Amylin Peptide: An Association with Feed
Intolerance in Preterm Neonates and Infants of
Diabetic Mothers
----------------------------------------------------------------------------------------------------------
Dr Venkatesh Ramulu Kairamkonda
----------------------------------------------------------------------------------------------------------
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
----------------------------------------------------------------------------------------------------------
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
----------------------------------------------------------------------------------------------------------
November 2012
----------
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
1
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
Signature:
2
Date: November 2012
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 (18.6544.27) pmol/L, p<0.0001] and postnatal day 5 [n=9, 23.4 (15.37-46.57) pmol/L,
p<0.0001].
4
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.
5
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.
6
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
7
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
5.2
Scale of the Problem............................................................................ 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
6.1
Scale of the Problem............................................................................ 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
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
9
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
10
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 brain 41
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
Figure 18. General study methodology ............................................................. 80
11
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
12
Figure 32. The correlation graph showing line of best fit to assess the degree of
association between paired umbilical cord insulin (x axis) and umbilical cord
amylin (Y axis) in healthy preterm and term infants ........................................ 114
Figure 33. The correlation graph showing line of best fit to assess the degree of
association between gestational age at birth (x axis) and umbilical cord amylin
(Y axis) ............................................................................................................ 115
Figure 34. The correlation graph showing line of best fit to assess the degree of
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
13
Figure 45. Comparison box-and-whisker plot of amylin levels at birth and
postnatal day 5 between preterm IMDM and healthy preterm infants (controls)
........................................................................................................................ 142
Figure 46. The correlation graph showing line of best fit to assess the degree of
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 feedintolerant (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 feedintolerant (nTOL) group ................................................................................... 161
Figure 53. The correlation graph showing line of best fit to assess the degree of
association between amylin concentration (y axis) and days to reach full enteral
feeds (x axis) in nTOL group ........................................................................... 162
Figure 54. The correlation graph showing line of best fit to assess the degree of
association between days to discharge (x axis) and amylin concentration (Y
axis) in the feed-intolerant (nTOL) group ........................................................ 163
Figure 55. The correlation graph showing line of best fit to assess the degree of
association between % GRV (x axis) and amylin levels (y axis) in feed-tolerant
(TOL) group .................................................................................................... 164
14
Figure 56. The correlation graph showing line of best fit to assess the degree of
association between days to full enteral feeds (x axis) and amylin levels (Y axis)
in feed-tolerant (TOL) group ........................................................................... 165
Figure 57. The correlation graph showing line of best fit to assess the degree of
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
15
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
Table 4. Summary statistic of amylin concentration and demographic
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
Table 6. Summary statistic of amylin concentration and demographic
characteristics at birth in healthy preterm infants ............................................ 101
Table 7. Summary statistic of amylin concentration and demographic
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
Table 11. Summary statistic of amylin concentration and demographic
characteristics at birth in term IMDM infants ................................................... 126
Table 12. Summary statistic of amylin concentration and demographic
characteristics on postnatal day 5 in term IMDM infants ................................. 128
Table 13. Demographic characteristic of term IMDM at birth and postnatal day 5
........................................................................................................................ 130
16
Table 14. Summary statistic of amylin concentration and demographic
characteristics at birth in preterm IMDM infants .............................................. 132
Table 15. Summary statistic of amylin concentration and demographic
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
17
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
18
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
Colony forming unit
CCK
Cholecystokinin
CRF
Case Report Form
DBS
Dried blood spot
19
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
20
MMC
Migrating motor complex
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
intolerance
TPN
Total parenteral nutrition
VLBW
Very low birth weight infants
21
feed
Chapter 1 - Overview & Background
Chapter 1-
Overview & Background
1.1 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
al. 1978; Lee-Parritz 2004), and consequently cost of treatment. It is reasonable
22
Chapter 1 - Overview & Background
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.
1.2 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.
1.3 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.
23
Chapter 1 - Overview & Background
1.4 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.
1.5 Study Objectives
1.5.1 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.
1.5.2 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
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.
24
Chapter 1 - Overview & Background
Table 1. Study Objectives & Outcome Variables
Primary Objective
To
Primary Outcome Variable
determine
serum
amylin Amylin (median and interquartile
concentrations
range) concentration in pmol/L.
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.
Secondary Objectives
Secondary Outcome Variables
To investigate the relationship between Correlation Coefficient
serum
amylin
concentrations
and
gestational age and birth weight
To determine the relationship between Correlation Coefficient
amylin and insulin levels to confirm cosecretion
To
determine
agreement
between Correlation Coefficient and Bland
paired umbilical arterial and venous Altman method comparison test
amylin levels
To investigate the relationship between Correlation Coefficient
serum
amylin
concentrations
and
influential covariates (gastric residual
volumes (GRV), time to full enteral
feeds and length of hospital stay).
25
Chapter 2 – Literature Review: Amylin Peptide
Chapter 2-
Literature Review: Amylin Peptide
2.1 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.
2.2 Amylin Synthesis, Sources, Release & Metabolism
Amylin is co-synthesized, co-packaged within the Golgi apparatus, and cosecreted within the secretory granule by the islet beta cells in response to
26
Chapter 2 – Literature Review: Amylin Peptide
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 cosecretion 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).
27
Chapter 2 – Literature Review: Amylin Peptide
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).
28
Chapter 2 – Literature Review: Amylin Peptide
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).
29
Chapter 2 – Literature Review: Amylin Peptide
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).
2.3 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,
30
Chapter 2 – Literature Review: Amylin Peptide
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%
31
Chapter 2 – Literature Review: Amylin Peptide
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).
2.4 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.
2.5 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
32
Chapter 2 – Literature Review: Amylin Peptide
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
33
Chapter 2 – Literature Review: Amylin Peptide
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.
2.6 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 coexpressed 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.
34
Chapter 2 – Literature Review: Amylin Peptide
2.7 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).
2.8 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.
35
Chapter 2 – Literature Review: Amylin Peptide
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, KautzkyWiller et al. 1997) and possibly by noncompetitively antagonizing the effect of
36
Chapter 2 – Literature Review: Amylin Peptide
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).
37
Chapter 2 – Literature Review: Amylin Peptide
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).
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Chapter 2 – Literature Review: Amylin Peptide
2.9 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.
39
Chapter 2 – Literature Review: Amylin Peptide
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.
40
Chapter 2 – Literature Review: Amylin Peptide
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).
41
Chapter 2 – Literature Review: Amylin Peptide
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.
2.10 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).
42
Chapter 2 – Literature Review: Amylin Peptide
2.11 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).
2.12 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 (betaamyloid), 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).
43
Chapter 2 – Literature Review: Amylin Peptide
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
44
Chapter 2 – Literature Review: Amylin Peptide
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.).
45
Chapter 2 – Literature Review: Amylin Peptide
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).
46
Chapter 2 – Literature Review: Amylin Peptide
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).
47
Chapter 2 – Literature Review: Amylin Peptide
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
2.13 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,
48
Chapter 2 – Literature Review: Amylin Peptide
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.
49
Chapter 3 – Literature Review: Gastric Emptying
Chapter 3-
Literature Review: Gastric Emptying
3.1 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 1821 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
50
Chapter 3 – Literature Review: Gastric Emptying
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.
3.2 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.
51
Chapter 3 – Literature Review: Gastric Emptying
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).
3.3 Role of Other
Emptying
Gastrointestinal
Peptides
on
Gastric
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,
52
Chapter 3 – Literature Review: Gastric Emptying
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)
3.4 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).
53
Chapter 3 – Literature Review: Gastric Emptying
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
54
Chapter 3 – Literature Review: Gastric Emptying
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.
3.5 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).
55
Chapter 3 – Literature Review: Gastric Emptying
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 13Coctanoate 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
56
Chapter 3 – Literature Review: Gastric Emptying
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
57
Chapter 3 – Literature Review: Gastric Emptying
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.
3.6 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
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Chapter 3 – Literature Review: Gastric Emptying
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 noninvasive 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.
59
Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
Chapter 4-
Literature Review: Feed Intolerance in
Preterm Infants
4.1 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).
4.2 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).
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Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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).
4.3 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;
61
Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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.
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Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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 preprandial 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.
4.4 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
63
Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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 nonpropagated 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
64
Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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.
4.5 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 gastroesophageal 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
65
Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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
66
Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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
67
Chapter 4 – Literature Review: Feed Intolerance in Preterm Infants
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.
68
Chapter 5 – Literature Review: Gestational Diabetes Mellitus
Chapter 5-
Literature Review: Gestational Diabetes
Mellitus
5.1 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)
5.2 Scale of the Problem
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).
5.3 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-6phosphate by the enzyme glucokinase regulates the rate of glucose oxidation
and subsequent insulin secretion.
69
Chapter 5 – Literature Review: Gestational Diabetes Mellitus
5.4 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).
5.5 Strategies
to
Hyperinsulinaemia
Prevent
Fetal
and
Neonatal
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
70
Chapter 5 – Literature Review: Gestational Diabetes Mellitus
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.
71
Chapter 6 – Literature Review: IMDM
Chapter 6-
Literature Review: IMDM
6.1 Scale of the Problem
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 insulindependent 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.
6.2 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
72
Chapter 6 – Literature Review: IMDM
(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 protein3 [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.
6.3 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-forgestational-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
73
Chapter 6 – Literature Review: IMDM
the fact that the aetiology and pathophysiology of feed intolerance in IMDM is
yet to be determined.
6.4 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.
74
Chapter 7 – Overview of Project Plan
Chapter 7-
Overview of Project Plan
7.1 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
75
Chapter 7 – Overview of Project Plan
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
76
Chapter 7 – Overview of Project Plan
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 case77
Chapter 7 – Overview of Project Plan
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.
7.2 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.
78
Chapter 7 – Overview of Project Plan
Figure 17. Study plan Gantt chart
79
Chapter 7 – Overview of Project Plan
PROJECT TITLE: AMYLIN PEPTIDE: AN ASSOCIATION WITH FEED INTOLERANCE IN PRETERM
NEONATES AND IMDM
Study A
Amylin levels at birth and
postnatal day 5 in healthy
preterm and term infants
Study B
Amylin levels at birth and
postnatal day 5 in preterm and
term IMDM
Study C
Amylin 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
Inclusion
≥ 24 week gestation infants
born without major antenatal
or labour complications
Neonatal intensive care unit
Jessop wing, Royal Hallamshire Hospital
& Doncaster General Hospital
Inclusion
≥ 24 weeks gestation born
to mothers with diabetes
mellitus (gestational, insulin
or non-insulin dependent)
Inclusion
24-36 weeks gestation admitted
to intensive care unit and
requiring gastric tube feeding
Prefeed GRV > 50% on 2
consecutive occasions
Yes=nTOL
No=TOL
Matched for birthweight, gestation, postnatal age
Parental consent
0.5-1.0 ml
blood collected from discarded umbilical
cord, during routine collection and Guthrie
0.5-1.0 ml
blood collected within an
hour
Plasma stored at -70 degrees
ELISA
Amylin levels
Insulin levels (where possible)
Figure 18. General study methodology
80
0.5-1.0 ml
blood collected at the next
routine collection
Chapter 7 – Overview of Project Plan
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.
7.3 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.
The study protocol and any amendments were submitted to a properly
constituted Ethics Committee (REC) for approval of the study conduct. The
81
Chapter 7 – Overview of Project Plan
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
over plasma was disposed immediately after analysis.
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Chapter 7 – Overview of Project Plan
(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.
7.4 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
per day) and time to discharge. Additionally, inflammatory markers such as C83
Chapter 7 – Overview of Project Plan
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.
7.5 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).
7.6 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
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Chapter 8 – Clinical Study A
Chapter 8Clinical Study A: Umbilical & Guthrie
Amylin in Preterm and Term Infants
8.1 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.
8.2 Methodology
8.2.1 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.
8.2.2 Consent
The Infants were recruited once informed consent was obtained from their
parents/guardians. Infants whose parents refused consent were excluded from
the study.
8.2.3 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
85
Chapter 8 – Clinical Study A
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.
8.2.4 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.
8.2.5 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
86
Chapter 8 – Clinical Study A
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.
8.2.6 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.
87
Chapter 8 – Clinical Study A
8.2.7 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.
8.2.8 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
88
Chapter 8 – Clinical Study A
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.
8.2.9 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).
8.2.10 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 chisquare 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
89
Chapter 8 – Clinical Study A
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.
8.2.11 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.
8.2.12 Study Summary
Table 2 highlights the key features of the study.
90
Chapter 8 – Clinical Study A
Table 2. Study A Summary
Title
Study Design
Study Duration
Objectives
Number of blood samples
Umbilical and Guthrie amylin levels in
healthy preterm and term infants
Single
centre,
Prospective
Observational Study
12 months
To determine plasma amylin levels in
healthy preterm and term infants
200 umbilical cord samples
/ Participants
Main Inclusion Criteria
Infants ≥ 24 weeks gestation
Infants
Main Exclusion Criteria
born
pregnancy
to
was
mothers
whose
complicated
by
diabetes
Median and interquartile ranges for
summary
Coefficient
Statistical Methodology
statistics.
to
Corelation
evaluate
the
relationship between variables. Bland
Altman test for method comparison
between
umbilical
umbilical
venous
arterial
blood
method
Outcome variable
Plasma amylin levels
Measurement Assay
ELISA
Figure 19 below shows the overall study plan in a Gantt chart.
91
and
sampling
Chapter 8 – Clinical Study A
Figure 19. Study A Gantt chart
8.3 Results
Figure 20 below shows participant flow and collection of blood samples.
92
Chapter 8 – Clinical Study A
196 mothers eligible
14 mothers excluded due to
significant perinatal complications
182 mothers consented
184 infants eligible
3 infants excluded
(2 Downs syndrome, 1 CDH)
Healthy Preterm infants (n=43)
Healthy Term infants (n=138)
Paired umbilical
cord arterial
blood where
possible (n=26)
Umbilical cord
venous blood
(n=138)
Umbilical cord
venous blood
(n=43)
Paired umbilical
cord arterial blood
where possible
(n=8)
18 infants
discharged on D1
124 infants
discharged on D1
14 infants were inpatients for
maternal reasons
25 infants were inpatient due
to problems relating to
prematurity
(Post caesarean section)
Guthrie Blood D5
(n=14)
ELISA
Guthrie Blood D5
(n=25)
Cord Venous Amylin levels (181)
Paired Cord Arterial Amylin levels (34)
Figure 20. Study A flow diagram of participants and blood samples
93
Chapter 8 – Clinical Study A
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.
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Chapter 8 – Clinical Study A
Table 3. Summary statistic of amylin concentration and demographic
characteristics at birth in healthy term infants
Healthy Term Infants
Birth weight
Gestation
Amylin
(Umbilical Cord Group)
(Kg)
(Weeks)
(pmol/L)
Median
3.28
39.00
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
1.86-4.64
37-42
0.50-27.40
-0.06
0.60
(p=0.74)
(p=0.00)
0.21
-0.43
(p=0.49)
(p=0.26)
0.75
0.00
<0.00
Accept
Reject
Reject
Min-Max
Coefficient of Skewness
Coefficient of Kurtosis
*D-P test for Normal
distribution
* D’Agostino-Pearson test
95
6.10
1.14
(p<0.00)
1.49
(p=0.01)
Chapter 8 – Clinical Study A
30
25
20
15
10
5
0
Healthy Term Infant Group (n=138)
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).
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Chapter 8 – Clinical Study A
Table 4. Summary statistic of amylin concentration and demographic
characteristics at postnatal day 5 in healthy term infants
Healthy Term Infants
Birth weight
Gestation
Amylin
(Guthrie Group)
(Kg)
(Weeks)
(pmol/L)
Median
3.40
38.5
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
-0.67
0.52
(p=0.24)
(p=0.35)
3.23
-1.27
(p=0.05)
(p=0.23)
*DP test for Normal
p=0.05
p=0.32
p=0.00
distribution
Reject
Accept
Reject
Coefficient of Skewness
Coefficient of Kurtosis
* D’Agostino-Pearson test
97
5.65
1.97
(p=0.00)
5.26
(p=0.00)
Chapter 8 – Clinical Study A
25
20
15
10
5
0
Healthy Term Infant
Guthrie Group (n=14)
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).
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Chapter 8 – Clinical Study A
Table 5. Demographic characteristics of healthy term infants at birth and
postnatal day 5
Healthy Term infants
Birth (Umbilical
Postnatal Day
Cord venous)
5 (Guthrie)
n
138
14
Amylin (pmol/L)
6.10
5.65
(3.30-9.70)
(3.10-8.20)
39
38.5
(38.0-40.0)
(38.0-41.0)
3.28
3.40
(2.98-3.67)
(3.12-3.58)
Male
71
7
Female
67
7
Gestation (wks)
Birth Weight (kg)
Gender
* NS- not significant
Median and interquartile range, Mann Whitney U test
99
p value
*NS
*NS
*NS
Chapter 8 – Clinical Study A
30
25
20
15
10
5
0
Umbilical Cord Group
Healthy Term Infants
(n=138)
Guthrie Group
Healthy 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).
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Chapter 8 – Clinical Study A
Table 6. Summary statistic of amylin concentration and demographic
characteristics at birth in healthy preterm infants
Healthy Preterm Infants
Birth weight
Gestation
Amylin
(Umbilical cord Group)
(Kg)
(Weeks)
(pmol/L)
Median
2.05
34.00
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
0.52
-0.87
0.35
(p=0.13)
(p=0.02)
0.20
-0.36
(p=0.60)
(p=0.47)
p=0.29
p=0.05
Accepted
Rejected
Coefficient of Skewness
Coefficient of Kurtosis
*DP test for Normal
distribution
* D’Agostino-Pearson test
101
4.60
(p=0.31)
-1.16
(p=0.12)
p=0.18
Accepted
Chapter 8 – Clinical Study A
12
10
8
6
4
2
0
Healthy Preterm Infant Group (n=43)
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.
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Chapter 8 – Clinical Study A
Table 7. Summary statistic of amylin concentration and demographic
characteristics at postnatal day 5 in healthy preterm infants
Healthy Preterm Infants
Birth weight
Gestation
Amylin
(Guthrie Group)
(Kg)
(Weeks)
(pmol/L)
Median
1.72
33.0
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
0.00
-0.86
0.93
(p=0.99)
(p=0.06)
-0.62
-0.32
(p=0.37)
(p=0.55)
p=0.67
p=0.15
Accepted
Accepted
Coefficient of Skewness
Coefficient of Kurtosis
*DP test for Normal
distribution
* D’Agostino-Pearson test
103
6.9
(p=0.04)
-0.08
(p=0.73)
p=0.13
Accepted
Chapter 8 – Clinical Study A
20
15
10
5
0
Healthy Preterm Infant Group
(n=25)
Figure 25. Box-and-whisker plot of amylin levels at postnatal day 5 in healthy
preterm infants
The central box represents the values from 25th to 75th percentile, middle line
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).
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Chapter 8 – Clinical Study A
Table 8. Demographic charateristics of healthy preterm infants at birth and
postnatal day 5.
Healthy Preterm
Birth (Umbilical
Postnatal Day 5
Cord venous)
(Guthrie)
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
Male
25
13
Female
18
12
infants
n
Gender
NS- not significant
Median and interquartile range, Mann Whitney U test
105
P value
Chapter 8 – Clinical Study A
20
15
10
5
0
Umbilical Cord Group
Healthy Preterm Infants
(n=43)
Guthrie Group
Healthy 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).
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Chapter 8 – Clinical Study A
Table 9. Comparison of demography and amylin levels in healthy term and
preterm infants at birth and postnatal day 5
Healthy
Term
Birth
Healthy
Preterm
Birth
Healthy
Term
Postnatal
Day 5
Heathy
Preterm
Postnatal
Day 5
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 (2.759.50)
*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
Gender
Male
Female
71
67
25
18
7
7
13
12
n
Median and interquartile range, Mann Whitney U test
107
p value
Chapter 8 – Clinical Study A
30
25
20
15
10
5
0
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).
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Chapter 8 – Clinical Study A
25
20
15
10
5
0
Guthrie Group
Healthy Preterm Infants
(n=25)
Guthrie Group
Healthy 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 (3.108.20) pmol/L) and healthy preterm infants (4.60 (1.90-8.30) and 6.90 (2.759.50)) pmol/L respectively.
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Chapter 8 – Clinical Study A
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).
25
20
15
10
5
0
0
5
10
15
20
25
Umbilical Cord Venous Amylin Concentration (pmol/L)
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)
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).
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Chapter 8 – Clinical Study A
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).
4
+1.96 SD
2.8
2
0
Mean
-0.8
-2
-4
-1.96 SD
-4.4
-6
-8
-10
0
5
10
15
20
25
AVERAGE of Cord Venous Amylin and Cord Arterial Amylin (pmol/L)
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).
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Chapter 8 – Clinical Study A
60
+1.96 SD
40
41.5
20
0
Mean
-20
-13.9
-40
-60
-1.96 SD
-80
-69.4
-100
-120
-140
0
5
10
15
20
25
AVERAGE of Cord Venous Amylin and Cord Arterial Amylin
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
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Chapter 8 – Clinical Study A
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.
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Chapter 8 – Clinical Study A
25
20
15
10
5
0
0
5
10
15
20
Umbilical cord blood insulin concentration (mu/L)
Figure 32. The correlation graph showing line of best fit to assess the degree of
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).
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Chapter 8 – Clinical Study A
30
25
20
15
10
5
0
25
30
35
40
45
Gestation at Birth (weeks)
Figure 33. The correlation graph showing line of best fit to assess the degree of
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 pvalue, and a 95% CI for rho (n=181, Spearman’s rho=0.20, 95% CI= 0.04-0.35,
p=0.012).
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Chapter 8 – Clinical Study A
30
25
20
15
10
5
0
0
1000
2000
3000
4000
5000
Birth Weight (Grams)
Figure 34. The correlation graph showing line of best fit to assess the degree of
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).
8.4 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
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Chapter 8 – Clinical Study A
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 2.4 on page 32, 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
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Chapter 8 – Clinical Study A
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
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Chapter 8 – Clinical Study A
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.
8.5 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.
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Chapter 9 – Clinical Study B
Chapter 9-
Clinical Study B: Umbilical and Guthrie
Amylin in Preterm and Term IMDM
9.1 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.
9.2 Methodology
9.2.1 Study Design & Setting
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.
9.2.2 Consent
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
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.
9.2.4 Exclusion Criteria
1. Deliveries associated with chorioamnionitis, or other significant antenatal
or labour complications.
2. Infants with congenital and chromosomal abnormality
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Chapter 9 – Clinical Study B
9.2.5 Study Procedure
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.
9.2.6 Study End Point
The infants were no longer followed up after collection of cord and postnatal
bloods.
9.2.7 Sample Collection & Storage
A 0.5 to 1 ml whole blood was collected and processed as described in Study A,
section 8.2.7 on page 88.
9.2.8 Sample Analysis
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
8.2.8 on page 88.
9.2.9 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
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Chapter 9 – Clinical Study B
90% power. The mean and standard deviation were derived from the data
published by (Mayer, Durward et al. 2002)
9.2.10 Statistical Analysis
Statistical analysis was performed using Medcalc version 11.4.4.0. 1993-2010.
as described in study A, section 8.2.10 on page 89.
9.2.11 Data Collection & Case Report Form
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.
9.2.12 Study Summary
Table 10 below highlights the key features of the study methodology.
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Chapter 9 – Clinical Study B
Table 10. Study B summary
Title
Umbilical and Guthrie amylin levels in
preterm and term IMDM
Study Design
Prospective Observational Study
Study Duration
12 months
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.
Outcome variable
Plasma amylin levels
Measurement Assay
ELISA
Figure 35 below provides details of the study plan in a Gantt chart.
123
Chapter 9 – Clinical Study B
Figure 35. Study B Gantt chart
124
Chapter 9 – Clinical Study B
9.3 Result
The Figure 36 below shows the participant flow and blood samples.
31 singleton mothers were eligible who consented
Term infants (n=17)
Umbilical cord venous blood
Paired umbilical cord
arterial blood (n=3)
Guthrie blood
(n=5)
Feed
intolerance
(n=4)
Preterm infants (n=14)
Umbilical cord venous blood
Paired umbilical cord
arterial blood (n=3)
Discharged on D1-4 (n=17)
Inpatients
Cord Amylin (n=31)
Guthrie Amylin (n=14)
Paired insulin (n=6)
Guthrie blood
(n=9)
Feed
intolerance
(n=8)
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).
125
Chapter 9 – Clinical Study B
Table 11. Summary statistic of amylin concentration and demographic
characteristics at birth in term IMDM infants
Term IMDM
Birth weight
Gestation
(umbilical cord Group)
(kg)
(weeks)
Median
3.74
39.00
95% CI
3.44-3.98
38.00 - 39.98
32.79-47.39
3.41 – 4.07
37.75- 40.00
28.35-50.00
2.51-4.85
37-42
22.10-70.10
-0.25
0.58
(p=0.62)
(p=0.26)
-0.13
-0.21
(p=0.71)
(p=0.66)
0.82
0.49
Accept
Accept
25-75 percentile (IQR)
Min-Max
Coefficient of Skewness
Coefficient of Kurtosis
*D-P test for Normal
distribution
* D’Agostino-Pearson test
126
Amylin
(pmol/L)
34.30
0.59
(p=0.25)
-0.65
(p=0.417)
0.38
Accept
Chapter 9 – Clinical Study B
80
70
60
50
40
30
20
IMDM Term Infant
Umbilical Cord Group (n=17)
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).
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Chapter 9 – Clinical Study B
Table 12. Summary statistic of amylin concentration and demographic
characteristics on postnatal day 5 in term IMDM infants
Term IMDM
Birth weight
Gestation
(Guthrie Group)
(kg)
(weeks)
Median
3.53
37
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
2.51-4.46
37-42
-0.54
2.23
0.85
-0.522
5.00
-0.988
(p=0.75)
(p=0.02)
Sample size too
Sample size
small
too small
Min-Max
Coefficient of Skewness
Amylin
(pmol/L)
25.20
17.10-60.00
(p=0.74)
Coefficient of Kurtosis
*D-P test for Normal
distribution
(p=0.59)
Sample size
too small
* D’Agostino-Pearson test
Four of five infants in the Guthrie group also had feed intolerance at the time of
blood collection.
128
Chapter 9 – Clinical Study B
60
55
50
45
40
35
30
25
20
15
IMDM Term Infant
Guthrie group (n=5)
Figure 38. Box-and-whisker plot of Guthrie amylin levels in term IMDM 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). The sample size was too
small to test normal distribution.
129
Chapter 9 – Clinical Study B
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 IMDM
Birth
Postnatal Day
(Umbilical Cord)
5 (Guthrie)
17
5
34.30
25.20
(28.35-50.00)
(22.20-48.75)
39
37
(37.75-40.00)
(37-38.25)
3.74
3.53
(3.41-4.07)
(3.21-4.43)
Male
8
2
*NS
Female
9
3
*NS
Group
n
Amylin (pmol/L)
Gestation (wks)
Weight
(kg)
p value
*NS
*NS
*NS
Gender
* NS-not significant
Median and interquartile range, Mann Whitney U test
130
Chapter 9 – Clinical Study B
80
70
60
50
40
30
20
10
Term IMDM
Umbilical Cord Group
(n=17)
Term IMDM
Guthrie 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.
131
Chapter 9 – Clinical Study B
In preterm IMDM, amylin level at birth (umbilical cord) was 32.0 (18.65-44.27)
pmol/L (Table 14 & Figure 40).
Table 14. Summary statistic of amylin concentration and demographic
characteristics at birth in preterm IMDM infants
Preterm IMDM
Birth weight
Gestation
Amylin
(umbilical cord group)
(kg)
(weeks)
(pmol/L)
Median
2.88
34
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
-0.19
-1.69
1.25
(p=0.73)
(p=0.00)
-0.56
2.91
(p=0.49)
(p=0.05)
0.74
0.00
<0.06
Accept
Reject
Accept
Coefficient of Skewness
Coefficient of Kurtosis
*D-P test for Normal
distribution
* D’Agostino-Pearson test
132
32.00
(p<0.04)
1.28
(p=0.24)
Chapter 9 – Clinical Study B
90
80
70
60
50
40
30
20
10
IMDM Preterm Infants
Umbilical Cord Group (n=13)
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
median, and the vertical line represents range. D’Agostino-Pearson test for
normal distribution of amylin levels was accepted (p=0.06).
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Chapter 9 – Clinical Study B
In preterm IMDM, amylin level on postnatal day 5 (Guthrie) was 23.4 (15.3746.57) pmol/L (Table 15 & Figure 41).
Table 15. Summary statistic of amylin concentration and demographic
characteristics at postnatal day 5 in preterm IMDM infants
Preterm IMDM
Amylin
Birth weight
(pmol/L)
(kg)
(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
4.90-92.00
0.82-4.21
26-36
1.38
-0.05
-1.45
(p<0.05)
(p=0.92)
0.38
-1.22
(p=0.64)
(p=0.30)
0.14
0.59
Accept
Accept
(Guthrie group)
Min-Max
Coefficient of Skewness
Coefficient of Kurtosis
*D-P test for Normal
distribution
* D’Agostino-Pearson test
134
Gestation
(p=0.03)
1.58
(p=0.22)
0.05
Accept
Chapter 9 – Clinical Study B
100
90
80
70
60
50
40
30
20
10
0
IMDM Preterm Infants
Guthrie Group (n=9)
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
median, and the vertical line represents range. D’Agostino-Pearson test for
normal distribution of amylin levels was accepted (p=0.14).
At the time of Guthrie blood collection, eight of nine preterm IMDM were
experiencing feed intolerance.
135
Chapter 9 – Clinical Study B
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
n
Amylin (pmol/L)
Gestation (wks)
Weight (kg)
Birth
Postnatal day 5
(Umbilical cord)
(Guthrie)
14
9
32.0
23.4
(18.65-44.27)
(15.37-46.57)
34
34
(33-35.25)
(38-40)
2.88
2.61
(1.93-3.35)
(1.96-3.92)
Gender
P value
*NS
*NS
*NS
*NS
Male
9
5
Female
5
4
* NS-not significant
Median and interquartile range, Mann Whitney U test
136
Chapter 9 – Clinical Study B
100
90
80
70
60
50
40
30
20
10
0
Umbilical Cord Group
Preterm IMDM
(n=13)
Guthrie Group
Preterm 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.
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Chapter 9 – Clinical Study B
Figure 43 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).
100
90
80
70
60
50
40
30
20
10
0
TermIMDMCord
PretermIMDMCord TermIMDMGuthrie PretermIMDMGuthrie
p=0.25
Figure 43.
p=0.64
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.
138
Chapter 9 – Clinical Study B
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
n
Amylin
(pmol/L)
Gestation
(wks)
Term
Controls
Birth
Term IMDM
Birth
Term
Controls
Postnatal
Day 5
Term IMDM
Postnatal
Day 5
138
17
14
5
5.65
(3.10-8.20)
**25.20
(22.20-48.75)
38.5
(38.00-41.00)
37.0
(37-38.25)
NS
NS
6.10
(3.30-9.70)
*34.30
(28.35-50.00)
39.00
39
(38.00-40.00) (37.75-40.00)
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)
Gender
Male
Female
71
67
8
9
7
7
2
3
NS-not significant
Median and interquartile range, Mann Whitney U test
139
p value
*p<0.00
**p<0.00
Chapter 9 – Clinical Study B
80
70
60
50
40
30
20
10
0
HealthyTermCord
TermIMDMCord
p<0.0001
HealthyTermGuthrie TermIMDMGuthrie
P<0.001
Figure 44. Comparison box-and-whisker plot of amylin levels at birth and
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.
140
Chapter 9 – Clinical Study B
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
IMDM
Birth
Preterm
Controls
Postnatal
Day 5
Preterm
IMDM
Postnatal
Day 5
43
14
25
9
4.60
(1.90-8.30)
*32.00
(18.65-44.27)
6.90
(2.75-9.50)
**23.40
(15.37-46.57)
Gestation
34
(wks)
(30.5 - 35.0)
34
(33-35.25)
33
(30.0 - 35.0)
34
(38-40)
NS
Weight
(kg)
2.88
(1.97-3.35)
1.72
(1.50 - 2.29)
2.61
(1.96-3.92)
NS
n
Amylin
(pmol/L)
Gender
Male
Female
2.05
(1.53-2.39)
p value
*p<0.00
**p<0.00
NS
25
18
9
5
13
12
NS-not significant
Median and interquartile range, Mann Whitney U test
141
5
4
Chapter 9 – Clinical Study B
90
80
70
60
50
40
30
20
10
0
HealthyPretermCord PretermIMDMCord HealthyPretermGuthrie TermIMDMGuthrie
p<0.0001
P<0.0001
Figure 45. Comparison box-and-whisker plot of amylin levels at birth and
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).
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Chapter 9 – Clinical Study B
100
80
60
40
20
0
0
5
10
15
20
25
Umbilical Cord Insulin Concentration (mu/L)
Figure 46. The correlation graph showing line of best fit to assess the degree of
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=0.290.74, 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.
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Chapter 9 – Clinical Study B
9.4 Discussion
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
144
Chapter 9 – Clinical Study B
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
145
Chapter 9 – Clinical Study B
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.
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Chapter 9 – Clinical Study B
9.5 Conclusions
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.
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Chapter 10 – Clinical Study C
Chapter 10- Clinical Study C: Amylin Levels in
Preterm infants with Feed Intolerance
10.1 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.
10.2 Methodology
10.2.1 Study Design & Setting
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.
10.2.2 Consent
Infants were recruited once informed consent was obtained from their
parents/guardians. Infants whose parents refused consent were excluded from
the study.
10.2.3 Inclusion Criteria
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.
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Chapter 10 – Clinical Study C
10.2.4 Exclusion Criteria
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.
10.2.5 Study Procedure
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
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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.
10.2.6 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
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the minimal feed volume at any given time was more than 2mls even in the
smallest infant.
10.2.7 Definition of feed tolerance
Infants with minimal (prefeed GRV <50%) or no GRV for > 24 hours were
identified as feed tolerant (TOL).
10.2.8 Study End Point
The infants were no longer followed up after collection of bloods.
10.2.9 Sample Collection & Storage
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 8.2.7 on page 88.
10.2.10
Sample Analysis
All samples were analysed as described previously in section 8.2.8 on page 88.
10.2.11
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)
10.2.12
Statistical Analysis
Statistical analysis was performed using Medcalc version 11.4.4.0. 1993-2010.
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
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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.
10.2.13
Data Collection & Case Report Form
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.
10.2.14
Study Summary
Table 19 & Figure 47 below highlight the key features of the study methodology
and Gantt chart respectively.
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Table 19. Study C summary
Title
Amylin levels in preterm infants with
feed intolerance
Study Methodology
Case-Control Study
Study Duration
12 months
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.
Coefficient
be
will
Correlation
performed
to
evaluate the relationship between
variables
Outcome variable
Plasma amylin levels
Measurement Assay
ELISA
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Chapter 10 – Clinical Study C
Figure 47. Study C Gantt chart
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Chapter 10 – Clinical Study C
10.3 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.
Consent obtained from parents of preterm neonates
(24-36 weeks) with gastric tube placement
n=70
Enteral feeds >30ml/kg or
>25% of total fluid intake
>50% prefeed GRV on two
consecutive occasions
None or <50% prefeed GRV
Study entry and consent
Study entry and consent
Feed-intolerant (nTOL)
(n=34)
Feed-tolerant (TOL)
(n=36)
n=6 excluded
Blood samples haemolysed &
therefore unsuitable for analysis
n=4 excluded
Blood samples haemolysed &
therefore unsuitable for analysis
Feed-tolerant (TOL)
(n=30)
Feed-intolerant (nTOL)
(n=30)
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.
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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
Gestation (wks)
34
29.5 (28-31)
36
30.0 (29-33)
0.33
Birth weight (kg)
1.30 (1.00-1.80)
1.30 (1.10-1.80)
0.62
Gender
Male (n)
Female (n)
23
11
24
12
0.64
0.62
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
22
7
5
24
7
5
0.12
0.26
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
6 (6-10)
6 (6-10)
0.51
Postnatal age (days)
Feed type
EBM (n)
Formula (n)
Mixed (n)
C reactive protein (mg/l)
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).
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Table 21. Serum amylin levels, % GRV, days to reach full feeds and length of
stay in feed-intolerant (nTOL) and feed-tolerant (TOL) neonates
n
Amylin (pmol/L)
GRV (%)
Time to full feeds (days)
Length of hospital stay
(days)
nTOL
TOL
P value
30
30
47.9 (21.4-79.8)
8.7 (5.7-16)
<0.00
150 (100-350)
5 (0-5)
<0.00
13.0 (10.0-16.0)
6.5 (4.0-9.0)
<0.00
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.
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Chapter 10 – Clinical Study C
120
100
80
60
40
20
0
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) (MannWhitney 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
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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.
450
400
350
300
250
200
150
100
50
0
TOL
(n=30)
nTOL
(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).
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Chapter 10 – Clinical Study C
50
40
30
20
10
0
nTOL
(n=30)
TOL
(n=30)
Figure 51. The box and whisker plot of days to reach full feeds (Y axis) in feedintolerant (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).
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Chapter 10 – Clinical Study C
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).
120
nTOL Group (n=30)
100
80
60
40
20
0
0
100
200
300
400
500
Percentage Gastric Residual Volume (%GRV)
nTOL Group (n=30)
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 feedintolerant (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.
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Chapter 10 – Clinical Study C
120
nTOL Group (n=30)
100
80
60
40
20
0
5
10
15
20
25
30
35
Days To Full Enteral Feeds
nTOL Group (n=30)
Figure 53. The correlation graph showing line of best fit to assess the degree of
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.
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Chapter 10 – Clinical Study C
120
nTOL Group (n=30)
100
80
60
40
20
0
0
20
40
60
80
100
120
Days To Discharge
nTOL Group (n=30)
Figure 54. The correlation graph showing line of best fit to assess the degree of
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.
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Chapter 10 – Clinical Study C
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).
80
70
TOL Group (n=30)
60
50
40
30
20
10
0
0
5
10
15
20
25
Percentage Gastric Residual Volume (%GRV)
TOL Group (n=30)
Figure 55. The correlation graph showing line of best fit to assess the degree of
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.
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Chapter 10 – Clinical Study C
80
70
TOL Group (n=30)
60
50
40
30
20
10
0
0
10
20
30
40
50
Days To Full Enteral Feeds
TOL Group (n=30)
Figure 56. The correlation graph showing line of best fit to assess the degree of
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.
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Chapter 10 – Clinical Study C
80
70
TOL Group (n=30)
60
50
40
30
20
10
0
0
20
40
60
80
100
120
Days To Discharge
TOL Group (n=30)
Figure 57. The correlation graph showing line of best fit to assess the degree of
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.
10.4 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
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Chapter 10 – Clinical Study C
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.
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Chapter 10 – Clinical Study C
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
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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 2.4 on page 32.
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 2.2 and previous study observations as highlighted in section
heading 8.4 and 9.4. Notably twenty-three of thirty nTOL bloods were collected
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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
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Chapter 10 – Clinical Study C
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 Creactive protein, white cell count and neutrophil count were comparable
between the groups and therefore not attributable to the observed difference in
the amylin levels.
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Chapter 10 – Clinical Study C
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
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Chapter 10 – Clinical Study C
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.
10.5 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.
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Chapter 11 – Conclusions of the dissertation
Chapter 11-
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 (1.90174
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.
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Chapter 12 – Implications for future research
Chapter 12-
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
attractive hypothesis that could be put to clinical test is that transient
176
Chapter 12 – Implications for future research
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 newborn infants with feed intolerance. There is an urgent need for validation of noninvasive 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.
177
Chapter 12 – Implications for future research
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.
178
Chapter 14 – Appendices
Chapter 13-
Publications arising from this Project
13.1 Amylin peptide levels are raised in infants of diabetic
mothers. Arch Dis Child. 2005 Dec; 90(12):1279-82
179
Chapter 14 – Appendices
180
Chapter 14 – Appendices
181
Chapter 14 – Appendices
182
Chapter 14 – Appendices
183
Chapter 14 – Appendices
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
185
Chapter 14 – Appendices
186
Chapter 14 – Appendices
187
Chapter 14 – Appendices
188
Chapter 14 – Appendices
189
Chapter 14 – Appendices
Chapter 14-
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) .
Analyte
Analytespecific
antibody
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.
190
Chapter 14 – Appendices
AntibodyAnalyte
Conjugate
Step 2. An enzyme-linked analyte-specific detection antibody binds to a second
epitope on the analyte forming the analyte-antibody complex.
Substrate-
Light
or
Colour
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.
Therefore the standard curve is described by equations for competitive binding.
191
Chapter 14 – Appendices
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.
192
Chapter 14 – Appendices
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
193
Chapter 14 – Appendices
inverting the plate and tapping it smartly onto absorbent towels several times.
Microtitre plate arrangements of wells is as shown below
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
Sam
ple
1
Sam
ple
1
Samp
le 2
Sam
ple
2
Sam
ple
3
Sa
mpl
e3
C
D
E
F
G
H
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.
194
Chapter 14 – Appendices
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).
195
Chapter 14 – Appendices
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:
196
Chapter 14 – Appendices
Sample No
1
Amylin Added pM
20
50
80
Within % CV
2.6
5.0
14.8
Between % CV
11.9
12.9
11.5
2
20
50
80
11.5
2.5
7.9
17.4
17.8
16.2
3
20
50
80
7.8
5.4
7.3
17.6
11.9
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
1
Sample
Concentration (pM)
3.25
Amylin
% of Recovery
20
50
80
104
99
91
2
2.56
20
50
80
96
89
89
3
11.23
20
50
80
101
87
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:
197
Chapter 14 – Appendices
Sample
No
1
Volume
Sampled
50
40
25
10
Expected pM
Observed pM
22.32
22.32
21.13
19.98
18.55
100
95
90
83
2
50
40
25
10
25.34
25.35
22.30
20.54
21.05
100
88
81
83
3
50
40
25
10
20.54
20.54
20.85
15.96
19.65
100
101
78
96
198
%of Expected
Chapter 14 – Appendices
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 CaseControl Study
Study Duration
12 months
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 nonparametric 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
199
Chapter 14 – Appendices
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).
200
Chapter 14 – Appendices
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 CPreterm 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)
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Chapter 14 – Appendices
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 1100 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.
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Chapter 14 – Appendices
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: 1721
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.
203
Chapter 14 – Appendices
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: S9S13
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.
204
Chapter 14 – Appendices
Appendix 5. Research protocol for ethics approval, Doncaster
Research Ethics Committee
Amylin peptide: Association with feed intolerance in preterm infants- A CaseControl 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 Duration
12 months
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 nonparametric 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.
205
Chapter 14 – Appendices
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 generelated 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).
206
Chapter 14 – Appendices
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
207
Chapter 14 – Appendices
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 feedintolerant (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.
208
Chapter 14 – Appendices
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: 1721
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: S9S13
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.
209
Chapter 14 – Appendices
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?
210
Chapter 14 – Appendices
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
211
Chapter 14 – Appendices
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.
212
Chapter 14 – Appendices
Appendix 7. Child participation information leaflet (Study C) Royal
Hallamshire Hospital
CHILD PARTICIPATION INFORMATION LEAFLET
Neonatal Intensive Care Unit, Jessop Wing, Royal Hallamshire Hospital
Study title: Amylin peptide: Association with feed intolerance in preterm infants.
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 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.
Why has my baby been chosen?
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
213
Chapter 14 – Appendices
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 0.5ml (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
What are the possible disadvantages and risks of taking part?
None
What are the possible benefits of taking part?
None
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
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.
214
Chapter 14 – Appendices
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.
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
Thank you for reading this information sheet.
215
Chapter 14 – Appendices
Appendix 8. Child participation information leaflet (Study C)
Doncaster Royal Infirmary
CHILD PARTICIPATION INFORMATION LEAFLET
Neonatal Intensive Care Unit, Doncaster Royal Infirmary, Doncaster
Study title: Amylin peptide: Association with feed intolerance in preterm infants.
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 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.
Why has my baby been chosen?
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
216
Chapter 14 – Appendices
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 0.5ml (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
What are the possible disadvantages and risks of taking part?
None
What are the possible benefits of taking part?
None
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 Anjum Deorukhkar on telephone 01302 366666 ext 3166).
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
217
Chapter 14 – Appendices
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 Women’s
Hospital Doncaster Royal Infirmary, Doncaster. There is no outside funding or
financial incentive to this study.
Who has reviewed the 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
Thank you for reading this information sheet.
218
Chapter 14 – Appendices
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
219
Chapter 14 – Appendices
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
220
Chapter 14 – Appendices
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.
221
Chapter 14 – Appendices
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)
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.
222
Chapter 14 – Appendices
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).
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
Chapter 14 – Appendices
Appendix 14. Case report form Study A
CASE REPORT FORM STUDY A
Study 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
Cord blood sample
Arterial
Venous
Both
Postnatal blood sample
yes
no
Guthrie sample
yes
no
APGAR
APGAR1
APGAR5
Resuscitation
yes
no
ETVentilation/CPAP
yes
no
Sepsis
yes
no
PROM
yes
no
Chorioamnionitis
yes
no
Antenatal steroids
yes
no
Date of blood sampling
Number of blood samples
Mode of delivery
Maternal Details
Maternal Diagnosis
Medications
224
APGAR10
Chapter 14 – Appendices
Appendix 15. Case report form Study B
CASE REPORT FORM STUDY B
Study 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
Cord blood sample
Arterial
Venous
Both
Postnatal blood sample
yes
no
Guthrie sample
yes
no
Mode of delivery
CS
Instrumental
Normal
APGAR
APGAR1
APGAR5
APGAR10
Resuscitation
yes
no
ETVentilation/CPAP
yes
yes
Sepsis
yes
no
Feed intolerance
yes
no
Diabetes Type
GDM
IDDM
PROM
yes
no
Chorioamnionitis
yes
no
Antenatal steroids
yes
no
Date of blood sampling
Number of blood samples
Maternal Details
Maternal Diagnosis
Medications
225
NIDDM
Chapter 14 – Appendices
Appendix 16. Case report form Study C
CASE REPORT FORM STUDY C
Study ID
Date
Baby details
Patient Sticker
Name
Male/Female
Date of Birth
Hospital Number
Address
Baby Details
DOA
DOD
Birth weight
Gestational age
Growth Type
AGA
SGA
LGA
Cord blood sample
Arterial
Venous
Both
Postnatal blood sample
yes
no
Guthrie sample
yes
no
Mode of delivery
CS
Instrumental
Normal
APGAR
APGAR1
APGAR5
APGAR10
Resuscitation
yes
no
ET Ventilation/CPAP
yes
no
Sepsis
yes
no
yes
no
Date of blood sampling
Number of blood samples
Medications
TPN
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
CRP
226
Chapter 14 – Appendices
Blood culture
Maternal Details
Maternal Diagnosis
Significant Antenatal/natal/postnatal
history
PROM
yes
no
yes
no
Chorioamnionitis
yes
no
Antenatal steroids
yes
no
Medications
227
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