day

5 in preterm IMDM was also significantly increased at 23.40 pmol/L compared

to 6.90 pmol/L in preterm healthy controls (p <0.0001) (Figure 45).

Table 18. Demographic characteristics and serum amylin levels of preterm

healthy controls and preterm IMDM at birth and postnatal day 5

Preterm Controls

Birth

Preterm IMDMBirth

Preterm ControlsPostnatal

Day 5

Preterm IMDM

Postnatal Day 5

p value

n 43 14 25 9

Amylin (pmol/L)

4.60(1.90-8.30)

*32.00(18.65-44.27)

6.90(2.75-9.50)

**23.40(15.37-46.57)

*p<0.00**p<0.00

Gestation (wks)

34(30.5 - 35.0)

34(33-35.25)

33(30.0 - 35.0)

34(38-40)

NS

Weight(kg)

2.05(1.53-2.39)

2.88(1.97-3.35)

1.72(1.50 - 2.29)

2.61(1.96-3.92)

NS

Gender NSMale 25 9 13 5Female 18 5 12 4

NS-not significantMedian and interquartile range, Mann Whitney U test

143


0

10

20

30

40

50

60

70

80

90

p<0.0001 P<0.0001

Am

ylin

Con

cent

ratio

n (p

mol

/L)

HealthyPretermCord PretermIMDMCord HealthyPretermGuthrie TermIMDMGuthrie


postnatal day 5 between preterm IMDM and healthy preterm infants (controls)

Mann-Whitney test for independent samples was significant, cord group p

<0.0001 and Guthrie group p<0.0001.

The null hypothesis was therefore rejected. Thus serum amylin levels are

significantly raised in preterm IMDM both at birth and postnatal day 5 when

compared to healthy preterm infants (controls).

Paired amylin and insulin levels were analysed from 3 IMDM preterm and 3

IMDM term infants from umbilical cord blood samples. The median insulin level

was 10.5 (8.0-16.5) mu/L. Increased positive correlation was observed when

this data were combined with paired amylin and insulin levels from healthy

preterm and term infants (n=39, Spearman’s rho= 0.56, 95% CI=0.29-0.74,

p<0.0001) (Figure 46).

144


0 5 10 15 20 250

20

40

60

80

100

Umbilical Cord Insulin Concentration (mu/L)

Um

bilic

al C

ord

Am

ylin

Con

cent

ratio

n (p

mol

/L)


association between umbilical cord insulin (x axis) and umbilical cord amylin (Y

axis) from paired umbilical cord blood samples of healthy and IMDM preterm

and term infants

Since the distribution of cord venous amylin levels were non-normal, data were

ranked in order of size, and calculations were based on the ranks of

corresponding values X and Y to provide Spearman correlation coefficient rho

with p-value, and a 95% CI for rho, (n=39, Spearman’s rho= 0.56, 95% CI=0.29-

0.74, p<0.0001).

Thus the median insulin level in preterm and term IMDM was twice (n=6, 10.5

(8.0-16.5) mu/L) the level observed in healthy preterm and term infants (n=33,

5.0 (3.00-8.25) mu/L) and its correlation with amylin levels confirms an

association and possibly co-secretion.

145


10.4Discussion

This single centre prospective observational study was the first to determine

plasma amylin concentrations in term and preterm IMDM at birth and fifth

postnatal day.

An alternative to observational methodology would have been case-control

study, IMDM with feed intolerance (cases) compared to IMDM without feed

intolerance (controls). Case-control study incorporating objective assessment of

gastric emptying in the study methodology may have added validity to the study

results. However not all infants with feed intolerance were admitted to the

neonatal unit or necessitated introduction of oro/nasogastric tube, a prerequisite

to identify infants with feed intolerance. Furthermore, the issues of funding,

technical expertise and ethical concerns of performing gastric emptying tests in

neonatal setup were considered as major limiting factors. Interestingly, 4/5 term

infants who required admission to the neonatal unit for monitoring of blood

sugars were also found to have feed intolerance with increased prefeed gastric

residual volumes and frequent vomiting. 8/9 preterm infants were also

experiencing feed intolerance at the time of Guthrie blood collection. Due to

small number of the admitted infants who were also experiencing feed

intolerance we could not draw meaningful relationship of amylin levels to the

degree of feed intolerance or resolution of symptoms of feed intolerance. In

addition we could not identify any obvious differences in the characteristics of

IMDM who experienced feed intolerance with those who did not. It is possible

that minor but important differences in relation to the degree of feed tolerance

and amylin levels were missed as we did not set out to study objective

measures of feed intolerance. Further studies are warranted to shed more light

on this topic.

In this study serum amylin levels obtained from cord and Guthrie blood were

significantly raised in infants born to mothers whose pregnancy was

complicated by diabetes and these levels were comparable to those reported in

critically ill children with feed intolerance (Mayer, Durward et al. 2002). We

suggest that these high serum amylin levels observed in IMDM may be partially

responsible for the feed intolerance seen in this population. This is in keeping

with the physiological action of amylin, as it is 15–20 times more potent than

146


other known inhibitors of gastric motility (Young 1997). There are a number of

possible explanations for such high levels observed in IMDM. Amylin is deficient

in type 1 diabetes mellitus and is present in excess in conditions in which insulin

is hyper-secreted (Rink, Beaumont et al. 1993). It would therefore seem

plausible that transient neonatal hyperinsulinaemia in IMDM as a consequence

of in-utero hyperglycaemia, may also stimulate hypersecretion of endogenous

amylin. This is further supported by evidence of co-secretion of amylin and

insulin, shown by the positive correlation between amylin and insulin in study A

and Study B (n=39, spearman’s rho= 0.56, 95% CI=0.29-0.74, p<0.0001) and

previous studies (Rink, Beaumont et al. 1993; Mayer, Durward et al. 2002).

Moreover, the median insulin level in preterm and term IMDM was twice (n=6,

10.5 (8.0-16.5) mu/L) the level observed in healthy preterm and term infants

(n=33, 5.0 (3.00-8.25) mu/L) suggesting possible fetal and neonatal

hyperinsulinaemia. Unfortunately we were unable to obtain sufficient sample

volumes for analysis of paired amylin and insulin on the fifth postnatal day.

It is plausible that the amylin levels observed on postnatal day 5 in preterm and

term IMDM in this study may have been influenced by the timing of blood

sampling in relation to the infants’ pre-prandial or post-prandial state. This is

less likely as the amylin levels were similar between the cord and Guthrie

groups in both preterm and term IMDM. We were unable to study diurnal

variation of amylin and the influence of feeding due to the limitation of the study

protocol to opportunistic blood collection.

Although, the median amylin levels on the fifth postnatal day were lower than

umbilical cord samples at birth in both preterm and term IMDM, they did not

reach statistical significance. This is most likely to be due to small sample size

and comparatively lower gestation and birth weight possibly due to a selection

bias towards blood collection of inpatient neonates in the Guthrie group.

However a more plausible explanation for this gradual decline in Guthrie amylin

levels in preterm and term IMDM is possibly explained by declining

hyperinsulinaemic environment. This would also coincide with the time course of

resolution of the feed intolerance observed in 4 term IMDM infants who were

discharged by day 7 established to full feeds. We did not set out to study these

infants in more detail and therefore cannot draw any meaningful conclusions

about specific factors relevant to the group of infants who displayed feed 147


Page reference here


intolerance. The observed serum amylin levels in preterm and term IMDMs on

the fifth postnatal day (23.4 and 25.20 pmol/L) were still above the normal range

observed in healthy preterm and term infants (6.90 and 5.65 pmol/L).

The high amylin levels in IMDM further support the theory that transplacental

passage either does not occur or is minimal as serum amylin levels are

subnormal in diabetic patients (Ludvik, Lell et al. 1991; Sanke, Hanabusa et al.

1991; Koda, Fineman et al. 1992; Akimoto, Nakazato et al. 1993). Thus the

observed increased amylin levels in IMDM would seem to represent

endogenous amylin production.

AC187 is a truncated amylin peptide antagonist that acts by selectively and

potently blocking amylin receptors (Young, Gedulin et al. 1994). The biological

actions of amylin in IMDM need to be elucidated prior to evaluation of

pharmacological blockade of this peptide as a potential therapeutic option.

The strength of the study was strict adherence to study protocol with regards to

sample processing and storage. However, a major limitation of the study was

selection bias to inpatient IMDM which also contributed to small numbers

especially in the Guthrie group. Although it was possible to collect serial blood

for amylin levels beyond first week of postnatal life, we did not include this in our

methodology fearing refusal by the ethics committee. Inclusion in the study

methodology of maternal amylin concentration and serial amylin measurements

in preterm and term IMDM may have helped understand the influence of

transplacental passage, diurnal variations, possible influence of feeds and

decay time of amylin. Moreover, this study did not attempt to correlate amylin

levels with objective measures of gastric emptying and feed intolerance or its

resolution. This is an important limitation of the study and may be considered in

the design of future studies.

In conclusion, we have presented evidence in support of our hypothesis that

amylin levels are raised in preterm and term IMDM which may contribute to the

observed feed intolerance.

148


10.5Conclusions

The amylin levels in term and preterm IMDM at birth and postnatal day 5 were

significantly higher than levels observed in healthy preterm and term controls.

Amylin by virtue of its potent inhibition of gastric emptying may play a role in the

pathogenesis of feed intolerance in this group of infants.

The cord insulin levels in preterm and term IMDM were twice the levels

observed in the cord blood from healthy preterm and term infants. These levels

correlated positively with amylin levels which suggest an association with insulin

secretion.

149

Chapter 10 – Clinical Study C

Chapter 11- Clinical Study C: Amylin Levels in Preterm infants with Feed Intolerance

11.1 Introduction

Amylin is a potent inhibitor of gastric emptying and its elevated levels are shown

to be associated with delayed gastric emptying in critically ill children with feed

intolerance (Mayer, Durward et al. 2002). We hypothesized that preterm

neonates with feed intolerance evidenced by increased prefeed GRV (surrogate

marker for delayed gastric emptying) may have high plasma amylin

concentrations. The main objective of this study was to determine serum amylin

concentrations in preterm neonates with and without feed intolerance.

11.2 Methodology


This two-centre case-control study was conducted at the University Hospitals of

Sheffield NHS Trust and Doncaster General Hospital neonatal departments,

over a one year period. Analyses of the blood samples were conducted in the

Academic Unit of Reproductive and Developmental medicine located at Jessop

Wing, Royal Hallamshire Hospital.

11.2.2 Consent

Infants were recruited once informed consent was obtained from their


the study.


1. Preterm infants between 24-36 weeks gestation at birth admitted to the

neonatal intensive care unit and requiring gastric tube placement and

2. Preterm infants who were receiving > 30 ml/kg or > 25% of total fluid intake

as enteral feeds through a gastric tube.

150


This should be in introductory chapters



1. Deliveries associated with intrauterine growth retardation, abnormal

antenatal Doppler assessment (risk factors for NEC which may influence

amylin levels), maternal infection, chorioamnionitis, pre-eclampsia and

significant labour complications (maternal inflammatory conditions which

may influence amylin levels).

2. Infants born to mothers whose pregnancy were complicated by diabetes

mellitus (due to conclusions of Study B).

3. Infants with congenital abnormalities, chromosomal abnormalities (possible

bias due to association with feed intolerance), structural gastrointestinal

abnormalities, necrotising enterocolitis, and sepsis (possible bias due to

association with feed intolerance and in addition may influence amylin

levels).

4. Infants requiring administration of prokinetic agents including erythromycin.

5. Exclusive breast feeding infants in who prefeed gastric residual volume

cannot be determined.

6. Haemoglobin less than 7 g/dl (due avoid further blood loss).

Rationale for study exclusion criteria: Amylin is raised in IMDM as shown in

study B, acute inflammatory conditions, maternal and neonatal sepsis, and in

rat intestinal ischaemic injury (possible role in necrotising enterocolitis). It

shares 50 % homology with CGRP which has anti-inflammatory and vasodilator

function and is also shown to be raised in patients with sepsis and soft tissue

injury (Joyce, Fiscus et al. 1990; Clementi, Caruso et al. 1995; Parida,

Schneider et al. 1998; Hall and Brain 1999; Onuoha and Alpar 1999; Phillips,

Abu-Zidan et al. 2001; Kairamkonda, Deorukhkar et al. 2005). This data from

animal and human studies of conditions which may influence amylin

concentrations informed the study exclusion criteria.


The feeding regime of the neonates was dictated by the local guideline.

Expressed breast milk (EBM) was encouraged, and formula feed was used if

breast milk was not available. The neonates were fed every hour and the 151


volumes increased as clinically tolerated but not more than 20mls/kg birth

weight/day. Every four hours pre-feed GRV were recorded as a percentage of

the previous feed volume. Abdominal girth, colour of gastric residuals and

emesis were also noted. If this GRV was greater than 50% on two consecutive

occasions, neonates were classified as feed-intolerant (nTOL). The further

management of these neonates was up to the attending neonatologist.

Researcher or responsible consultant identified preterm infants who were

receiving > 30 ml/kg or > 25% of total fluid intake as enteral feeds through a

gastric tube. Parents were approached for consent and information leaflets

provided. In neonates with feed intolerance (nTOL), 0.5-1 ml blood was

collected from capillary, vein, or indwelling arterial catheters as deemed

necessary for routine collection; within 2 hours of the second big (> 50%)

prefeed GRV. Feed-tolerant (TOL) neonates served as controls. The

researchers identified TOL infants matched for gestational age and birth weight

and wherever possible postnatal age with nTOL infants. 0.5-1 ml blood was

collected from these neonates at the time of next routine collection. All blood

handling was performed as per guidance for the minimization of infection risk to

both participants and investigators.

11.2.6 Definition of feed intolerance

Infants whose Gastric Residual Volume (GRV) was greater than 50% on two

consecutive occasions were identified as feed-intolerant (nTOL). We chose this

definition of feed intolerance partly due to personal and published observation

by Mayer et al (Mayer, Durward et al. 2002) who defined critically ill children as

feed-intolerant on the basis of GRV greater than 125% 4 hour after a feed

challenge which correlated with three objective measures of gastric emptying. In

addition, based on the assumption that stomach empty’s every 2 hours, we

concluded that in infants in our study who were fed every hour, GRV of more

than 50% on 2 consecutive occasions would improve the sensitivity of the

definition. This definition was different from previously reported in that the target

GRV had to be proved on 2 consecutive occasions which we felt would improve

the accuracy and sensitivity to the definition. It was also acknowledged that the

proportion of feed that may be aspirated is strongly influenced by the size of the

feed and hence the inclusion criteria was limited to those preterm infants who

were receiving > 30 ml/kg or > 25% of total fluid intake as enteral feeds. Thus 152


the minimal feed volume at any given time was more than 2mls even in the

smallest infant.

11.2.7 Definition of feed tolerance

Infants with minimal (prefeed GRV <50%) or no GRV for > 24 hours were

identified as feed tolerant (TOL).


The infants were no longer followed up after collection of bloods.


A 0.5 to 1 ml whole blood was drawn into ice-cooled EDTA tube containing 500

IU of aprotinin. The separation of plasma and storage was performed as

explained in Study A, section 9.2.7 on page 90.


All samples were analysed as described previously in section 9.2.8 on page 90.

11.2.11 Sample Size

To detect a minimum difference of 15 pmol/L between the feed tolerant (TOL)

and intolerant (nTOL) groups, with a power of 90%, assuming a standard

deviation of 13, a total number of 20 neonates per group had to be recruited for

significance levels of 5% (a=0.05). The mean and standard deviation for the

sample size calculation was based on data from previous studies in neonates

and children (Mayer, Durward et al. 2002; Kairamkonda, Deorukhkar et al.

2005)



Individual group data were assessed for normal distribution by applying

coefficient of Skewness and Kurtosis and D’Agostino-Pearson test. The

coefficient of Skewness and Kurtosis is a measure for the degree of symmetry

and peakedness/flatness respectively in the variable distribution. The

D'Agostino-Pearson test (Sheskin 2003) computes a single p-value for the

153


combination of the coefficients of Skewness and Kurtosis. The tests for normal

distribution were accepted when the p value was > 0.05. Normal data were

analysed for group differences with chi-square or Fisher’s exact tests for the

categorical variables and with t-tests for the continuous variables. Median and

interquartile range (25th–75th centile) and non-parametric tests such as

Wilcoxon rank sum test & Mann-Whitney U test were employed where data

were non-normal. Pearson’s correlation coefficient (r) or Spearman’s rank test

(rho) was used to assess correlation between variables with parametric and

non-parametric distribution respectively. A p value of less than 0.05 was

considered significant.


The Case report form (Appendix 16) of infants recruited into the study consisted

of demography, clinical details, and time of blood sampling. Maternal details

and clinical history were also entered on a CRF.


Table 19 & Figure 47 below highlight the key features of the study methodology

and Gantt chart respectively.

154


Table 19. Study C summary

Title Amylin levels in preterm infants with

feed intolerance

Study Methodology Case-Control Study


Study Centres Two Centre

Objectives To determine plasma amylin levels in

preterm infants with feed intolerance

Number of Participants 20 nTOL and 20 TOL

Main Inclusion Criteria Preterm infants (24-36 weeks)

admitted to the neonatal intensive

care unit and requiring gastric tube

feeding

Statistical Methodology Median and interquartile ranges and

non-parametric tests. Correlation

Coefficient will be performed to

evaluate the relationship between

variables



155


Figure 47. Study C Gantt chart

156

Enteral feeds >30ml/kg or>25% of total fluid intake

Consent obtained from parents of preterm neonates (24-36 weeks) with gastric tube placement

n=70

None or <50% prefeed GRV

Study entry and consent

Feed-tolerant (TOL)(n=30)

Feed-intolerant (nTOL)(n=30)

n=4 excluded Blood samples haemolysed &

therefore unsuitable for analysis

n=6 excluded Blood samples haemolysed &

therefore unsuitable for analysis

Feed-intolerant (nTOL)(n=34)

Feed-tolerant (TOL)(n=36)

>50% prefeed GRV on two consecutive occasions

Study entry and consent


11.3 Results

Figure 48 below shows study participant flow and timing of blood collection. 70

preterm infants were eligible for the study once they met the selection criteria.

Blood was collected from 34 feed intolerant (nTOL) and 36 feed tolerant (TOL)

infants. 4 blood samples in the nTOL and 6 in the TOL group were heavily

hemolysed and therefore discarded. Thirty blood samples in each group were

available for further analysis.

Figure 48. Study C-Flow diagram of participants and blood samples

The null hypothesis of the study was that serum amylin levels in preterm infants

with feed intolerance were no different to preterm infants without feed

intolerance.

157


There was no significant difference between the nTOL and TOL group with

regards to baseline characteristics such as gestation, birth weight, gender,

postnatal age, ventilation days, CPAP days, type of feed, blood glucose, white

cell count and CRP (Table 20).

Table 20. Baseline characteristics at the time of blood sampling of preterm

neonates defined as feed-intolerant (nTOL) and feed-tolerant (TOL)

nTOL (n=34) TOL (n=36) p value

n 34 36Gestation (wks) 29.5 (28-31) 30.0 (29-33) 0.33

Birth weight (kg) 1.30 (1.00-1.80) 1.30 (1.10-1.80) 0.62

GenderMale (n) 23 24 0.64Female (n) 11 12 0.62

Postnatal age (days) 7.5 (4-10) 9.5 (5-12) 0.16

Ventilation (days) 1 (0-4) <1 (0-3) 0.21

CPAP (days) 3 (0-4) 2 (0-5) 0.41

Feed typeEBM (n) 22 24 0.12Formula (n) 7 7 0.26Mixed (n) 5 5 0.18

Blood glucose (mmol/l) 4.2 (3.5-4.9) 3.9 (3.1-5.0) 0.23

White cell count (109/l) 9.0 (7.5-10.4) 8.3 (6.3-10.4) 0.08

C reactive protein (mg/l) 6 (6-10) 6 (6-10) 0.51

Data are presented as median (interquartile range)

Amylin concentration, % GRV and time to full enteral feeds were significantly

raised in nTOL compared to TOL group. However, there was no difference in

length of hospital stay between nTOL and TOL group (Table 21).

158


Table 21. Serum amylin levels, % GRV, days to reach full feeds and length of

stay in feed-intolerant (nTOL) and feed-tolerant (TOL) neonates

nTOL TOL P value

n 30 30

Amylin (pmol/L) 47.9 (21.4-79.8) 8.7 (5.7-16) <0.00

GRV (%) 150 (100-350) 5 (0-5) <0.00

Time to full feeds (days) 13.0 (10.0-16.0) 6.5 (4.0-9.0) <0.00

Length of hospital stay (days)

52.5 (21-69) 37 (20-50) 0.08

Data are presented as median [interquartile range]GRV (%)-percentage gastric residual volume prior to next scheduled feed measured prior to amylin sampling

In particular, median amylin level was significantly raised in nTOL group at 47.9

pmol/L compared to 8.7 pmol/L in TOL group. The null hypothesis was therefore

rejected. Thus amylin levels in preterm infants with feed intolerance are

significantly raised compared to preterm infants without feed intolerance.

Figure 49, Figure 50 & Figure 51 show box and whisker plots of plasma amylin,

% GRV and time to reach full enteral feeds in nTOL and TOL group.

159


Am

ylin

Con

cent

ratio

n (p

mol

/L)

0

20

40

60

80

100

120

nTOL(n=30)

TOL(n=30)

Figure 49. Box and whisker plot of amylin levels (y-axis) in feed-intolerant

(nTOL) and feed-tolerant (TOL) neonates (x-axis)

D’Agostino-Pearson test for normal distribution for amylin levels was accepted

for nTOL (p=0.21) and rejected for TOL (p<0.0001) group. The central box

represents 25th to 75th percentile, middle line represents the median, and the

vertical line, range, excluding "far out" values (red circles). Median amylin level

was significantly raised in nTOL compared to TOL group (47.9 vs 8.7) (Mann-

Whitney test for independent samples was significant, p value <0.0001).

The details of the 2 outliers in the TOL group are as follows. The male infant

with amylin level 76.1 pmol/L was born at 29+5 weeks gestation with birth

weight 1.44 kg to a 33 year mother without any medical problems. He was fed

expressed breast milk and was on caffeine at the time of blood collection on day

12. The male infant with amylin level 67.7 pmol/L was born at 29 weeks

gestation with birth weight 1.14 kg to a 26 year mother who presented to labour

ward with premature rupture of membranes for > 12 hours and abnormal

cardiotocograph. The infant had initial features of hypoxic ischaemic

encephalopathy which settled in three days. He was also treated for coagulase

negative staphylococcal infection and abdominal distension with feed

160


intolerance until day 12. He was fed nutriprem milk with occasional vomiting and

was on caffeine at the time of blood collection on day 28.

Per

cent

age

Gas

tric

Res

idua

l Vol

ume

0

50

100

150

200

250

300

350

400

450

nTOL(n=30)

TOL(n=30)

Figure 50. Box and whisker plot of percentage GRV (y axis) in feed-intolerant

(nTOL) and feed-tolerant (TOL) neonates (x axis)

D’Agostino-Pearson test for normal distribution was accepted for nTOL

(p=0.105) and rejected for TOL (p<0.0001). The central box represents 25th to

75th percentile, middle line represents the median, and the vertical line

represents range, excluding "outside" (black circles) and "far out" (red circles)

values which are displayed as separate points. %GRV was significantly raised

in nTOL compared to TOL (150% vs 5%) (Mann-Whitney test for independent

samples was significant, p value <0.0001).

161


Day

s To

Ful

l Ent

eral

Fee

ds

0

10

20

30

40

50

nTOL(n=30)

TOL(n=30)

Figure 51. The box and whisker plot of days to reach full feeds (Y axis) in feed-

intolerant (nTOL) and feed-tolerant (TOL) neonates (X axis)

D’Agostino-Pearson test for normal distribution was rejected for nTOL and TOL

(p<0.001). The central box represents 25th to 75th percentile, middle line

represents the median, and the vertical line represents range, excluding

"outside" (black circles) and "far out" (red circles) values which are displayed as

separate points. Days to reach full enteral feeds was significantly increased in

nTOL compared to TOL group (13 days vs 5.5 days) (Mann-Whitney test for

independent samples was significant, p value <0.0001).

162


In the nTOL group, plasma amylin levels correlated with percentage gastric

residual volumes (Figure 52), days to reach full enteral feed volume of 150

ml/kg/day (Figure 53) and days to discharge (Figure 54).

0 100 200 300 400 500

0

20

40

60

80

100

120

Percentage Gastric Residual Volume (%GRV)nTOL Group (n=30)

Am

ylin

Con

cent

ratio

n (p

mol

/L)

nTO

L G

roup

(n=3

0)

Figure 52. Correlation graph showing line of best fit to assess the degree of

association between amylin concentration (y axis) and % GRV (x axis) in feed-

intolerant (nTOL) group

D’ Agostino-Pearson test for normal distribution was accepted for both % GRV

(p=0.10) and amylin levels (p=0.21). n=30, Pearson correlation coefficient,

r=0.78, p<0.0001, 95%CI 0.59-0.89.

163


add curve


incorrect


incorrect


These figure numbers are incorrect


5 10 15 20 25 30 35

0

20

40

60

80

100

120

Days To Full Enteral FeedsnTOL Group (n=30)

Am

ylin

Con

cent

ratio

n (p

mol

/L)

nTO

L G

roup

(n=3

0)


association between amylin concentration (y axis) and days to reach full enteral

feeds (x axis) in nTOL group

D’ Agostino-Pearson test for normal distribution was rejected for days to reach

full enteral feeds (p=0.001) and accepted for amylin levels (0.21). n=30,

Spearmans coefficient of rank correlation rho=0.56, p=0.001, 95% CI 0.26-0.77.

164


add curve


add curve


0 20 40 60 80 100 120

0

20

40

60

80

100

120

Days To DischargenTOL Group (n=30)

Am

ylin

Con

cent

ratio

n (p

mol

/L)

nTO

L G

roup

(n=3

0)


association between days to discharge (x axis) and amylin concentration (Y

axis) in the feed-intolerant (nTOL) group

D’ Agostino-Pearson test for normal distribution was accepted for days to

discharge (p=0.49) and amylin levels (0.21). n=30, Pearson’s correlation

coefficient r=0.43, p=0.015, 95%CI 0.092-0.68.

165


In the TOL group, plasma amylin levels did not correlate with percentage gastric

residual volumes (Figure 55), days to reach full enteral feed volume of 150

ml/kg/day (Figure 56), and days to discharge (Figure 57).

0 5 10 15 20 25

0

10

20

30

40

50

60

70

80

Percentage Gastric Residual Volume (%GRV)TOL Group (n=30)

Am

ylin

Con

cent

ratio

n (p

mol

/L)

TOL

Gro

up (n

=30)


association between % GRV (x axis) and amylin levels (y axis) in feed-tolerant

(TOL) group

D’Agostino-Pearson test for normal distribution was rejected for % GRV and

amylin (p<0.001). n=30, Spearman’s coefficient of rank correlation rho=0.33,

p=0.068, CI -0.025-0.622.

166


0 10 20 30 40 50

0

10

20

30

40

50

60

70

80

Days To Full Enteral FeedsTOL Group (n=30)

Am

ylin

Con

cent

ratio

n (p

mol

/L)

TOL

Gro

up (n

=30)


association between days to full enteral feeds (x axis) and amylin levels (Y axis)

in feed-tolerant (TOL) group

D’Agostino-Pearson test for normal distribution was rejected for days to reach

full enteral feeds and amylin (p<0.001). n=30, Spearman’s coefficient of rank

correlation rho=-0.003, p=0.98, CI -0.363-0.357.

167


add curve


0 20 40 60 80 100 120

0

10

20

30

40

50

60

70

80

Days To DischargeTOL Group (n=30)

Am

ylin

Con

cent

ratio

n (p

mol

/L)

TOL

Gro

up (n

=30)


association between days to discharge (x axis) and amylin levels (Y axis) in the

feed-tolerant (TOL) group

D’Agostino-Pearson test for normal distribution was accepted for days to

discharge (p=0.11) and rejected for amylin (p<0.001). n=30, Spearman’s

coefficient of rank correlation rho=-0.33, p=0.07, -0.61-0.03.

In conclusion the study results confirm the hypothesis that plasma amylin levels

are raised in preterm infants with feed intolerance.

11.4 Discussion

Up to 67% preterm very low birth infants struggle to attain full enteral nutrition in

the first few weeks of life (Reddy, Deorari et al. 2000; ElHennawy, Sparks et al.

2003; Patole, Rao et al. 2005; Hay 2008). Feed intolerance is the common

cause of this problem and is characterised by large gastric residual volumes

(GRV) with or without associated clinical manifestations (Jadcherla and

Kliegman 2002; Mihatsch, von Schoenaich et al. 2002; Patole 2005). Reliance

on total parenteral nutrition during this period is usually not enough to maintain

protein and lipid intake (Grover, Khashu et al. 2008; Hay 2008) and this often 168


You can’t use all these figures without a text explanatation and proper legends


leads to problems with infection and hepatic damage (Donnell, Taylor et al.

2002; Kaufman, Gondolesi et al. 2003). Notably these infants may lag behind in

protein and calorie intake by up to 6-8 weeks by the time they are ready to go

home (Grover, Khashu et al. 2008) which may have adverse effects on their

postnatal growth and neurodevelopment (Frank and Sosenko 1988; Hack,

Breslau et al. 1991; Ehrenkranz, Younes et al. 1999; Hack, Schluchter et al.

2003; Cooke, Ainsworth et al. 2004; Hay 2008). Delayed gastric emptying,

intestinal immaturity, ileus of prematurity, and gastro-esophageal reflux may all

play a role. It is not clear whether one or all of these mechanisms contribute to

the observed feed intolerance. This is the first study to evaluate the role of

amylin as an inhibitor of gastric motility in feed intolerance in the neonatal

intensive care setting.

The results of this study support the hypothesis that plasma amylin levels are

increased in neonates with delayed gastric emptying compared to controls and

these levels correlate with increasing GRV. We propose that although poor

gastric motility in premature neonates is probably multifactorial in origin, amylin

may play a role in its pathophysiology. The amylin levels in nTOL were

approximately 3.5 times that of TOL controls. These levels were comparable to

those reported in critically ill children with feed intolerance (Mayer, Durward et

al. 2002) and are amongst the highest reported levels in humans. This is in

keeping with the physiological action of amylin, as it is 15-20 times more potent

than other known inhibitors of gastric motility (Young 1997). The amylin levels in

the TOL group (8.7 pmol/L) were comparable to the Guthrie levels in healthy

preterm neonates (6.90 pmol/L). Although the median postnatal timing (9.5

days) of amylin sampling was delayed in TOL controls, this difference was not

significant compared to nTOL group (7.5 days). The main reason for this delay

was because the routine collection of blood in TOL controls was less frequent.

However the blood samples from nTOL and TOL were treated in the same way

and therefore did not bias the observed results.

The proposed mechanism of action of amylin is outlined below in Figure 58. It is

plausible that amylin may play a similar role on gastric emptying in preterm

neonates with feed intolerance.

169


repetition


See Chapter? Give page refs


page refs


Figure 58. Proposed pathway of amylin action

Nutrient intake into stomach not only stimulates insulin release but also amylin

release which inhibits gastric emptying through brain centres regulating gastric

motility. Amylin also inhibits glucagon release and further food intake, thus

reducing endogenous glucose production in the postprandial period.

The reason for increased amylin levels in nTOL neonates is unclear. We could

only speculate the possible reasons for this hyperamylinemia in the nTOL

group. As amylin is co-secreted with insulin under normal physiological

conditions, an attractive hypothesis is that transient hyperinsulinemia and

relative insulin resistance in nTOL infants may stimulate amylin release. This is

based on the observation that many extremely preterm infants develop

hyperglycemia in the first week of life during continuous glucose infusion due to

relative insulin resistance and defective islet beta cell processing of proinsulin

(Mitanchez-Mokhtari, Lahlou et al. 2004). Furthermore compared with term

170


neonates; proinsulin and C-peptide levels were higher in normoglycemic

preterm infants less than 30 weeks (23.2 +/- 0.9 vs 18.9 +/- 2.71 pmol/L and

1.67 +/- 0.3 vs 0.62 +/- 0.12 nmol/L, respectively). In addition, among low birth

weight infants, fat mobilization and beta oxidation of fatty acids in the liver is not

suppressed by glucose stimulated insulin secretion suggesting decreased

insulin sensitivity (Hemachandra and Cowett 1999). Interestingly the associated

blood glucose abnormalities were not observed in the study infants and

furthermore the glucose levels were comparable between nTOL and TOL

group. We speculated that the reason for this observation was because insulin

secretion in neonates is less closely linked to circulating blood glucose

concentrations (Hawdon, Aynsley-Green et al. 1993; Mitanchez-Mokhtari,

Lahlou et al. 2004). However we could not speculate the reasons for the

absence of hyperinsulenemia and insulin resistance in the TOL infants. It is

known that large intravenous dextrose loads may promote amylin release

(Mitsukawa, Takemura et al. 1990); however the study neonate’s received

standard glucose maintenance and remained euglycemic. Although the major

site of amylin biosynthesis is within pancreatic islet β cells, amylin secretion

other than nutrient mediated stimulation cannot be ruled out. In addition, other

potential physiological mediators of gastric emptying including GLP-1 and CCK

may potentiate the actions of amylin (Young 1997). The effects of possible

interaction of amylin with other gut peptides remain to be studied in preterm

population. Transplacental passage giving rise to raised amylin levels in nTOL

group seems unlikely as discussed previously in section 3.4 on page 34.

Studies A and B and a previous study (Mayer, Durward et al. 2002) showed

correlation between serum amylin and insulin levels indicating a degree of

preservation of pancreatic hormonal co-release. Although ideally we would like

to have repeated this in this study we were limited ethically by the blood volume

we could take in very low birth weight neonates. Unfortunately we were also

unable to measure insulin resistance.

It is plausible that the amylin levels observed in nTOL and TOL group in this

study may have been influenced by the blood sampling time and relationship to

the infants’ prandial state. This is less likely due to the observations highlighted

in section heading 3.2 and previous study observations as highlighted in section

heading 9.4 and 10.4. Notably twenty-three of thirty nTOL bloods were collected 171


page refs


repetition


when the infants were nil by mouth and 7/30 before the next scheduled feed

(reduced or same volume). Thus the observed amylin levels in nTOL could be

considered as baseline and pre-prandial in the nTOL group. We did not observe

an association of amylin levels to the timing of blood collection in the TOL group

probably due to the short interval between scheduled feeds (1-2 hours).

The mean stomach half emptying time in infants fed expressed breast milk was

shorter than formula fed infants (Ewer, Durbin et al. 1994). Additionally, gastric

distension (which may be influenced by bolus or continuous milk feeds) may

stimulate amylin release and resultant slowing of gastric emptying (Reda,

Geliebter et al. 2002). The types of milk feed were comparable between the

groups and the neonates in both the study groups received intermittent bolus

feeds.

The percentage GRV is influenced by the absolute feed volume and feeding

interval. It could be argued that the difference in % GRV simply reflects much

smaller absolute feed volume in the nTOL group and therefore the amount of

aspirate would be a much greater percentage. We do not think that the absolute

feed volumes or intervals influenced % GRV observed between the groups as

only 3 infants in the nTOL group had absolute feed volume less than 2 mls/hr.

Amylin levels correlated with percentage GRV and time to reach full enteral

feeds which were significantly increased in the nTOL group. Although it is

conceivable that gestational age or birth weight might have been responsible for

this association, a similar observation was not demonstrated in the TOL group.

Furthermore both gestational age at birth and birth weight were not statistically

different between nTOL and TOL groups. This has clinical significance since

increased length of time to reach full enteral feeding is associated with a poorer

neurological outcome (Patole 2005). Amylin may thus be used as an early

marker of delayed enteral nutrition. Although the length of stay was increased in

nTOL compared to TOL group, it did not reach statistical significance even after

stratification by gestation and birth weight.

Studies investigating feed intolerance in preterm population could be facilitated

by the availability of a consistently reliable clinical or biochemical marker of feed

intolerance. Percentage prefeed GRV to define feed intolerance has been

172


This should be in introduction



validated in children and adults against more objective measures of gastric

emptying including paracetamol absorption test, ultrasonography and

scintigraphic techniques (Mayer, Durward et al. 2002; ElHennawy, Sparks et al.

2003; Gomes, Hornoy et al. 2003; Pozler, Neumann et al. 2003). We formulated

a pragmatic definition of feed intolerance based on consensus opinion,

physiology of gastric emptying in preterm infants and previously published

definitions. Our definition of feed intolerance is similar to those used in previous

trials (Ng, So et al. 2001; Salhotra and Ramji 2004). Also the plasma amylin

levels correlated with % GRV in the nTOL group adding validity to the definition

of feed intolerance in this study. Thus more than 50% prefeed GRV may be

used as a clinical indicator of delayed gastric emptying.

With the prevailing fear of NEC and its association with feed intolerance most

caregivers are often persuaded to investigate colour of prefeed GRV, abdominal

distension, vomiting, the presence of blood in stool and apnoea and

bradycardia. However the clinical significance of each of these criteria to

establish feeding strategies and for feeding intolerance has yet to be

determined. Most clinicians and nurses determine daily feeding strategy based

on greenish aspirates, vomiting or abdominal girth. We also monitored and

recorded abdominal girth, colour of the gastric residuals and emesis. Notably

green gastric residuals in ELBW infants were not negatively correlated with

feeding volume on day 14 (Mihatsch, von Schoenaich et al. 2002). Only seven

infants in this study showed vomiting and abdominal distension (more than

2cm) at the time of sample collection (4 nTOL and 3 TOL). Although this is a

small sample size, we did not see an association with NEC, a delayed time to

achieve full enteral feeds, PDA or amylin levels.

Amylin is raised in IMDM, and may be affected by intestinal ischaemic injury

and acute inflammatory conditions (Joyce, Fiscus et al. 1990; Clementi, Caruso

et al. 1995; Parida, Schneider et al. 1998; Hall and Brain 1999; Onuoha and

Alpar 1999; Phillips, Abu-Zidan et al. 2001; Kairamkonda, Deorukhkar et al.

2005) influencing our exclusion criteria. The inflammatory markers such as C-

reactive protein, white cell count and neutrophil count were comparable

between the groups and therefore not attributable to the observed difference in

the amylin levels.

173


Amylin was found to be significantly elevated in the ischaemia-reperfusion

group that had clamping of the superior mesenteric artery for 60 min followed by

15 min reperfusion (median 39 pM, range 30-44) compared with the Sham

operated group that underwent laparotomy and isolation (without clamping) of

the superior mesenteric artery (19 pM, range 15-45; Mann-Whitney U, p < 0.05)

and the Control group that underwent no surgery (24 pM, range 15-55; p < 0.

01) (Phillips, Abu-Zidan et al. 1999). The same researchers observed a positive

correlation between the histologic score of the intestinal injury and the

measured plasma amylin concentration (R = 0.48, P =.007) (Phillips, Abu-Zidan

et al. 2001). Thus amylin is increased in intestinal ischeamia and its levels are

related to the degree of intestinal ischaemic injury in rats. However none of the

infants in nTOL and TOL groups experienced necrotising enterocolitis or

significant abdominal pathology. In addition, amylin levels were not associated

with vomiting and abdominal distension (more than 2cm) in 4 nTOL and 3 TOL

infants at the time of sample collection.

Objective assessment of gastric emptying may have added validity to this study,

however with intra-observer variability, safety, ethical issues, and the need for

repeated blood sampling in our preterm population render these assessments

prohibitive. Serial measurement of amylin levels may have helped to establish

the critical amylin level associated with resolution of feed intolerance. However

multiple samples could not be taken from this population. A degree of selection

bias was unavoidable, as we aimed to include neonates with and without gastric

stasis, reflective of our intensive care population. The strengths of the study

were the adherence to sample processing and storage protocol and the blood

samples were obtained during times when blood was collected for routine

clinical monitoring which minimised any extra discomfort to the infant.

This study has implications for future research in preterm infants with feed

intolerance. In particular, further studies are warranted to establish beyond

doubt that amylin is responsible for delayed gastric emptying in preterm

neonates. Furthermore, although we established that neonates with feed

intolerance have high amylin levels; it is unclear why amylin is increased in

these preterm but well neonates. The possible areas of interest are the role of

hyperinsulinemia, insulin resistance, amylin receptor insensitivity and other gut

peptides. This study may also stimulate future research into the effect of bolus 174


versus continuous feeds on amylin secretion (stretch receptors) and gastric

emptying in preterm population.

Amylin receptor antagonists are being developed for animal studies. It would be

premature to discuss pharmacological blockade of amylin peptide as one of the

therapeutic option in feed intolerance until further studies firmly establish the

conclusions of this study. In addition considerable caution is warranted as it is

not known if amylin by virtue of its association with feed intolerance in preterm

infants plays a protective role on the immature gut. In addition there is a

possibility of adverse effects due to the blockade of amylin’s other biological

functions.

11.5 Conclusions

Amylin by virtue of its inhibitory action on gastric emptying may play a role in its

etiology.

The study results confirmed the hypothesis that serum amylin levels are

increased in preterm infants with feed intolerance compared to preterm infants

without feed intolerance.

Serum amylin concentration, percentage gastric residual volume and time to

reach full enteral feeds were significantly raised in nTOL group compared to

TOL group. Thus measurement of serum amylin level in preterm infants may

have clinical and revenue implications.

Definition of feed intolerance in preterm infants (> than 50% prefeed GRV prior

to the next scheduled feed) correlated with amylin levels. Increased GRV may

be associated with feed intolerance and may be a useful surrogate marker for

feed intolerance in preterm neonates.

175

Chapter 11 – Conclusions of the dissertation

Chapter 12- Conclusions of the Dissertation

Amylin is a potent inhibitor of gastric emptying and plays an important role in

glucose homeostasis.

Feed intolerance is common in preterm infants and infants born to mothers

whose pregnancy was complicated by diabetes.

We therefore set out to test the hypothesis that serum amylin levels are

increased in preterm infants with feed intolerance. We also hypothesized that

serum amylin levels are raised in infants born to mothers whose pregnancy was

complicated by diabetes.

It was evident from literature that there was no uniform validated definition of

feed intolerance in neonatal population. Additionally a gold standard test for

gastric emptying in new-born population especially preterm infants was not

available. Therefore a pragmatic definition of feed intolerance was formulated

based on consensus opinion, physiology of gastric emptying in preterm infants

and previous published literature.

The primary aims of the study were to (i) establish normal serum amylin

concentrations at birth and postnatal day 5 in healthy preterm and term infants

(ii) determine amylin levels in infants of diabetic mothers and (iii) determine

amylin levels in preterm infants with feed intolerance. The secondary aims were

to (i) investigate if amylin and insulin were indeed co-secreted (ii) investigate

agreement of serum amylin levels in paired umbilical cord arterial and venous

blood (iii) investigate the relationship between serum amylin concentrations and

gastric residual volumes (GRV) (iv) investigate the relationship between serum

amylin concentrations and length of hospital stay.

To meet the aims and objectives, three studies were designed and conducted in

parallel. Prospective observational and case control study designs were

adopted to answer the hypotheses.

The median serum amylin levels in term infants at birth and postnatal day 5

were 6.10 (3.30-9.70) and 5.65 (3.10-8.20) pmol/L respectively. The median

serum amylin in preterm infants at birth and postnatal day 5 were 4.60 (1.90-

176

Chapter 11 – Conclusions of the dissertation

8.30) and 6.90 (2.75-9.50) pmol/L respectively. These levels were comparable

to those seen in children and adults.

Amylin like insulin may have ontogenetic significance as it correlated with both

birth weight and gestation.

Paired amylin levels agreed in umbilical cord arterial and venous blood. Either

sampling method could therefore be used in future research involving amylin

levels in cord bloods.

Amylin and insulin levels correlated in paired blood samples suggesting an

association between these peptides and possible co-secretion.

Amylin levels were significantly increased at birth and postnatal day 5 in

preterm (32.00 and 23.40) and term (34.30 and 25.20) infants born to mothers

whose pregnancy was complicated by diabetes which may explain the observed

feed intolerance.

Amylin levels were significantly raised in preterm neonates with delayed gastric

emptying compared to controls (47.9 vs 8.7 pmol/L).

The serum amylin levels correlated with gastric residual volumes providing

validity of GRV as a marker of feed intolerance due to delayed gastric emptying.

Thus the study definition of feed intolerance (prefeed residual gastric volumes

of more than 50% on 2 consecutive occasions) may be considered to be

reliable for future studies.

The serum amylin levels in preterm neonates also correlated with time to reach

full enteral feeds and days to discharge which has clinical and revenue

implications.

Overall the study has achieved its intended aims and objectives. The study

made original contribution to establishing normative data of amylin levels in the

new-born population and explored its significance in infants born to mothers

whose pregnancy was complicated by diabetes and preterm infants with feed

intolerance.

177


You say not significant on p136


One or two places of decimals

Chapter 12 – Implications for future research

Chapter 13- Implications for Future Research

There are several areas that I was able to identify during the course of this

study that have implications for future research.

One of the major handicaps to analytical studies in preterm population is the

volume of blood required for ELISA and similar tests. Our study methodology

was therefore restricted to opportunistic blood collections due to the volume of

blood required especially in preterm infants. Dried blood spot (DBS) technology

has been shown to be useful in pharmacokinetic-pharmacodynamic (PK-PD)

studies including preterm population. Although this technology is expensive,

future study designs limited by volume of blood collected in preterm infants

should explore this technique.

It is important to establish placental passage of amylin to make sense of the

postnatal amylin concentrations we established in new-born population. Future

studies of this hormone should investigate maternal amylin levels prior to birth

of their baby. This is especially relevant in infants of diabetic mothers in addition

to measurement of amylin levels from umbilical cord blood and serial postnatal

samples. Alternatively studies on perfused human placenta at various amylin

concentrations may help answer this question conclusively.

Amylin is increased in rat intestinal ischaemia (Phillips, Abu-Zidan et al. 2001)

and may have a biologic role in sepsis (Parida, Schneider et al. 1998). Future

research exploring the role of amylin in necrotising enterocolitis and sepsis in

the neonatal population may benefit from normal values established by our

study.

It is theorised that exposure to maternal diabetes and alterations in fetal

glucose, insulin, or other factors results in altered fetal adaptation and changes

in normal fetal programming (Pettitt, Aleck et al. 1988). Evidence of altered fetal

programming (reduced insulin sensitivity) is shown to be present before birth in

large for gestation neonates (Dyer, Rosenfeld et al. 2007). Thus high amylin

levels in IMDM could be explained by hyperinsulinaemia. However the reason

for increased amylin levels in feed intolerant preterm neonates is unclear. As

amylin is co-secreted with insulin under normal physiological conditions, an

178


attractive hypothesis that could be put to clinical test is that transient

hyperinsulinemia and relative insulin resistance may stimulate amylin release.

The adult gold standard of euglycemic clamp to study insulin resistance would

not be feasible and safe in neonatal population due to the invasive nature of the

procedure. However it is possible to study glucose/insulin and insulin/cortisol

ratios (Gesteiro, Bastida et al. 2009) to assess insulin resistance in neonatal

population. Other possible areas of interest are the role of amylin receptor

insensitivity and interaction with other gut peptides such as CCK, GLP-1, leptin

and ghrelin.

Other potential physiological mediators of gastric emptying including GLP-1 and

CCK may potentiate the actions of amylin (Young 1997). Therefore studies

investigating the interaction with other gut peptides including ghrelin may help

understand the conundrum of the pathophysiology of gastric emptying in new-

born infants with feed intolerance. There is an urgent need for validation of non-

invasive techniques for gastric emptying assessments in new-born infants and

preterm extremely low birth weight infants. Employment of these validated tests

of gastric emptying in future studies may add validity to the biological role of

amylin and other gut peptides in the pathophysiology of feed intolerance in

humans.

Amylin release is also known to be influenced by gastric distension and stretch

receptors. The results of this study may stimulate future research into the effect

of bolus versus continuous feeds on amylin secretion (stretch receptors) and

gastric emptying in preterm population.

Amylin receptor antagonists are being developed for research purposes in

animals. Amylin receptor blockade would seem to be one of the potential

therapeutic options for feed intolerance in preterm infants. However

considerable caution is warranted as it is not known if amylin by causing feed

intolerance in preterm neonates has a protective role on the immature

gastrointestinal tract. As highlighted before amylin has other important biological

function in addition to gastric emptying and glucose homeostasis. Thus

pharmacological blockade of this peptide as a potential therapeutic option could

expose the preterm infant to greater risk of necrotising enterocolitis in addition

to other possible adverse effects.

179


Obesity is a worldwide problem with significant health and economic problems.

Amylin by virtue of its central and peripheral action on the stomach acts as an

anorectic hormone. Thus synthetic analogues of amylin may be developed as

anorectic agents that may have therapeutic implications in the management of

childhood and adult obesity.

180

Chapter 14 – Appendices

Chapter 14- Publications arising from this Project

14.1 Amylin peptide levels are raised in infants of diabetic mothers. Arch Dis Child. 2005 Dec; 90(12):1279-82

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185


14.2 Amylin peptide is increased in preterm neonates with feed intolerance. Arch Dis Child Fetal Neonatal Ed. 2008 Jul; 93(4):F265-70

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Chapter 15- Appendices

Appendix 1.Basic principle of luminescent ELISA

The basic steps in any luminescent ELISA are as follows:

1. Antibodies or antigens are passively adsorbed to solid surfaces such as

commercially available 96-well format microtiter plates for use in ELISA.

2. As one of the reactants in the ELISA is attached to a solid-phase, the

separation of bound and free reagents is made by simple washing

procedures.

3. The result of an ELISA is a color reaction that can be observed by eye

and read rapidly using specially designed multichannel

spectrophotometers allowing data to be stored and analysed statistically

(Crowther 1995) .

Step 1. Analyte-specific antibody (capture antibody) is pre-coated onto a

microplate. The sample is added and any analyte present is bound by the

immobilized antibody.

192

Analyte

Analyte-specific antibody


Step 2. An enzyme-linked analyte-specific detection antibody binds to a second

epitope on the analyte forming the analyte-antibody complex.

Step 3. Substrate is added and converted by the enzyme, thereby producing a

colored product or light emission in direct proportion to the amount of analyte

bound in the intial reaction (signal increases as concentration increases).

Step 4. A standard curve is prepared from the data produced from the serial

dilutions with concentration on the x axis (log scale) versus absorbance on the

Y axis (linear). The concentration of the sample is interpolated from this

standard curve.

Nonlinear regression is often used to fit standard curves generated by ELISA

assay which is based on competitive binding. The assaying compound

competes for binding to an enzyme or antibody with a labeled compound.

193

Antibody-Analyte Conjugate

Substrate- Light or Colour


Therefore the standard curve is described by equations for competitive binding.

One-site competitive binding curve is tried first. If that doesn’t fit the data well,

sigmoid curves are tried, by entering the X values as the logarithms of

concentrations. Choice of an equation is important when using nonlinear

regression, if the equation does not describe a model that makes scientific

sense; the results of nonlinear regression won’t make sense either. With

standard curve calculations, the choice of an equation is less important because

all one has to do is assess visually that the curve nicely fits the standard points.

194


Appendix 2. Human Amylin (Total) ELISA KIT and Assay Procedure

The Human Amylin ELISA is a monoclonal antibody-based sandwich

immunoassay for determining total amylin levels in human plasma. The capture

antibody recognizes reduced human amylin and human amylin acid

(deamidated amylin) but not the 1-20 fragment of amylin. The detection

antibody binds to reduced or unreduced human amylin but not amylin acid and

is complexed with streptavidin-alkaline phosphatase to form a sandwich. The

substrate, 4-Methylumbelliferyl Phosphate (MUP), is applied to the completed

sandwich and the fluorescent signal, monitored at 355 nm/460 nm, is

proportional to the amount of amylin present in the sample.

After receipt of the kit, all components of the kit were stored at 2-8 degrees

centigrade. The assay is run in duplicate using 50 µl of assay buffer and 50 µl of

Standard, Control, or Sample in each well. The assay procedure is as follows:

The concentrated TBS (Tris Buffered Saline) wash buffer is diluted 10 fold by

mixing the entire contents of the 10 X wash buffer with 270 ml deionized water.

Standards and QCI and QC2 (lyophilized) vials is reconstituted with 250 µl of

deionoized water. The solution is mixed gently for at least 5 minutes to assure

complete reconstitution.

The microtitre assay plate is removed from the foil pouch and each well is filled

with 300 µl of diluted TBS wash buffer which is incubated at room temperature

for 10 minutes without shaking.

Wash buffer is decanted from the plate and the residual amount removed from

all wells by inverting the plate and tapping it smartly onto absorbent towels

several times. The well is not allowed to dry before proceeding to the next step.

50 µl of assay buffer is added to each well. 50 µl Standards, Samples and

Controls are added in duplicates. The wells are orientated as highlighted below.

The plate is sealed and incubated at room temperature on the shaker for one

hour. It is important to note that the incubation start time is the time as plate was

loaded on the shaker, not from the time one started loading the plate with

samples. The residual amount from all wells is decanted and removed by

195


inverting the plate and tapping it smartly onto absorbent towels several times.

Microtitre plate arrangements of wells is as shown below

The plate is washed 3 times with 300 µl per well TBS wash buffer. The plate is

decanted and tapped after each wash to remove residual buffer.

100 µl detection antibody is added to each well, plate is covered with sealer,

and incubated on the shaker at room temperature for 2 hours.

Near the completion of this incubation step, the substrate is hydrated by adding

1 ml deionized water to 10 mg, mixed well, and allowed to stand for 15 minutes

with occasional mixing to assure complete dissolution. 105 µl is removed from

the reconstituted substrate and added to the 21 ml vial of substrate diluent,

mixed well, referred to as substrate solution from here on.

Detection antibody is decanted and the residual amount removed from all wells

by inverting the plate and tapping it smartly onto absorbent towels several

times.

196

1 2 3 4 5 6 7 8 9 10 11 12

A 0 pM

0 pM

1 pM

1 pM

2 pM

2 pM

5 pM

5 pM

10 pM

10 pM

40 pM

40 pM

B 100 pM

100 pM

QC1 QC1 QC2 QC2 Sample 1

Sample 1

Sample 2

Sample 2

Sample 3

Sample 3

C

D

E

F

G

H


The plate is washed 3 times with 300 µl per well TBS wash buffer, decanted

and tapped after each wash, to remove residual buffer.

100 µl substrate solution is added to each well and incubated for 15 minutes at

room temperature in the dark without shaking.

A reading is taken and the RFU (Relative Fluorescence Unit) of the top

standard point is noted; when the reading is 2000 RFU or greater, 50 µl stop

solution is added, gently mixed, and read on the fluorescence plate reader after

5 minutes.

The RFU can be fitted directly to the concentration. If the curve fitting software

is available, the best fit can be obtained with a linear-linear spline fit. Since this

assay is a direct ELISA, the RFU is directly proportional to the concentration of

total human amylin in the sample. It is important to note that when sample

volumes assayed differ from 50 µl, an appropriate mathematical adjustment

must be made to accommodate for the dilution factor (e.g. if 25 µl of sample is

used, then calculated data must be multiplied by 2).

197


Appendix 3. Characteristics of Human Amylin (Total) ELISA Kit

The human amylin ELISA kit had the following assay characteristics:

Sensitivity: The lowest level of total human amylin that could be detected by this

assay is 1 pM (50 µl plasma sample size).

Performance: The parameters expressed as mean ± standard deviation of

assay performance are:

Parameters Mean ± SD

ED 80 82 ± 2 pM

ED 50 54 ± 5 pM

ED 20 2 6± 4 pM

Crossreactivity: The cross reactivity of the assay is as follows:

Peptides Percentage

Human Glucagon <1%

Human GLP-1 <1%

Human Insulin <1%

Human Pancreatic Polypeptide <1%

Human Adrenomedullin 1%

Human Calcitonin <1%

Calcitonin Gene Related Peptide <1%

Precision: The assay variation of Linco Human Amylin ELISA kits were studied

at three different spiked concentrations of amylin in three different human

plasma samples. The within variations was the mean from four duplicate

determinations in a single assay. The between variation was the mean value of

the mean of four duplicate determinations in each plasma across four assays.

Within and between assay variation is as follows:

198


Sample No Amylin Added pM Within % CV Between % CV1 20 2.6 11.9

50 5.0 12.980 14.8 11.5

2 20 11.5 17.450 2.5 17.880 7.9 16.2

3 20 7.8 17.650 5.4 11.980 7.3 13.0

Recovery: Varying concentrations of Human Amylin were added to three human

plasma samples and the amylin content was determined in four different ELISA

assays. The % of recovery=observed amylin concentration/expected amylin

concentration x 100%. Spike and recovery of total human amylin in human

plasma was as follows:

Sample Sample Concentration (pM)

Amylin % of Recovery

1 3.25 20 10450 9980 91

2 2.56 20 9650 8980 89

3 11.23 20 10150 8780 90

Linearity: Three human plasma samples with the indicated sample volumes

were assayed in four different assays. Required amount of assay buffer was

added to compensate for lost volumes below 50 µl. The resulting dilution factors

of 1.0, 1.25, 2.0, and 5.0 representing 50 µl, 40 µl, 25 µl, and 10 µl sample

volumes assayed, respectively, were applied. The effect of plasma dilution was

as follows:

199


Sample No

Volume Sampled

Expected pM Observed pM %of Expected

1 50 22.32 22.32 10040 21.13 9525 19.98 9010 18.55 83

2 50 25.34 25.35 10040 22.30 8825 20.54 8110 21.05 83

3 50 20.54 20.54 10040 20.85 10125 15.96 7810 19.65 96

200


Appendix 4. Research protocol for ethics approval, South Sheffield

Research Ethics Committee

Title: Amylin peptide: Association with feed intolerance in preterm infants and

IMDM- Observational & Case-Control Study

Study Sponsor: University Hospitals of Sheffield NHS Trust

Study Summary

Title Amylin peptide: Association with feed

intolerance in preterm infants & IMDM

Short Title Amylin peptide

Methodology Prospective Observational Study and Case-

Control Study


Study Centres Single Centre (Study A & B ) & Two Centre

(Study C)

Objectives To determine serum amylin levels in well

preterm and term infants (Study A), infants of

diabetic mothers (Study B) and preterm

infants with feed intolerance (Study C).

Number of Samples/Participants Study A=200 samples

Study B= 21 IDM

Study C= 20 nTOL and 20 TOL

Main Inclusion Criteria 1. Infants ≥ 24 weeks gestation

2. Infants of diabetic mothers

3. Preterm infants (24-36 weeks) admitted to

the neonatal intensive care unit and requiring

gastric tube feeding

Statistical Methodology Median and interquartile ranges and non-

parametric tests. Correlation Coefficient will

be performed to evaluate the relationship

between variables. Bland altman test will be

applied for method comparison between

umbilical arterial and umbilical venous blood

sampling method.

Outcome variable Serum amylin levels in pmol/l

201


Background: The goal of neonatal nutrition is the attainment of normal growth

and development. However impairment of gastric motility is common in critically

ill patients (1) and failure to establish enteral feeds in neonates necessitates the

use of parenteral nutrition which carries a significant increased risk of morbidity

and mortality (2). There is scant information on the mechanisms relating to feed

intolerance in these patients, therefore limiting the therapeutic options.

Insight into the regulation of gastric motility has been advanced by the recent

discovery of ‘amylin’, a novel 37 amino-acid peptide hormone from the

calcitonin gene-related peptide family. As a potent inhibitor of gastric emptying it

plays an important physiological role in the control of carbohydrate absorption

by regulating the efflux from the stomach to the small bowel. The

pathophysiological role of amylin is well documented in adult patients with

diabetes mellitus (1), where inappropriately high levels have been reported with

delayed gastric emptying.

There is no published data on amylin levels and its role in neonatal population.

We aim to determine normal amylin levels in neonatal population and

investigate its role in infants born to mothers whose pregnancies were

complicated by diabetes (IMDM) & preterm infants with feed intolerance.

Rationale and significance of the study: Up to 67% of infants weighing less than

1500 g (1-3) experience feed intolerance resulting in prolonged parenteral fluid

therapy, increased hospital stay, increased risk of sepsis and consequently

increased cost of treatment (4). Feed intolerance is also a major problem in

IMDM (5). An increased prefeed gastric residual volume is a commonly used

measure of feed intolerance in newborn infants.

Decreased gastrointestinal motility can be multifactorial in origin, we

hypothesize that amylin may have an important role to play in its

pathophysiology. Amylin is novel “brain-gut peptide” which is co-secreted with

insulin from the pancreas (6). In conjunction to playing a role in glucose

homeostasis, it is a potent inhibitor of gastric emptying (7). A study in critically ill

infants and children showed elevated amylin levels which correlated with

delayed gastric emptying and feed intolerance (8).

202


We aimed to investigate the role of amylin in newborn infants with feed

intolerance which could potentially form the basis of a subsequent clinical trial.

Research question: Is amylin elevated in neonates with feed intolerance

(delayed gastric emptying)?

Aims and Objectives: 1. To determine reference range of amylin at birth

(umbilical cord) and at 5-7 days (Guthrie) in term and preterm neonates (Study

A). 2. To determine amylin levels in IMDM (Study B). 3. To determine amylin in

preterm neonates with feed intolerance (study C).

Study design: Prospective observational and case-control study to be

conducted simultaneously as 3 sub-studies. Study A- To establish normal

ranges of cord and postnatal amylin levels, mothers will be recruited antenatally

for umbilical cord blood and inpatient Guthrie blood. Study B- All infants of

diabetic mothers will be recruited for cord bloods, Guthrie bloods, and

opportunistic bloods taken at the time of routine glucose measurement. Study

C- Preterm infants admitted to neonatal intensive care unit and requiring tube

feeding will be recruited. Amylin levels of feed intolerant infants (case) will be

compared to feed tolerant infants (control)

Study population: Study A-Term and preterm infants > 24 weeks of gestation,

Study B-Term IMDM and preterm IMDM > 24 weeks of gestation and Study C-

Preterm infants > 24 weeks of gestation

Exclusion Criteria

1. Infants born less than 24 weeks gestation

2. Congenital malformation

3. Chromosomal anomaly

4. Structural gastrointestinal abnormalities and necrotising enterocolitis

5. Haemoglobin less than 7 g/dl

6. Breast feeding where feed volume cannot be determined

Primary outcome measure: Serum amylin levels (pmol/l)

203


Definition of feed tolerance: Feed intolerance will be defined as prefeed gastric

residual volumes of greater than 50% of the previous feed on 2 consecutive

occasions (4).

Method of collection and analysis: 0.5 ml-1ml blood will be drawn into a tube

containing EDTA with 500 IU of aprotinin per millilitre of blood and kept on ice

before separation and stored at -70˚C. All samples will be analysed for total

amylin levels using monoclonal antibody-based sandwich immunoassay. The

amylin ELISA kit is validated for the measurement of total amylin from EDTA

human plasma samples. It has sensitivity to 1 pM and a standard range of 1-

100 pM. Were possible, plasma immunoreactive insulin will be measured by

ELISA. Blood glucose will be measured by the glucose oxidase method. The

assays will be performed by technical team blinded to the patient data led by Mr

R Fraser.

Setting: Labour ward and neonatal intensive care unit, Jessop wing, Royal

Hallamshire Hospital. Study C will also be conducted at the neonatal intensive

care unit, Doncaster General Hospital.

Consent: Parents will be recruited prior to delivery and written consent obtained.

Study Plan: Estimated sample volume is 0.5-1.0 ml. Prefeed gastric aspirates

are recorded as part of routine neonatal practise. Data will be collected with

reference to method of nutrition, demographic data and medication at the time

of sample collection.

Study A: Mothers of babies having normal, preterm and LSCS deliveries will be

recruited. Cord blood will be obtained immediately after delivery of these

babies. A repeat sample will be taken at the same time as routine Guthrie from

babies who are inpatients. These will be analysed for amylin and insulin.

Study B: Cord and Guthrie blood and a daily sample taken at the time routine

blood sugar (hypoglycaemia protocol) of infants of diabetic mothers, and will be

analysed for amylin and insulin.

Study C: Preterm infants with apparent feed intolerance will be recruited when

delayed gastric emptying is observed. Samples for amylin, insulin and glucose

will be taken at the next routine phlebotomy. 204


Analysis: A sample size of 200 in study A and 20 each in study B and study C

will have a 90% power to detect a difference in means of 15 (the difference

between a group 1 mean of 37.7 and group 2 mean of 22.7) assuming that the

common standard deviation is 13 using a two group t-test with a 0.05 two sided

significance level.

Benefits and risks: There will be no benefit to the babies included in the trial.

However there are no known risks involved. The information we get from this

study may help us to understand the mechanisms of delayed emptying of the

stomach.

Confidentiality: Patient data will be allocated a unique number and only the

research team will have access to family identities. All patient information will be

stored in locked filing cabinet and patient information will be stored on password

protected PC’s at Jessop wing, Royal Hallamshire hospital.

Resource Requirements

Amylin Kits £270/Kit £1080

Serum tubes £50

Technical staff time £0

Trasylol £10

Dissemination and Outcome: The data will be presented at local, national and

international meetings. The results will be published in pediatrics research

journals.

References

1. Dive A et al. Crit Care Med 1994;22:441-447

2. Yu VY. Acta Med Port 1997;10(2-3):185-196

3. Premji SS et al. Cisapride: A review of the evidence supporting its use in

premature infants with feeding intolerance. Neonatal Netw 1997; 16: 17-

21

4. Gross SJ. Growth and biochemical response of preterm infants fed

human milk or modified infant formula. N Engl J Med 1983; 308:237-241.

5. Gross SJ et al. Feeding the low birth weight infant. Clin Perinatol 1993;

20: 193-209.205


6. Reddy PS et al. Double blind placebo controlled study on prophylactic

use of cisapride on feed intolerance and gastric emptying in preterm

neonates. Indian Pediatr 2000; 37: 837-844

7. Kitzmiller J L. Am J Obstet Gynecol 1978; 131: 560.

8. Ludvig B et al. Amylin: History and overview. Diabetic Med 1997; 14: S9-

S13

9. Macdonald IA. Amylin and the gastrointestinal tract. Diabetic Med 1997;

14: S24-S28.

10.Mayer AP. Amylin is associated with delayed gastric emptying in critically

ill children. Intensive Care Medicine. 2002; 28(3): 336-340.

206


Appendix 5. Research protocol for ethics approval, Doncaster

Research Ethics Committee

Amylin peptide: Association with feed intolerance in preterm infants- A Case-

Control Study

Study Sponsor: Doncaster General Hospital NHS Trust

Study Summary

Title Amylin peptide: Association with feed

intolerance in preterm infants

Short Title Amylin peptide

Methodology Case-Control Study


Study Centre Single Centre

Objectives To determine serum amylin levels in preterm

infants with feed intolerance.

Number of Samples/Participants 20 nTOL and 20 TOL

Main Inclusion Criteria Preterm infants (24-36 weeks) admitted to the

neonatal intensive care unit and requiring

gastric tube feeding

Statistical Methodology Median and interquartile ranges and non-

parametric tests. Corelation Coefficient will be

performed to evaluate the relationship

between variables.

Outcome variable Serum amylin levels in pmol/l

Background: The goal of neonatal nutrition is the attainment of normal growth

and development. However impairment of gastric motility is common in critically

ill patients (1). Failure to establish enteral feeds in neonates necessitates the

use of parenteral nutrition which carries significantly increased risk of morbidity

and mortality (2). There is scant information on the mechanisms relating to feed

intolerance in these patients, therefore limiting the therapeutic options.207


Insight into the regulation of gastric motility has been advanced by the discovery

of ‘amylin’, a novel 37 amino-acid peptide hormone from the calcitonin gene-

related peptide family. As a potent inhibitor of gastric emptying it plays an

important physiological role in the control of carbohydrate absorption by

regulating the efflux from the stomach to the small bowel. The

pathophysiological role of amylin is well documented in adult patients with

diabetes mellitus (1), where inappropriately high levels have been reported with

delayed gastric emptying.

We have previously established normal serum amylin levels in umbilical cord

and Guthrie blood samples from healthy preterm and term new-born infants.

This study is planned to investigate the role of amylin in preterm infants with

feed intolerance.

Rationale and significance of the study: Feed intolerance has been reported in

up to 67% of infants weighing less than 1500 g (1-3) resulting in prolonged

parenteral fluid therapy, increased hospital stay, increased risk of sepsis and

consequently increased cost of treatment (4).

Decreased gastrointestinal motility can be multifactorial in origin, we

hypothesize that amylin may have an important role to play in its

pathophysiology. Amylin is a novel “brain-gut peptide” which is co-secreted with

insulin from the pancreas (6). In conjunction to playing a role in glucose

homeostasis, it is a potent inhibitor of gastric emptying (7). A study examining

critically ill infants and children with feed intolerance showed elevated serum

amylin levels (8).

We aim to establish the association of feed intolerance to elevated amylin levels

in the neonatal population which could potentially form the basis of a

subsequent clinical trial using a commercially available amylin antagonist.

Research question: Is amylin elevated in neonates with delayed gastric

emptying?

Aim and Objectives: To establish amylin levels in preterm neonates with

delayed gastric emptying (feed intolerance).

208


Study design: This is a case-control study. The cases will be preterm infants

admitted to neonatal intensive care unit who subsequently develop feed

intolerance. Preterm infants without feed intolerance matched for birth weight,

gestation and postnatal age will be controls.

Study population: Preterm infants between 24-36 weeks of gestation

Exclusion Criteria

1. Congenital malformation

2. Chromosomal anomaly

3. Structural gastrointestinal abnormalities and necrotising enterocolitis

4. Breast feeding where feed volume cannot be determined

5. Infant born to mother whose pregnancy is complicated by diabetes

6. Primary outcome: Serum amylin levels in infants with and without feed

intolerance.

Definition of feed intolerance: In this population feed intolerance will be defined

as, prefeed gastric residual volume (GRV) of greater than 50% of the previous

feed (4), increase in the abdominal girth from baseline by more than 2 cm in the

region of the epigastrium over 24 hours by regular monitoring and vomiting.

Method of collection and analysis: Blood for amylin and insulin will be drawn

into a tube containing EDTA with 500 IU of aprotinin per millilitre of blood and

kept on ice before separation and stored at -70˚C. The total amylin levels in

human plasma are assayed using an ELISA monoclonal antibody-based

sandwich immunoassay. The amylin ELISA kit is validated for the measurement

of total amylin from EDTA human plasma samples. It has sensitivity to 1 pM and

a standard range of 1-100 pM. Plasma immunoreactive insulin will be measured

by ELISA. Blood glucose will be measured by the glucose oxidase method. The

assays will be done by technical team under R Fraser who will be blinded to the

patient data.

Setting: Neonatal intensive care unit, Doncaster Royal Infirmary

209


Consent: Parents of infants admitted to the neonatal unit and requiring tube

feeding will be approached for consent.

Study Plan: All the infants admitted to the neonatal intensive care unit and

requiring tube feeding will be eligible for the study. The feeding regime of the

neonates will be dictated by the local guideline. Expressed breast milk (EBM) is

encouraged, and formula feed is used if breast milk is not available. The

neonates are fed every hour and the volumes increased as clinically tolerated

but not more than 20mls/kg birth weight/day. Every four hours pre-feed GRV will

be recorded as a percentage of the previous feed volume. Abdominal girth,

colour of gastric residuals and emesis will also be noted. If this GRV is greater

than 50% on two consecutive occasions, neonates will be classified as feed-

intolerant (nTOL). The further management of these neonates is up to the

attending neonatologist. Researcher or responsible consultant will identify

preterm infants introduced to gastric tube feeding who are receiving > 30 ml/kg

or > 25% of total fluid intake as enteral feeds. Parents will be approached for

consent and information leaflets provided. Blood for amylin will be collected

within 2 hours of the second big (> 50%) prefeed GRV in nTOL neonates. 0.5

ml blood will be collected from capillary, vein, or indwelling arterial catheters as

deemed necessay for routine collection. Feed-tolerant (TOL) neonates matched

for gestational age, birth weight and postnatal age will serve as controls. 0.5 ml

blood for amylin in these neonates will be collected during their next routine

collection. Data will be collected with reference to method of nutrition,

demographic data and medication at the time of sample collection.

Analysis: A sample size of 20 each in nTOL and TOL will have a 90% power to

detect a difference in means of 15 (the difference between a group 1 mean of

37.7 and group 2 mean of 22.7) assuming that the common standard deviation

is 13 using a two group t-test with a 0.05 two sided significance level.

Benefits and risks: There will be no benefit to the babies included in the trial.

However there are no known risks involved. The information we get from this

study may help us to understand the mechanisms of delayed emptying of the

stomach.

210


Confidentiality: Patient data will be allocated a unique number and only the

research team will have access to family identities. All patient information will be

stored in locked filing cabinet and patient information will be stored on password

protected PC’s at Women’s Hospital, Doncaster Royal Infirmary

Resource Requirements

Amylin Kits £270/Kit £270

Serum tubes £50

Trasylol £10

Dissemination and Outcome: The data will be presented at local, national and

international meetings. The results will be published in paediatrics research

journals.

References

1. Dive A et al. Crit Care Med 1994;22:441-447

2. Yu VY. Acta Med Port 1997;10(2-3):185-196

3. Premji SS et al. Cisapride: A review of the evidence supporting its use in

premature infants with feeding intolerance. Neonatal Netw 1997; 16: 17-

21

4. Gross SJ. Growth and biochemical response of preterm infants fed

human milk or modified infant formula. N Engl J Med 1983; 308:237-241.

5. Gross SJ et al. Feeding the low birth weight infant. Clin Perinatol 1993;

20: 193-209.

6. Reddy PS et al. Double blind placebo controlled study on prophylactic

use of cisapride on feed intolerance and gastric emptying in preterm

neonates. Indian Pediatr 2000; 37: 837-844

7. Kitzmiller J L. Am J Obstet Gynecol 1978; 131: 560.

8. Ludvig B et al. Amylin: History and overview. Diabetic Med 1997; 14: S9-

S13

9. Macdonald IA. Amylin and the gastrointestinal tract. Diabetic Med 1997;

14: S24-S28.

10.Mayer AP. Amylin is associated with delayed gastric emptying in critically

ill children. Intensive Care Medicine. 2002; 28(3): 336-340.

211


Appendix 6. Child participation information leaflet (Study A & B)

CHILD PARTICIPATION INFORMATION LEAFLET

Neonatal Intensive Care Unit, Jessop Wing, Royal Hallamshire Hospital

Study title: Amylin peptide: Association with feed intolerance in preterm infants

and IMDM.

What is this study about?

You and your baby are being invited to take part in a research study. Before you

decide whether to take part, it is important for you to understand why the

research is being done and what it will involve. Please take time to read the

following information carefully and discuss it with others if you wish. Ask us if

there is anything that is not clear or you would like more information. Please

take time to decide whether or not you wish to take part.

What is the purpose of the study?

Many critically ill babies admitted to the intensive care unit have major problems

tolerating normal milk feeds. This is especially common in those babies born

early (prematurely) and babies born to mothers with sugar intolerance (diabetes

mellitus). We are presently investigating possible reasons why this may occur.

One explanation is that a substance called AMYLIN present in the blood stream

may be responsible. Amylin is a newly discovered hormone that potently inhibits

the function of the stomach. Because good nutrition is crucial to the healing

process in sick babies we are conducting a study to measure Amylin levels in

the hope that this may shed some light on this difficult problem. If a relationship

can be shown between blood Amylin levels and poor stomach function we could

potentially develop treatments in the future to promote feeding, improve nutrition

and hopefully hasten recovery time in critically ill babies. To study this chemical

we aim to measure it in all babies born at this hospital at the time of routine

blood taking for neonatal screening.

Why has my baby been chosen?

212


Your baby has been chosen because if he/she is not tolerating feeds or is slow

to feed, we are trying to find out if the feed intolerance is due to increased blood

amylin level.

Do I have to take part?

It is up to you to decide whether or not to take part. If you do decide to take part

you will be given this information sheet to keep and be asked to sign a consent

form. If you decide to take part you are still free to withdraw at any time and

without giving a reason. This will not affect the standard of care your baby

receives.

What will happen if my baby takes part?

If you decide to take part in the study the only change from routine management

is that 0.5 ml (about 8 drops) of your baby’s blood would be taken during routine

blood sampling, so causing no added distress or pain. Additionally a sample

would be taken from umbilical cord after delivery (this causes no added distress

or discomfort to your baby as the sample is taken after the cord is cut).

What are the possible disadvantages and risks of taking part?

We will collect no more than 0.5 ml blood. This is safe amount to take from your

baby.

What are the possible benefits of taking part?

There will be no direct health benefit to your baby.

What if something goes wrong?

If you have any cause to complain about any aspect of the way in which you

have been approached or treated during the course of this study, the normal

National Health Service complaints mechanisms are available to you and are

not compromised in any way because you have taken part in a research study.

If you have any complaints or concerns please contact the project co-ordinator:

(Dr Coombs on telephone 01142268387). Otherwise you can use the normal

213


hospital complaints procedure and contact the following person: (Chris Welsh,

Medical Director STH, 8 Beech Hill Road, Sheffield, S10 2SB, 0114 271 2178).

Will my baby’s taking part in this study be kept confidential?

All information, which is collected, about your baby during the course of the

research will be kept strictly confidential. Any information about your baby,

which leaves the hospital/surgery, will have your name and address removed so

that you cannot be recognised from it.

What will happen to the results of the research study?

When the study finishes all the results will be analysed, which may lead to a

publication in a medical journal. At no time will any identifying information will be

published and all records will remain confidential. If you wish we will forward

you the published report of the study.

Who is organising and funding the research?

The research is being organised by the neonatal intensive care unit at Jessops

Wing, Royal Hallamshire hospital. There is no outside funding or financial

incentive to this study.

Who has reviewed the study?

The study has been reviewed and approved by South Sheffield Ethics

Committee.

Who can I contact if I want to know more about the study?

Drs V Kairamkonda & A Deorukhkar, Neonatal intensive care unit, Jessop

Wing, Royal Hallamshire Hospital, Sheffield S10 2SF, Tel: 01142268322, Fax:

01142268449.

Thank you for reading this information sheet.

214


Appendix 7. Child participation information leaflet (Study C) Royal

Hallamshire Hospital


Neonatal Intensive Care Unit, Jessop Wing, Royal Hallamshire Hospital

Study title: Amylin peptide: Association with feed intolerance in preterm infants.












potentially in the future use drugs to block the action of Amylin in order to

promote feeding, improve nutrition and hopefully hasten recovery time in

critically ill babies. To study this chemical we aim to measure it in all preterm

babies admitted to the intensive care with feed intolerance or who subsequently

develop feed intolerance.




amylin level.




form. If you decide to take part you are still free to withdraw at any time and 215



receives.



is that 0.5ml (about 8 drops) of your baby’s blood would be taken during routine

blood sampling, so causing no added distress or pain.

What are the side effects of taking part?

None


None


None







(Dr Coombs on telephone 01142268387). Otherwise you can use the normal

hospital complaints procedure and contact the following person: (Chris Welsh,

Medical Director STH, 8 Beech Hill Road, Sheffield, S10 2SB, 0114 271 2178).






216




publication in a medical journal. At no time will any identifying information will be




The research is being organised by the neonatal intensive care unit at Jessops

Wing, Royal Hallamshire Hospital. There is no outside funding or financial

incentive to this study.


The study has been reviewed and approved by South Sheffield Ethics

Committee.

Contacts for Further Information

Drs V Kairamkonda & A Deorukhkar, Neonatal intensive care unit, Jessop

Wing, Royal Hallamshire Hospital, Sheffield S10 2SF, Tel: 01142268322, Fax:

01142268449


217


Appendix 8. Child participation information leaflet (Study C)

Doncaster Royal Infirmary


Neonatal Intensive Care Unit, Doncaster Royal Infirmary, Doncaster

Study title: Amylin peptide: Association with feed intolerance in preterm infants.












potentially in the future use drugs to block the action of Amylin in order to

promote feeding, improve nutrition and hopefully hasten recovery time in

critically ill babies. To study this chemical we aim to measure it in all preterm

babies admitted to the intensive care with feed intolerance or who subsequently

develop feed intolerance.




amylin level.




form. If you decide to take part you are still free to withdraw at any time and 218



receives.



is that 0.5ml (about 8 drops) of your baby’s blood would be taken during routine

blood sampling, so causing no added distress or pain.

What are the side effects of taking part?

None


None


None







(Dr Anjum Deorukhkar on telephone 01302 366666 ext 3166).








publication in a medical journal. At no time will any identifying information will be 219





The research is being organised by the neonatal intensive care unit at Women’s

Hospital Doncaster Royal Infirmary, Doncaster. There is no outside funding or

financial incentive to this study.


The South Sheffield Ethics Committee and the Doncaster Research Ethics

Committee.

Contacts for Further Information

Dr. A Deorukhkar, Neonatal intensive care unit, Women’s Hospital DRI

Doncaster, Telephone: 01302 366666 ext 3166


220


Appendix 9. Study A & B Consent form

CONSENT FORM FOR PARTICIPATION OF CHILD

Title of Project: Amylin peptide: Association with feed intolerance in preterm

infants and IMDM.

Patient Identification Number: Centre Number: Study Number:

Name of Researchers: V Kairamkonda, A Mayer, A Deorukhkar, R Coombs, R

Fraser

Please initial box

I confirm that I have read and understand the information sheet Version 14/8/00 for the above study and have had the opportunity to ask questions.

I understand that my participation is voluntary and that I am free to withdraw at any time, without giving any reason, without my medical care or legal rights being affected.

I understand that sections of any of my medical notes may be looked at by responsible individuals from research team or from regulatory authorities where it is relevant to my taking part in research. I give permission for these individuals to have access to my records.

I agree to take part in the above study.

________________________ _______________ ___________________Name of Patient Date Signature

_________________________ ________________ ___________________Name of Person taking consent Date Signature

Original to be kept in baby’s medical notes; 1 copy to parent; 1 copy for research file

221


Appendix 10. Study C Consent form

CONSENT FORM FOR PARTICIPATION OF CHILD

Title of Project: Amylin peptide: Association with feed intolerance in preterm

infants.

Patient Identification Number: Centre Number: Study Number:

Name of Researchers: V Kairamkonda, A Mayer, A Deorukhkar, R Coombs, R

Fraser

Please initial box

I confirm that I have read and understand the information sheet Version 14/8/00 for the above study and have had the opportunity to ask questions.

I understand that my participation is voluntary and that I am free to withdraw at any time, without giving any reason, without my medical care or legal rights being affected.

I understand that sections of any of my medical notes may be looked at by responsible individuals from research team or from regulatory authorities where it is relevant to my taking part in research. I give permission for these individuals to have access to my records.

I agree to take part in the above study.

________________________ _______________ ___________________Name of Patient Date Signature

_________________________ ________________ ___________________Name of Person taking consent Date Signature

Original to be kept in baby’s medical notes; 1 copy to parent; 1 copy for research file

222


Appendix 11. Advert for Study A

AMYLIN STUDY

Dear Parents

Feed intolerance is a common problem in babies admitted to neonatal intensive

care unit. Amylin is a newly discovered hormone that potently inhibits the

function of the stomach. We are conducting a study to measure amylin levels in

the hope that this may shed some light on this difficult problem.

The study involves taking blood from your baby at birth from discarded umbilical

cord and at day 5 routine Guthrie (few drops)

If you would like further information or interested in taking part, please contact

staff on the unit OR Dr Venkatesh Kairamkonda, Specialist Registrar

0114228456/68356 OR Dr Anjum Deorukhkar, clinical research fellow

0114228456/68356.

223


Appendix 12. Advert for Study B

AMYLIN STUDY

Dear Parents

Feed intolerance is a common problem in babies born to mothers whose

pregnancy was complicated by diabetes. Amylin is a newly discovered hormone

that potently inhibits the function of the stomach. We are conducting a study to

measure amylin levels in the hope that this may shed some light on this difficult

problem.

The study involves taking blood from your baby at birth from discarded umbilical

cord and at day 5 routine Guthrie (few drops)




0114228456/68356.

224


Appendix 13. Advert for Study C

AMYLIN STUDY

Dear Parents

Feed intolerance is a common problem in babies admitted to neonatal intensive

care unit. Amylin is a newly discovered hormone that potently inhibits the

function of the stomach. We are conducting a study to measure amylin levels in

the hope that this may shed some light on this difficult problem.

The study involves taking a small sample of your baby’s blood during routine

collection (few drops).




0114228456/68356.

225


Appendix 14. Case report form Study A

CASE REPORT FORM STUDY AStudy ID

Date

Baby details Patient Sticker

Name

Male/Female

Date of Birth

Hospital Number

Address

Baby Details

Birth weight

Gestational age

Growth Type AGA SGA LGA

Date of blood sampling


Cord blood sample Arterial Venous Both

Postnatal blood sample yes no

Guthrie sample yes no

Mode of delivery

APGAR APGAR1 APGAR5 APGAR10

Resuscitation yes no

ETVentilation/CPAP yes no

Sepsis yes no

Maternal Details

Maternal Diagnosis

PROM yes no

Chorioamnionitis yes no

Antenatal steroids yes no

Medications

226


Appendix 15. Case report form Study B

CASE REPORT FORM STUDY BStudy ID

Date


Name

Male/Female

Date of Birth

Hospital Number

Address

Baby Details

Birth weight

Gestational age







Mode of delivery CS Instrumental Normal



ETVentilation/CPAP yes yes

Sepsis yes no

Feed intolerance yes no

Maternal Details

Maternal Diagnosis

Diabetes Type GDM IDDM NIDDM

PROM yes no



Medications

227


Appendix 16. Case report form Study C

CASE REPORT FORM STUDY CStudy ID

Date


Name

Male/Female

Date of Birth

Hospital Number

Address

Baby Details

DOA

DOD

Birth weight

Gestational age







Mode of delivery CS Instrumental Normal



ET Ventilation/CPAP yes no

Sepsis yes no

Medications

TPN yes no

Age at feed intolerance

Type of feed

Volume of feed

Method of feed

% aspirate

Colour of aspirate

Days to full feeds

Days to discharge

WCC (differential count)

Platelet count

228


CRP

Blood culture

Maternal Details

Maternal Diagnosis

Significant Antenatal/natal/postnatal history

yes no

PROM yes no



Medications

229

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