Thesis 20Jan17

221 Pages·2017·28.58 MB·English

Characterisation of the role of vitamin D
in paediatric non-alcoholic fatty liver
disease

By

Philippa Gibson

Supervisors:
Dr. J. Bernadette Moore
Dr. Kathryn H. Hart
Prof. Susan A. Lanham-New

Submitted for the degree of Doctor of Philosophy

Department of Nutritional Sciences
Faculty of Health and Medical Sciences
University of Surrey

July 2016
Declaration of Originality
This thesis and the work to which it refers are the results of my own efforts. Any
ideas, data, images or text resulting from the work of others (whether published
or unpublished) are fully identified as such within the work and attributed to
their originator in the text, bibliography or in footnotes. This thesis has not been
submitted in whole or in part for any other academic degree or professional
qualification. I agree that the University has the right to submit my work to the
plagiarism detection service TurnitinUK for originality checks. Whether or not
drafts have been so-assessed, the University reserves the right to require an
electronic version of the final document (as submitted) for assessment as above.

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Summary
Previous research has suggested a role for vitamin D in non-alcoholic
fatty liver disease (NAFLD) pathogenesis. Several observational studies have
observed low vitamin D status (25OHD) with poorer histological findings. The
principal aims of this study were to assess diet and lifestyle, 25OHD status, gene
variants in vitamin D metabolism in UK children, and separately examine the
effect of vitamin D in an in vitro NAFLD model.
Dietary results from the case control study (n=32) indicated vitamin D
intakes of paediatric patient with biopsy-proven NAFLD and ultrasound-cleared
obese patients were 1.7μg/day and 3.5μg/day, respectively, well below the new
UK recommendation. Children failed to meet current UK government
recommendations for physical activity.
In our UK paediatric biopsy-proven NAFLD cohort (n=103), the majority
of patients presented with deficient (<25nmol/L, 25.5%) or insufficient
(<50nmol/L, 80.8%) mean serum 25OHD levels. Furthermore, patients had
significantly lower 25OHD levels during winter months in comparison to
summer (p=0.0001) and autumn (p=0.0026), while 25OHD levels were non-
significantly lower in NASH compared to non-NASH patients (p=0.0576). We
observed that single nucleotide polymorphisms (SNPs) involved in vitamin D
metabolism were associated with poorer liver histology grading; specifically,
three SNPs were associated with increased steatosis and one with increased
inflammation score in Caucasian patients.
Finally, LX-2 cells, an immortalised human hepatic stellate cell line,
demonstrated significantly reduced cell proliferation (p=0.0005) with increasing
doses of 1α,25(OH) D after 10 days of incubation in clonogenic assays.
2 3
In conclusion, we found that NAFLD children have extremely low levels of
25OHD throughout the year, with little dietary contribution. In addition, several
vitamin D related SNPs were associated with poorer histological findings. These
novel data suggest an important role for vitamin D in the pathogenesis and
progression of NAFLD in a paediatric population.
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Acknowledgements

First and foremost, I would like to thank my supervisors Bernadette
Moore, Kath Hart and Sue Lanham-New, for their invaluable support and
guidance throughout this PhD. They have always been there when I needed them,
for which I am extremely grateful. In addition, my collaborators at King’s College
Hospital Emer Fitzpatrick, Anil Dhawan, Martha Ford-Adams, Ragai Mitri and
Deepa Patel who have given clinical guidance over the past four years. Also thank
you to everyone in the extended team, namely Dr. Ruan Elliott who sat through
countless presentations, gave endless advice and asked somewhat daunting
questions.
My thanks also go to the Children’s Liver Disease Foundation who funded
this studentship, and to Anil, who gave funding for the final 3-month extension.
Dr. Ciaran Fisher deserve thanks for his help in the laboratory and for the
patience he displayed when teaching me new techniques. Particular thanks go to
Alberto for the analysis of biopsy slides often to a tight abstract deadline, and to
the nursing team at King’s who collected blood and anthropometrics from my
patients. Thanks also go to the undergraduate students who assisted on the diet
and lifestyle study as part of their final year projects: Joanna, Josie, Nicola,
Charlotte, Erika, Marisa, Katie, Georgina, Yi Qing and Marianne were all involved
in recruitment and dietary analysis, and Chloe, Hazel, and Amy who compiled the
food-based analysis. My study participants deserve special thanks for agreeing to
take part in my studies, allowing us into their homes and inspecting utensils and
crockery to get a better idea of food portion sizes.
My fellow PhD students and postdocs, past and present, deserve mention
for making my PhD such an enjoyable experience. In particular Elaina, Eleanor
and Sarah, whom were founding members of the 16AY03 tea and cake club.
Finally, I would like to thank my family and friends for their
encouragement throughout my PhD, particularly my cousin Han who
transformed images into thesis figures. You have all been supportive and
understanding during tough times. I could not have done it without you.
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Statement of Contributions
Philippa Gibson undertook the studies with the assistance of those below and
wrote this entire thesis and conference abstracts.
General Contributions
• General supervision was conducted by Dr. J. Bernadette Moore, Dr. Kathryn
Hart and Professor Susan Lanham-New
• Proofreading of thesis manuscript was conducted by Dr. J. Bernadette Moore,
Dr. Kathryn Hart, Prof. Susan Lanham-New, Dr. Ruan Elliott and Eleanor
Healing
• Staff at King’s College Hospital were responsible for patient anthropometrics
(weight, height and BMI). Additional anthropometric measurements (waist
circumference, mid-upper arm circumference and triceps skinfold) were
conducted by Philippa Gibson or undergraduate dietetic students.
• Measurement of blood biomarkers and 25OHD status were measured by
King’s College Hospital Department of Clinical Biochemistry
Specific Chapter Contributions
Chapter 1 – Systematic Review
• Sarah Lang devised and co-conducted the review with Philippa Gibson.
Chapter 2 – Diet and Physical Activity Levels in Paediatric NAFLD Patients
• Recruitment of participants was supported by Dr. Emer Fitzpatrick, Dr. Sanjay
Bansal, Dr. Martha Ford-Adams, and undergraduate dietetic students.
• Home visits were carried out by undergraduate dietetic students.
• Diet diaries were analysed by 2 independent student dieticians, with results
checked and verified by Philippa Gibson before analysis
• Amy Sturtivant compiled the food-based analysis data.
• Sarah Lang conducted statistical analysis and checked by Philippa Gibson with
the exception of the food-based analysis, which was conducted by Philippa
Gibson.
Chapter 3 – Assessing Vitamin D Status and Associated Genetic Polymorphisms
in a UK Paediatric Population
• Dr. Alberto Quaglia was the pathologist responsible for all biopsy analysis.
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• Dr. Huihai Wu adapted the Sachs vitamin D model for predicted August
vitamin D serum levels.

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Table of Contents
Declaration of Originality ............................................................................................... ii
Summary ............................................................................................................................ iii
Acknowledgements .......................................................................................................... iv
Statement of Contributions ............................................................................................ v
General Contributions ...................................................................................................................................... v
Specific Chapter Contributions .................................................................................................................... v
List of Tables ....................................................................................................................... x
List of Figures ..................................................................................................................... xi
Abbreviations ................................................................................................................. xiii
Chapter 1: Introduction ................................................................................................... 1
1.1 Research Relevance Overview .......................................................................................................... 1
1.1.1 Current Relevance ............................................................................................................................... 2
1.2 Non-Alcoholic Fatty Liver Disease .................................................................................................. 3
1.2.1 History of Disease .................................................................................................................................... 3
1.2.2 NAFLD Diagnosis ..................................................................................................................................... 5
1.2.3 NAFLD Prevalence ................................................................................................................................... 9
1.2.4 NAFLD Pathogenesis ........................................................................................................................... 13
1.2.5 The Role of Nutrition in NAFLD Treatment .............................................................................. 14
1.2.6 The Role of Physical Activity in NAFLD Management .......................................................... 17
1.3 Systematic Review .................................................................................................................................. 18
1.3.1 Literature Search .................................................................................................................................. 18
1.3.2 Data Extraction and Study Quality ............................................................................................... 19
1.3.3 Primary Outcome Measures ............................................................................................................. 20
1.3.4 Results ........................................................................................................................................................ 20
1.3.5 Study Characteristics .......................................................................................................................... 26
1.3.6 Study Findings ........................................................................................................................................ 27
1.3.7 Study Quality .......................................................................................................................................... 31
1.3.8 Discussion ................................................................................................................................................. 32
1.3.9 Conclusions .............................................................................................................................................. 36
1.4 Rationale for Study ................................................................................................................................. 37
1.5 Hypotheses, Aims and objectives ..................................................................................................... 37
1.5.1 Hypotheses ............................................................................................................................................... 37
1.5.2 Aims ............................................................................................................................................................ 37
1.5.3 Objectives ................................................................................................................................................. 38
Chapter 2: Diet and Physical Activity Levels in Paediatric NAFLD Patients: a
Case Control Study ......................................................................................................... 39
2.1 Background ................................................................................................................................................ 39
2.1.1 Physical Activity .................................................................................................................................... 39
2.1.2 Dietary Fructose .................................................................................................................................... 40
2.1.3 Dietary Fat ............................................................................................................................................... 40
2.1.4 Vitamin D ................................................................................................................................................. 41
2.1.5 Aims ............................................................................................................................................................ 42
2.1.6 Hypotheses ............................................................................................................................................... 42
2.1.7 Objectives ................................................................................................................................................. 43
2.2 Materials and Methods .......................................................................................................................... 44
2.2.1 Participants ............................................................................................................................................. 44
2.2.2 Demographic and Anthropometric Data ................................................................................... 44
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2.2.3 Dietary Assessment .............................................................................................................................. 45
2.2.4 Food-Based Dietary Analysis ........................................................................................................... 46
2.2.5 Physical Activity Assessment ........................................................................................................... 47
2.2.6 Statistical Analysis ............................................................................................................................... 48
2.3 Results .......................................................................................................................................................... 49
2.3.1 Study Population ................................................................................................................................... 49
2.3.2 Dietary Intakes ...................................................................................................................................... 50
2.3.3 Eating Styles ........................................................................................................................................... 51
2.3.4 Food Based Analysis ............................................................................................................................ 55
2.3.5 Physical Activity Levels ...................................................................................................................... 58
2.4 Discussion ................................................................................................................................................... 62
2.4.1 Participant Demographics ............................................................................................................... 62
2.4.2 Anthropometrics ................................................................................................................................... 62
2.4.3 Macro- and Micronutrient Intake ................................................................................................. 64
2.4.4 Under-reporting of Dietary Intake ................................................................................................ 66
2.4.5 Eating Behaviours ................................................................................................................................ 67
2.4.6 Food Group Based Analysis .............................................................................................................. 68
2.4.7 Physical Activity .................................................................................................................................... 68
2.4.8 Study Strengths and Limitations ................................................................................................... 69
2.5 Conclusions ................................................................................................................................................. 70
Chapter 3: Assessing Vitamin D status and Associated Genetic
Polymorphisms in a UK Paediatric Population .................................................... 72
3.1 Background ................................................................................................................................................ 72
3.1.1 Vitamin D ................................................................................................................................................. 72
3.1.2 Vitamin D Status ................................................................................................................................... 73
3.1.3 Vitamin D and NAFLD ........................................................................................................................ 74
3.1.4 Aims ............................................................................................................................................................ 75
3.1.5 Hypothesis ................................................................................................................................................ 75
3.1.6 Objectives ................................................................................................................................................. 76
3.2 Materials and Methods .......................................................................................................................... 76
3.2.1 Patients ..................................................................................................................................................... 76
3.2.2 Measurement of 25OHD Serum Levels ........................................................................................ 77
3.2.3 Genotyping ............................................................................................................................................... 77
3.2.4 Statistical Analysis ............................................................................................................................... 78
3.3 Results .......................................................................................................................................................... 78
3.3.1 Study Population ................................................................................................................................... 78
3.3.2 Vitamin D Status ................................................................................................................................... 80
3.3.3 Association of Genetic Variants with Liver Histology ........................................................... 83
3.4 Discussion ................................................................................................................................................... 85
Chapter 4: Examining the In Vitro Effects of Vitamin D ..................................... 91
4.1 Background ................................................................................................................................................ 91
4.1.1 Hepatic Stellate Cells ........................................................................................................................... 91
4.1.2 The Vitamin D Receptor ..................................................................................................................... 91
4.1.3 Vitamin D Treatment in Murine Models ..................................................................................... 92
4.1.4 In Vitro Treatment of Vitamin D .................................................................................................... 93
4.1.5 Aims ............................................................................................................................................................ 94
4.1.6 Hypotheses ............................................................................................................................................... 95
4.1.7 Objectives ................................................................................................................................................. 95
4.2 Materials and Methods .......................................................................................................................... 95
4.2.1 Chemicals ................................................................................................................................................. 95
4.2.2 Cell Culture .............................................................................................................................................. 96
4.2.3 Fatty Acid Treatment .......................................................................................................................... 96
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4.2.4 MTT Assay/Cell Viability ................................................................................................................... 97
4.2.5 Intracellular Lipid Quantification ................................................................................................. 97
4.2.6 Protein Analysis ..................................................................................................................................... 97
4.2.7 Vitamin D Treatment .......................................................................................................................... 98
4.2.8 RNA Isolation .......................................................................................................................................... 98
4.2.9 Clonogenic Assays ................................................................................................................................. 99
4.2.10 Statistical Analysis ............................................................................................................................ 99
4.3 Results ........................................................................................................................................................ 100
4.3.1 Fatty Acid Treatment ....................................................................................................................... 100
4.3.2 Vitamin D Receptor ........................................................................................................................... 102
4.3.3 Vitamin D Treatment ....................................................................................................................... 102
4.4 Discussion ................................................................................................................................................. 106
Chapter 5: Discussion .................................................................................................. 111
5.1 Thesis Aims .............................................................................................................................................. 111
5.2 Overview of Main Findings ................................................................................................................ 112
5.3 Strengths and Limitations .................................................................................................................. 114
5.4 Future Directions ................................................................................................................................... 116
5.5 Unique Contribution of this Thesis ................................................................................................ 117
Published Work ............................................................................................................. 119
Published Papers ........................................................................................................................................... 119
Published Abstracts ..................................................................................................................................... 119
References ...................................................................................................................... 120
Appendix A.1: Case-Control Study Information Sheet (For Parents/
Guardians) ...................................................................................................................... 147
Appendix A.2: Case-Control Study Information Sheet (For Participants) . 149
Appendix A.3: Case-Control Study Consent Form .............................................. 151
Appendix B: National Health Service Ethics Service (10/H0808/122) ...... 152
Appendix C: University of Surrey Ethics Committee (EC/2010/115/FHMS)
............................................................................................................................................ 155
Appendix D: Case-Control Study Food and Activity Diary .............................. 157
Appendix E: Dutch Eating Behaviour Questionnaire ........................................ 167
Appendix F: Youth Physical Activity Questionnaire ......................................... 170
Appendix G: Children’s Physical Activity Questionnaire ................................ 176
Appendix H: HepG2 Clonogenic Assay ................................................................... 182
Appendix I: LX-2 Vitamin D Cell Proliferation Comparison ........................... 183
Appendix J : Published Paper ................................................................................... 184
Appendix K: Published Abstracts ............................................................................ 197
Appendix L: Poster Presentations .......................................................................... 204

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List of Tables
Table 1.1: Summary of randomized control trials retrieved…………..……………….22
Table 1.2: Quality rating and risk of bias determined using the American
Dietetic Association Quality Criteria Checklist………………………………………………..32
Table 2.1: Population characteristics of NAFLD and obese control groups…..….50
Table 2.2: Nutritional intakes recorded vita participant completed 7-day
food diary of 24-hour recall expressed as absolute intakes……………………………..52
Table 2.3: Nutritional intakes recorded vita participant completed 7-day
food diary of 24-hour recall expressed as % DRV……………………………………………53
Table 2.4: Percentage of energy derived from macronutrients recorded
via participant completed 7-day food diary…………………………………………………….54
Table 2.5: Dietary intakes of food groups via participant completed 7-day
food diary……………………………………………………………………………………………………..55
Table 2.6: Physical activity levels recorded using 7-day activity diary…..………..59
Table 3.1: Population Characteristics……………………………………………………..……..79
Table 3.2: Genotype Distributions………………………………………………………..……….83

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