Vitamin D in Critically Ill Patients

Thesis/Dissertation Sheet

Surname/Family Name : Nair Given Name/s : Priya Abbreviation for degree as give in the University calendar : PhD Faculty : Medicine School : St. Vincent’s Clinical School Thesis Title : Vitamin D in critically ill patients

The pleiotropic functions of vitamin D, the clinical impact of deficiency and effect of supplementation have been a research focus in many specialties of ambulatory medicine. However, its prevalence in critical illness was not known. The parathyroid-vitamin D- calcium axis is the only endocrine axis that had not been previously described in critically ill patients. To study the effects of correction of deficiency, the dose, route and method of vitamin D supplementation needed investigation. Critically ill patients are heterogeneous and response to supplementation may not be consistent in all cohorts. This information is key to inform the design of a randomised controlled trial of supplementation. This thesis was a program of research that included a prospective observational study to describe serial changes in the parathyroid-vitamin D-calcium axis and the association with clinical outcomes in a sample of critically ill patients. This was followed by a randomised trial comparing two doses of intramuscular vitamin D to study the effectiveness and safety of supplementation. Subsequently, a sample of patients requiring life- sustaining extracorporeal support (ECMO) was studied to study pharmacokinetic profiles of vitamin D in patients with extreme disease severity. The observational study found marked changes in the parathyroid-vitamin D-calcium axis, which was associated with adverse outcomes. The supplementation study showed that a single intramuscular injection of cholecalciferol corrected vitamin D deficiency safely in critically ill patients. Vitamin D repletion was accompanied by a reduction in pro-inflammatory responses. The study on ECMO patients found them to be almost universally deficient in vitamin D. Supplementation by the single intramuscular dose corrected deficiency in only half these patients. Vitamin D deficiency is common in critical illness and is associated with adverse outcomes. Supplementation can be achieved effectively and safely in most patients. Groups of critically ill patients, such as those on ECMO are more susceptible to vitamin D deficiency and supplementation in this group is not easily achieved. This novel work and increasingly emerging evidence in the international literature provide a basis to inform the design of a high-quality comparative effectiveness randomised controlled trial of vitamin D supplementation in critically ill patients.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

…………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.

FOR OFFICE USE ONLY Date of completion of requirements for Award:

Vitamin D in critically ill patients

Fall Priya Nair 08

A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy

St. Vincents Clinical School

Faculty of Medicine

August 2018

INCLUSION OF PUBLICATIONS STATEMENT

UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure.

Publications can be used in their thesis in lieu of a Chapter if: • The student contributed greater than 50% of the content in the publication and is the "primary author", ie. the student was responsible primarily for the planning, execution and preparation of the work for publication • The student has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third partythat would constrain its inclusion in the thesis

Please indicate whether this thesis contains published material or not. □ Some of the workdescribed in this thesis has been published and it has been □ documented in the relevant Chapters with acknowledgement - This thesis has publications (eitherpublished or submitted forpublication) incorporatedinto it in lieu of a chapter and the details are presented below

CANDIDATE'S DECLARATION I declare that: • I have complied with the Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Name Signature Date (16107/18}

Priya Nair

dinator's Declaration ( �)

I declare that: • the information below is accurate • where listed publication(s) have been used in lieu of Chapter(s), their use complies with the Thesis Examination Procedure • the minimum requirements for the format of the thesis have been met.

PGC's Name PGC's Signature Date (dd/mm/yy) For each publication incorporated into the thesis in lieu of a Chapter, provide all of the requested details and signatures required

Details of publication #1: Fulltitle: Significant perturbationof vitamin D-parathyroid-calciumaxis and adverse clinical outcomes in critically illpatients Authors: PriyaNair, Paul Lee, Claire Reynolds, Nguyen Dinh Nguyen, John A. Myburgh, John A. Eisman, Jacqueline R. Center Journal or book name: Intensive Care Medicine Volume/page numbers: 39:267-274 Date accepted/published:9 Sep 2012113 Oct 2012 Status I Published x Accepted and In In progress I I press I I (submitted) I The Candidate's Contribution to the Work The candidate led the study -this included study design, ethics application, patient enrolment, data collection, analysis, drafting of the manuscript and responding to reviewers' comments. Co-authors assisted with data collection, analysis and interpretation. Supervisors (JRC and JAM) supervised and guided the candidate through the process from the concept of the study to publication. Location of the work In the thesis and/or how the work is incorporated in the thesis: This was a study conducted in primarily the St Vincents Hospital ICU along with collaboration from the Royal Prince Alfred and St George ICUs. Primary Supervisor's Declaration I declare that: . the information above is accurate . this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have aQreed to its veracity by siQninQ a 'Co-Author Authorisation' form. Supervisor'sname Supervisor's Date (16107/18) Prof. Jacqueline R Center signature

Details of publication #2: a Fulltitle: A Randomized Study of Single Dose of Intramuscular Cholecalciferol in Critically f/1Adults Authors: Priya Nair, Bala Venkatesh, Paul Lee, Stephen Kerr, Dominik J. Hoechter, Goce Dimeski, Jeffrey Grice, John A Myburgh, Jacqueline R. Center, Journal or book name: Critical Care Medicine Volume/page numbers:43:2313-2320 Date published: November 2015 Status x Published Accepted and In In progress press (submitted) The Candidate's Contribution to the Work The candidate led the study -this included study design, ethics application, patient enrolment, data collection, analysis, drafting of the manuscript and responding to reviewers' comments. Co-authors assisted with data collection, laboratory support, analysis and interpretation. Supervisors (JRC and JAM) supervised and guided the candidate throu h the recess from the conce t of the stud to ublication. Location of the work in the thesis and/or how the work is incorporated in the thesis: Work for this trial was erformed at St Vincent's Hos ital, S dne . Primary Supervisor's Declaration I declare that:

·------. the information above is accurate . this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter . All of the co-authors of the publication have reviewed the above information and have aareed to its veracity by signinQ a 'Co-Author Authorisation' form. Supervisor's name Supervisor's signature Date (16/07118) Prof. Jacqueline R Center

Details of publication #3: Fulltitle: Vitamin D status and supplementation in adult patients receiving Extracorporeal Membrane Oxygenation (ECMO) Authors: Priya Nair, Balasubramaniam Venkatesh, Dominik J Hoechter, Hergen Buscher, Stephen Ken; Jacqueline R Center John A Myburgh Journalor book name: Anaesthesia and lntensWe Care Volume/page numbers: not allocated Date accepted: 617/18 Status Published Accepted and In x In progress I I I press (submitted) I The Candidate's Contribution to the Work I I The candidate led the study -this included study design, data collection, analysis, drafting of the manuscript and responding to reviewers' comments. Co-authors assisted with data analysis and interpretation. Supervisors (JRC and JAM) supervised and guided the candidate throuQh the process from the concept of the study to publication. Location of the work in the thesis and/or how the work is incorporated in the thesis: St Vincents Intensive Care Unit Primary Supervisor's Declaration I declare that: . the information above is accurate . this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter . All of the co-authors of the publication have reviewed the above information and have aareed to its veracity bv sionino a 'Co-Author Authorisation' form. Supervisor's name Supervisor's signature Date (16/07118) Prof. JacquelineR Center

iii

I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International.

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.

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Date ……………………………………………......

AUTHENTICITY STATEMENT

I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.

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Date ……………………………………………......

ORIGINALITY STATEMENT

I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.

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ACKNOWLEDGEMENTS

I would like to dedicate this thesis to Dr Paul Lee, a beloved friend and colleague who sadly passed away during my candidature. It was Paul’s innovative mind and leadership that first introduced concept of the impact of vitamin D in the ICU which resulted in this research programme. He was always willing to devote time and share ideas, which was of immense help to me. Without his outstanding intellect and dedication, the world is a lesser place.

I would like to thank my supervisors, Jackie Center for her patience and attention to detail at every step and John Myburgh for his very prompt response times and importantly his constant encouragement and belief in me.

Bala Venkatesh has been incredibly generous with his time and expertise, which has been extremely helpful.

Claire Reynolds our Research Coordinator has been efficient and meticulous with all of our studies, going above and beyond for which I thank her very much.

My colleagues at work have been understanding and supportive throughout.

A massive thank you to Pierre for his presence, friendship and help with formatting and presenting this manuscript.

My parents and brother have made me the person that I am, have always encouraged me to reach for the stars and I am forever grateful for them. I lost my father, my biggest fan and ally during my candidature and I miss him deeply.

Jenny and Joan, my dear friends have kept me sane and positive over this time.

The fur kids, Gizmo and Kiara have been at my feet providing companionship and affection through many long nights and days of writing and this has been a huge comfort.

Finally, I thank John for his endless kindness, support and love that continues to be a source of inspiration.

RELEVANT PUBLICATIONS AND PRESENTATIONS

Publications-

• Lee P, Nair P, Eisman JA, Center JR. Vitamin D deficiency in the intensive care unit: an invisible accomplice to morbidity and mortality? Intensive Care Med. 2009;35(12):2028-32.

• Nair P, Lee P, Reynolds C, Nguyen ND, Myburgh J, Eisman JA, et al. Significant perturbation of vitamin D-parathyroid-calcium axis and adverse clinical outcomes in critically ill patients. Intensive Care Med. 2013;39(2):267- 74.

• Nair P, Venkatesh B. Vitamin D in ICU: time for its place in the sun. Critical Care and Resuscitation (2012) Volume 14 Issue 4.

• Krishnan A, Nair P, Venkatesh B. Vitamin D and the critically ill patient: An update for the intensivist. Handbook of Intensive Care and Emergency Medicine, Brussels 2013.

• Venkatesh B, Nair P. Hypovitaminosis D and morbidity in critical illness: is there proof beyond reasonable doubt? Critical Care 2014, 18:138

• Nair P, Venkatesh B, Lee P et al Randomised trial of single dose intramuscular cholecalciferol in critically ill adults. Crit Care Med 2015:43(11):2313-20

• Lee P, Ng C, Nair P, Eisman JAE, Center JR Preadmission bisphosphonate associated with reduced in-hospital mortality in critically ill patients. Journal of Clinical Endocrinology and Metabolism Jan 2016 101(5): jc20153467

• Lee P, Nair P, Eisman J, Center JR. Bone Failure in Critical Illness. Crit Care Med 2016; 44:2270–2274

• Nair P, Venkatesh B, Hoechter DJ, Buscher H, Kerr S, Center JR, Myburgh JA. Vitamin D status and supplementation in adult patients receiving Extracorporeal Membrane Oxygenation (ECMO); accepted for publication July 2018; Anaesthesia and Intensive Care.

• Nair P, Venkatesh B, Center JR. Vitamin D deficiency and supplementation in critical illness- the known knowns and known unknowns. Accepted for publication; September 2018; Critical Care.

• Venkatesh B and Nair P. Disorders of Calcium Metabolism; book chapter in Oh’s Intensive Care Manual- in press.

Presentations-

• “Fun in the Sun- the virtues of vitamin D in the ICU”-invited presentation- National Annual scientific meeting, College of Intensive Care Medicine. 2011

• Bi-national ANZICS Clinical Trial Group meetings, Noosa- Vitamin D in ICU research programme- 2010, 2011, 2013 & 2014.

• “Vitamin D in critical illness”- invited presentation-Australian Parenteral and Enteral Nutrition Society (AUSPEN) national meeting November 2013, Sydney

• Nair P, Venkatesh B et al. Vitamin D dosing study in patients with SIRS- oral presenation, best paper prize. National ASM, College of Intensive Care Medicine June 2014

• Nair P, Venkatesh B et al. Vitamin D dosing study in patients with SIRS- oral fast forward presentation, St Vincents Research Symposium, 2014

• Nair P, Vitamin D dosing study- oral presentation- Scandinavian Society of Anaesthesia and Intensive Care Annual Scientific Meeting, Iceland, June 2015.

• Nair P, Vitamin D status & dosing in ECMO patients- Poster Presentation- ANZICS ASM- Melbourne & ELSO conference Ann Arbor, Michigan-October 2014.

• Woollacott A, Nair P. Vitamin D status in parenteral nutrition patients-–oral presentation-SVH research symposium Sept 2014, ANZICS ASM October 2014.

• Vitamin D in critically ill patients- 2nd and 3rd year PhD presentations (2012 and 2014)- Garvan Institute for Medical Research

• Vitamin D and Bone Biology- invited presentation- Singapore ANZICS meeting, May 2017

• Vitamin D in critically ill patients- programme summary- St Vincents Hospital Research Week grand rounds, September 2018.

TABLE OF CONTENTS

Copyright Statement ……………………………………………………………..…. 3 Authenticity Statement ……………………………………………………………… 4 Originality Statement ……....……………………………………………………….. 5 Acknowledgements …………………………………………………………………. 6 Relevant publications and presentations …………...……….………………….... 7

Chapter 1: Background and Introduction ...... 12 1.1 Background ...... 12 1.2 Introduction ...... 13 1.2.1 Overview of vitamin D biology and metabolism ...... 13 1.2.2 Vitamin D production ...... 13 1.2.3 Vitamin D metabolism ...... 16 1.2.4 Vitamin D binding protein ...... 17 1.2.5 Vitamin D receptors ...... 19 1.2.6 Parathyroid Hormone response ...... 20 1.3 Functions of vitamin D ...... 22 1.3.1 Classic Functions ...... 22 1.3.2 Non-classic Functions ...... 25 1.4 Assessment of vitamin D status and deficiency ...... 27 1.4.1 Which metabolite to assay? ...... 28 1.4.2 Vitamin D assays ...... 28 1.4.3 Defining vitamin D deficiency ...... 29 1.5 Correction of deficiency and dosing strategies ...... 30 1.5.1 Recommendations ...... 31 1.6 Clinical significance of vitamin D deficiency ...... 33 1.7 Manifestations of deficiency in ambulatory patients ...... 33 1.7.1 Skeletal effects ...... 33 1.7.2 Extra-skeletal effects ...... 35 1.8 Potential implications for critically ill patients...... 40

Chapter 2: Research hypothesis and thesis outline ...... 47 2.1 Hypothesis ...... 47 2.2 Aim ...... 47 2.3 Objectives ...... 47 2.4 Outline of the thesis ...... 48

Chapter 3: Significant perturbation of vitamin D-parathyroid- calcium axis and adverse clinical outcomes in critically ill patients- an observational study114 ...... 51 3.1 Background ...... 51 3.2 Specific aims ...... 51 3.3 Methods ...... 52 3.4 Key results ...... 53 3.5 Interpretation ...... 53 3.5.1 Principal findings ...... 54 3.5.2 Comparison with previous and subsequent studies ...... 54 3.5.3 Key strengths ...... 59 3.5.4 Key limitations ...... 59 3.5.5 Clinical implications and implications for future research ...... 60 3.6 Publication details ...... 60 3.7 My role ...... 61 3.8 Declaration ...... 62 3.9 Manuscript ...... 62

Chapter 4: Randomised trial comparing two doses of Intramuscular Cholecalciferol in Critically Ill Adults152 ...... 71 4.1 Background ...... 71 4.2 Specific aims ...... 72 4.3 Methods ...... 72 4.4 Key results ...... 73 4.5 Interpretation ...... 74 4.5.1 Principal Findings ...... 74 4.5.2 Comparison with other studies ...... 74 4.5.3 Key strengths ...... 76 4.5.4 Key limitations ...... 77 4.5.5 Clinical Implications and implications for future research ...... 77 4.6 Publication details ...... 78 4.7 My role ...... 78 4.8 Declaration ...... 79 4.9 Manuscript ...... 79

Chapter 5: Vitamin D status and supplementation in adult patients receiving Extracorporeal Membrane Oxygenation (ECMO) ...... 88 5.1 Background ...... 88 5.2 Specific aims ...... 88 5.3 Methods ...... 89 5.4 Key results ...... 89

5.5 Interpretation ...... 90 5.5.1 Principal findings ...... 90 5.5.2 Comparison with other studies ...... 90 5.5.3 Key strengths ...... 91 5.5.4 Key limitations ...... 91 5.5.5 Clinical implications and implications for future research ...... 91 5.6 Publication details ...... 92 5.7 My role ...... 92 5.8 Declaration ...... 93 5.9 Manuscript ...... 93

Chapter 6: Summary of current knowledge and future considerations ...... 96 6.1 Introduction ...... 96

References ...... 99

Chapter 1: Background and Introduction

1.1 Background

Vitamin D is well known for its central role in calcium homeostasis and its contribution to bone health. Recognised manifestations of deficiency include hypocalcaemia, rickets in children and osteomalacia in adults.

In more recent times, the understanding of the extended role of vitamin D beyond just bone and the impact of deficiency has become a topic of research focus. This relates to the discovery that most tissues and cells in the body, not only bone, have a vitamin

D receptor. A number of these have the enzymatic ability to convert the major circulating form 25-hydroxyvitamin D, to the active form, 1,25-dihydroxyvitamin D locally at tissue level. This has provided new insights into the pleiotropic function of this vitamin. Indeed, this has led to its characterisation as a hormone, rather than a vitamin.

The role it may play in decreasing the risk of many chronic illnesses, including common cancers, autoimmune diseases, infectious diseases, and cardiovascular disease has been explored by the respective specialty groups over the last decade.

More recently however, its potential role in the contribution to acute critical illness and multi-organ failure, particularly sepsis has been of interest. It is noteworthy that the prevalence of low vitamin D levels in the critically ill medical and surgical populations is markedly higher than that seen either in the general population or non-critically ill hospitalised patients.

This suggests that deficiency in this vulnerable population could be relevant and that measuring and supplementing low vitamin D levels in critical illness should be considered, something that has not been incorporated into current clinical practice.

This relates to justified scepticism about whether deficiency is merely a marker of ill health. The higher prevalence in critically ill patients may therefore be a result of illness

severity and consequently of limited clinical significance.

Along the same lines, low vitamin D levels may mirror the phenomenon of observed low levels of other hormones in critical illness such as growth hormone, thyroid and cortisol likely being an adaptive mechanism.

If this is the case, vitamin D supplementation at best may provide no benefit and may even be harmful, as observed in studies of supplementation of these other hormones during critical illness.

This thesis is designed to address the knowledge gap that exists around the implications of vitamin D deficiency and its supplementation in critically ill patients.

1.2 Introduction

The aim of this chapter is to provide an introduction to the biology and metabolism of vitamin D. It then describes the classic and non-classic or pleiotropic functions and the assessment of vitamin D status and supplementation. Finally, it discusses the clinical significance of deficiency, with a focus on critically ill patients.

1.2.1 Overview of vitamin D biology and metabolism

To understand the clinical implications of vitamin D deficiency, it is important to review its formation, metabolism and mechanism of action.

Unlike other vitamins, it acts more like a hormone, mediating gene expression to perform many physiological functions within the body. Thus, it is important to understand how it is transported, its action through tissue vitamin D receptors and the body’s response to deficiency.

1.2.2 Vitamin D production

Vitamin D3 is produced in the skin from 7-dehydrocholesterol in a dual-stage process where the B ring is broken under ultraviolet rays (e.g. sunlight), and the pre-D3 formed

1 in this process isomerises to D3 in a thermo-sensitive but non-catalytic process.

The formation of pre-D3 is rapid, reaching a maximum within hours. Both the intensity of light rays and level of pigmentation in the skin regulate the rate of pre-D3 formation but not the maximum level reached.

With continued exposure, pre-D3 is converted to the biologically inactive lumisterol.

Tachysterol is also formed but, like pre-D3, does not accumulate with extended ultraviolet ray exposure. The formation of lumisterol and tachysterol is reversible and can be converted back to pre-D3 as pre-D3 levels fall. Thus, even prolonged exposure to sunlight does not result in D3 toxicity because of the photo-conversion of pre-D3 to lumisterol and tachysterol.2-4 (Figure 1)

Melanin in the epidermis, by absorbing ultraviolet rays, reduces D3 production. The intensity of ultraviolet rays from sunlight varies according to season and latitude, so the higher the latitude the person resides, the less time of the year is solar exposure

5 6 available to produce D3. Clothing and sunscreen prevent D3 production in the covered areas.

UVB Heat Diet 10-15% 7-dehydrocholesterol Pre-vitamin

Lumisterol/Tachysterol Vitamin D3

PTH & FGF-23 control

25-hydroxylase (CYP27A1) 1-alpha hydroxylase 1,25 Dihydroxy-D (CYP27B1)

25-Hydroxy vitamin D 24-hydroxylase DBP 24 hydroxylase 1,24,25hydroxy D

(inactive) 24,25hydroxy D

(inactive)

Tissue alpha hydroxylase Target Tissues

Local tissue Tissue 1,25 Dihydroxy-D paracrine actions

Figure 1: Activation and metabolism of Vitamin D PTH- Parathyroid Hormone, FGF-23- Fibroblast Growth Factor-23

1.2.3 Vitamin D metabolism

The three main steps in vitamin D metabolism- 25-hydroxylation, 1a-hydroxylation, and

24-hydroxylation are all performed by cytochrome P450 mixed-function oxidases

(CYPs). These enzymes are located either in the endoplasmic reticulum (ER) (e.g.,

CYP2R1) or in the mitochondria (e.g., CYP27A1, CYP27B1, and CYP24A1).7

The first step towards activation is conversion of vitamin D to 25-hydroxy vitamin D (25- hydroxy-D). In addition to UV activation small amounts of vitamin D, either as D2 or D3, can enter the body from its intestinal absorption from dietary intake and progress towards activation by hydroxylation.

While several mitochondrial as well as microsomal cytochrome P450 enzymes are capable of 25-hydroxylation8, CYP27A1 has been the one most extensively described.

This activation occurs principally, but not exclusively in the liver and these enzymes have a high capacity for substrate vitamin D. 25-hydroxy-D production is primarily substrate dependent, so serum 25-hydroxy-D is a reliable indicator of vitamin D status.9

The next step toward full activation is into 1, 25-dihydroxy vitamin D (1, 25-dihydroxy-

D) via CYP27B1 (also known as 1-alpha hydroxylase), a mitochondrial P450 enzyme in the proximal renal tubule of the kidney.

In addition to 1, 25-dihydroxy-D, the kidney also produces 24, 25-dihydroxy-vitamin D3

(24, 25 (OH)2D)- a relatively inactive metabolite.

25-hydroxyvitamin D 24 hydroxylase (24(OH)-ase also known as CYP24) can hydroxylate both 25-hydroxy-D and 1, 25-dihydroxy-D.

Thus, 24(OH)-ase limits the amount of 1, 25-dihydroxy-D in target tissues both by accelerating the catabolism of 1, 25-dihydroxy-D to 1, 24, 25 (OH)3D resulting in calcitroic acid. Alternatively, it might produce 24,25(OH)2D, thus decreasing the pool of

25-hydroxy-D available for 1-hydroxylation.10 (Figure 1)

The activation of 25-hydroxy-D to 1,25-dihydroxy-D was thought to occur

predominantly in the kidney. However, the enzyme 1-alpha hydroxylase is also found in a number of extra-renal sites such as immune cells, tissue epithelia of multiple tissues, bone, and parathyroid glands11. It functions to provide 1,25-dihydroxy-D for local tissue level consumption as an intracrine or paracrine factor.

It is the understanding of this function that has enabled studies of the clinical applications of the functions of vitamin D at tissue level.

Regulation of CYP27B1 in the proximal renal tubule is controlled by parathyroid hormone (PTH) and fibroblast growth factor (FGF)-23, which stimulate and inhibit, respectively, its expression. CYP27B1 expression is also inhibited by 1,25-dihydroxy-D through a negative feedback loop mechanism.12

Additionally, 1,25-dihydroxy-D negatively regulates its own levels by inducing CYP24, that catabolises both 1,25-dihydroxy-D and 25-hydroxy-D.13

Furthermore, macrophages may express a non-functional alternatively spliced form of

CYP24 located in the cytoplasm that potentially interferes with substrate access to the mitochondrial CYP2414 thus reducing both 25-hydroxy-D and 1,25-dihydroxy-D catabolism in these cells.

Another example is the keratinocyte, which also contains CYP27B1. Akin to the macrophage the enzyme can be induced by activation of specific TLRs.15

Both TNF-alpha and interferon (IFN)-alpha stimulate 1,25-dihydroxy-D production by keratinocytes16, suggesting that the keratinocyte like the macrophage uses 1,25- dihydroxy-D for host defence. Unlike the macrophage however, the keratinocyte has a fully functional CYP24, the mechanism by which 1,25-dihydroxy-D limits its own levels in the epidermis.17

1.2.4 Vitamin D binding protein

Vitamin D–binding protein (DBP) is the primary vitamin D carrier protein, which binds

85-90% of total circulating 25-hydroxy-D. It is a polymorphic single chain serum glycoprotein that is primarily produced by the liver. The unbound fraction (bioavailable

25-hydroxy-D) consists primarily of albumin-bound 25-hydroxy-D (10 to 15% of total

25-hydroxyvitamin D), with less than 1% in the free form.18

Binding of DBP impairs delivery of 25-hydroxy-D to vitamin D-activating 1-alpha- hydroxylase in target cells. Therefore, it is the action of unbound, free 25-hydroxy-D that exerts its physiologic activity.

The epithelial renal cells in the proximal tubule express both megalin and cubulin, which facilitate the entry of the 25-hydroxy-D-DBP complexes into the cell. Once internalised, through endocytosis, 25-hydroxy-D dissociates from DBP and then is metabolised to the free, biological active form.19

DBP has three main roles: (i) protecting vitamin D from biodegradation, (ii) limiting its access to target tissues, and (iii) reabsorbing vitamin D in the kidneys.

In addition to stabilising vitamin D concentrations, however, DBP slows the action of vitamin D in the intestine and reduces uptake by the liver. Additionally, other studies have demonstrated that DBP diminishes the action of vitamin D on target tissues such as monocytes and keratinocytes.

These findings have supported the application of the free hormone hypothesis to vitamin D. This model postulates that only hormones that are not bound to the carrier proteins are free to enter cells and induce biological actions.

Following the identification of binding coefficients of 25-hydroxy-D and 1,25-dihydroxy-

D with albumin and DBP, relative fractions in bound and unbound states may be calculated from their total concentrations and the concentrations of albumin and DBP.

Vitamin D behaves like some steroid hormones, where it binds much more strongly to its own binding protein rather than to albumin. Albumin-bound hormone can almost be considered similar to free hormone, as it is available to act on target tissues, while DBP bound hormone cannot. Indeed, given this weak binding, fluctuations in albumin level

(such as in critical illness) should not markedly affect vitamin D bioactivity.

Standard clinical assays measure the level of total 25-hydroxy-D without distinguishing the bound fraction. This makes the definition of true vitamin D deficiency challenging.

Common genetic polymorphisms in the DBP gene produce variant proteins that differ in their affinity for vitamin D. The prevalence of these polymorphisms differs between racial groups. For example, Black-Americans have low levels of measured 25-hydroxy-

D, but additionally have lower levels of DBP resulting in equivalent free levels of 25- hydroxy-D, implying they may indeed not be functionally deficient. This is consistent with their higher bone mineral density (BMD) and reduced incidence of fractures compared to equivalent non-black populations.20

Likewise, other factors such as medications (e.g. anti-retroviral drugs, oral contraceptive pills, aspirin), smoking, hormones (e.g. sex hormones, growth hormone,

PTH, insulin) and disease states (e.g. end-stage liver disease, renal disease, critical illness) all affect DBP levels.21

Although ideally DBP should be measured routinely to overcome these issues, there is a lack of standardisation of its measurement. As a result, it is difficult to compare the different assays and the studies describing these.

This inability to accurately measure DBP and hence interpret true deficiency is likely to be relevant in a critically ill population with heterogeneous conditions and disease states.

1.2.5 Vitamin D receptors

The diverse biological actions of 1,25-dihydroxy-D take place via specific changes in gene expression, which are mediated by an intracellular vitamin D receptor (VDR).22

The more recent discovery that these receptors are present in most organs and tissues such as breast, prostate, colon and immune cells among others has enhanced the understanding of the extra-skeletal, pleiotropic functions of vitamin D.

1,25-dihydroxy-D-occupied VDR hetero-dimerises with the retinoid X receptor and

interacts with vitamin D response elements (VDREs) in target genes and mediates their transcription. The receptor is composed of two domains: a domain that binds to the hormone and a domain that binds to DNA.23

In most target cells, these actions trigger the expression of networks of target genes whose functional activities combine to orchestrate specific biological responses.

These responses are tissue-specific and range from highly complex actions essential for homeostatic control of mineral metabolism (the traditionally recognised role) to more recently revealed focal actions that control the growth, differentiation and functional activity of numerous cell types. These include those of the immune system, skin, the pancreas as well as many other targets. These functions are described in the next section.

Ongoing clinical studies combined with evolving laboratory techniques have improved the understanding of the sophisticated mechanisms that underlie these varied biological responses.24

1.2.6 Parathyroid Hormone response

Parathyroid hormone (PTH) is another key regulator of calcium metabolism. In the presence of vitamin D deficiency, low ionised calcium concentrations stimulate the parathyroid glands to release PTH (secondary hyperparathyroidism).

High levels of PTH raise serum calcium concentrations through an increase in (i) renal calcium reabsorption, (ii) mobilisation of calcium from the skeleton and (iii) indirectly through a rise in 1,25-dihydroxy-D levels, which results in the absorption of calcium from the intestine.25

In fact, the PTH response has been used to define vitamin D deficiency. Optimal vitamin D status has been considered to be a 25-hydroxy-D level that maximally suppresses PTH secretion. A plateau in suppression of PTH occurs when the 25- hydroxy-D level reaches approximately 75nmol/L. This has therefore been chosen by

some as the cut-off to define optimal vitamin D status.26 However, many patients have very low 25-hydroxy-D values without an increase PTH, and conversely, others have

25-hydroxy-D levels greater than 75nmol/L, which does not result in PTH suppression.

The reasons behind this variability in PTH secretion are unclear but certain factors such as magnesium deficiency may play an important role.

Another factor may be gender. A study by Gunnarsson et al demonstrated PTH response to low circulating 25-hydroxy-D is modified differently between the sexes.

They found that men in the lowest PTH quartile were significantly younger, had less energy intake, lower body mass index (BMI) and better kidney function compared with the highest PTH quartile.

They had also higher ionised calcium, insulin‐like growth factor (IGF1) and testosterone and were more likely to smoke. Women within the lowest PTH quartile were younger, had lower BMI and magnesium values and higher IGF1 levels and were more likely to smoke. Stepwise multivariate regression showed that IGF1, testosterone and BMI were significantly associated with PTH in men (R2=0.472) whilst the factors identified in women were smoking, BMI and kidney function (R2=0.362).27

In a study of elderly subjects with vitamin D deficiency, Chen et al28 demonstrated that a number of patients do not respond with secondary hyperparathyroidism, despite marked vitamin D deficiency. These ‘non-responders’ consequently had lower rates of bone turnover and reduced mortality when compared with patients who elevated their

PTH in response to vitamin D deficiency.

High bone turnover has been shown to be associated with higher mortality. In a prospective study of elderly subjects, Sambrook et al29 found that the rate of bone turnover measured by markers of bone remodelling was independently associated with all-cause mortality. The mechanism of the effect of bone turnover on mortality was mainly manifested in deaths from cardiovascular causes. Another study in older

patients with heart failure found that elevated PTH levels were associated with peri- operative myocardial injury and in-hospital all-cause mortality, and that elevated PTH level contributes to both disturbed bone metabolism and poor outcomes.30

Therefore, there may be a link between PTH-related bone turnover with mortality and adverse outcomes, which may be relevant in a critically ill population.28

1.3 Functions of vitamin D

This section describes the functions of vitamin D in the different organs and tissues of the body. This will facilitate the future discussion on the clinical consequences of deficiency in ambulant and critically ill populations.

These functions have been divided into classic functions, which vitamin D has been known to undertake since its discovery and the non-classic paracrine functions carried out by tissue level activation, which have been described more recently.

1.3.1 Classic Functions

Calcium, Phosphorous metabolism and Bone Health

The principal function of 1,25-dihydroxy-D in the maintenance of calcium homeostasis is to increase calcium absorption from the intestine. The vitamin D receptor (VDR) is expressed in all segments of the small and large intestine and active 1,25-dihydroxy-D calcium absorption has been reported in the distal as well as the proximal intestine.

1,25-dihydroxy-D has been reported to regulate every step of intestinal trans-cellular calcium transport. It induces the expression of the apical membrane calcium channel

TRPV6, the calcium-binding protein calbindin-D9k and the plasma membrane Calcium

ATP-ase, PMCA1b. (plasma membrane calcium ATP-ase 1b)

Figure 2

Thereby, 1,25-dihydroxy-D exerts its control in the intestine on calcium entry, calcium binding, and basolateral extrusion of calcium.31,32 In the absence of vitamin D, only 10 to 15% of dietary calcium and about 60% of phosphorus is absorbed. The interaction of

1,25-dihydroxy-D with the VDR increases the efficiency of intestinal calcium absorption to 30 to 40% and phosphorus absorption to approximately 80%.

If normal serum calcium cannot be maintained by intestinal calcium absorption alone, then 1,25-dihydroxy-D acts together with PTH to increase calcium reabsorption from the renal distal tubule and to resorb calcium from bone.

1,25-dihydroxy-D is recognised by its receptor in osteoblasts, causing an increase in the expression of the receptor activator of nuclear factor-κB ligand (RANKL).

RANK, the receptor for RANKL on pre-osteoclasts, binds RANKL, which induces pre- osteoclasts to become mature osteoclasts. Mature osteoclasts remove calcium and phosphorus from the bone, maintaining calcium and phosphorus levels in the blood.

2+ 2− Adequate calcium (Ca ) and phosphorus (HPO4 ) levels promote the mineralisation of the skeleton.9

When there is vitamin D deficiency, the parathyroid glands are stimulated, resulting in

secondary hyperparathyroidism.

PTH increases the metabolism of 25OH D to 1,25-dihydroxy-D, which further exacerbates the vitamin D deficiency. PTH also causes phosphaturia, resulting in low serum phosphorus levels. Without an adequate calcium–phosphorus product, mineralisation of the collagen matrix is compromised, leading to classic signs of rickets in children and osteomalacia in adults.

Osteoporosis and Fractures

Osteoporosis is a systemic skeletal disease characterised by decreased bone strength and increased risk of fractures.

Although osteoporosis affects both aging men and women, it is more frequently observed in post-menopausal women.33 In addition to low estradiol, low serum 25- hydroxy-D is also associated with adverse skeletal outcomes.

The secondary hyperparathyroidism caused by vitamin D deficiency, which is common in the elderly, can result in decreased bone density and increased risk of fracture.34

Osteoporosis is a commonly encountered problem, unlike osteomalacia, which is only seen with severe vitamin D deficiency.

Muscle function

Vitamin D deficiency causes muscle weakness. Skeletal muscles have a vitamin D receptor and may require vitamin D for maximum function.35,36

In a randomised controlled trial, nursing home residents who received 800 IU of vitamin

D per day plus calcium had a 72% reduction in the risk of falls as compared with the placebo group (adjusted rate ratio, 0.28%; 95% CI, 0.11 to 0.75)37

A meta- analysis of five randomised trials (with a total of 1237 subjects) demonstrated that increased vitamin D intake reduced the risk of falls by 22% (pooled corrected odds ratio, 0.78; 95% CI, 0.64 to 0.92) as compared with only calcium or placebo.36

1.3.2 Non-classic Functions

The non-classic actions of vitamin D have been broadly categorised into three general effects: regulation of immune function, regulation of hormone secretion and regulation of cellular proliferation and differentiation.

However, the effects of 1,25-dihydroxy-D on any given tissue may involve actions in more than one of these categories1. Directly or indirectly, 1,25-dihydroxy-D controls more than 200 genes.

Regulation of immune function

Nearly all immune cells including B and T lymphocytes and activated macrophages display the vitamin D receptor (VDR) and 1α-hydroxylase. The immune modulatory functions of vitamin D are summarised below:

(i) Stimulates innate immunity (resistance to infection)- a) Facilitates neutrophil motility and phagocytic function. b) Stimulates the expression of potent antimicrobial peptides, cathelicidin (LL37) by macrophages, which is involved in bacterial killing.

(ii) Reduces inflammation - Modulates cytokine responses, such as interleukin 6 (IL-6)

(iii) Modulates adaptive immunity resulting in reduced auto-immunity and reduced antigen stimulation of the immune response. Inhibits proliferation of T helper 1 cells consequently impairing production of IL-2, tumor necrosis factor and interferon (IFN)], as well as T helper 17 (Th17) cells, skewing cytokine production toward a T helper 2

(Th2) phenotype.

Thus, the presence of adequate 25-hydroxy-D for local production of tissue 1,25- dihydroxy-D by cells of the immune system should enhance the capacity of white cells to repel invading pathogens at the same time as reducing their tendency to attack normal tissues with a consequent reduction in inflammation.38 39

Regulation of hormone secretion

In addition to its effect on PTH secretion described above, vitamin D stimulates insulin secretion. Although the mechanism is not well defined, VDR and calbindin-D28k are found in pancreatic beta cells.40

A number of case control and observational studies have suggested that vitamin D deficiency contributes to an increased risk for type 2 diabetes.41

Another hormone, FGF-23 is produced primarily by bone, and in particular by osteoblasts and osteocytes. 1,25-dihydroxy-D stimulates this by an unclear mechanism. FGF23 is a bone-derived hormone that regulates systemic phosphate homeostasis, vitamin D metabolism and α-Klotho expression through a novel bone- kidney axis.

FGF-23 inhibits renal tubular reabsorption of phosphate through mechanisms independent of PTH. As described previously, it also reduces circulating 1,25- dihydroxy-D through its dual effects to suppress CYP27B1 production and to stimulate

CYP24 catabolism of 1,25-dihydroxy-D.

FGF-23 also suppresses the gene transcription of α-klotho by the kidney, which exists as a membrane and soluble protein. Membrane Klotho acts as a co-receptor for and dictates organ specificity of FGF-23, whereas soluble Klotho act as an endocrine factor that regulates activity of cell surface glycoproteins and receptors in multiple tissues.42

As FGF-23 inhibits 1,25-dihydroxy-D production by the kidney, this feedback loop like that for PTH secretion maintains a balance in the levels of these important hormones.43

Regulation of cell proliferation and differentiation

The potential anti-cancer activity of vitamin D has been studied in both animal and cell studies for a number of years.

A large number of malignant cells have been demonstrated to express the VDR. The

basis for 1,25-dihydroxy-D in the prevention and treatment of malignancy includes its anti-proliferative, pro-differentiating effects.

In particular 1,25-dihydroxy-D stimulates the expression of cell cycle inhibitors p21 and p27 and the expression of the cell adhesion molecule E-cadherin and inhibits the transcriptional activity of beta-catenin1. Although vitamin D has shown to be protective in a number of cancers, most attention has been paid to cancers of the breast, colon, and prostate.

In keratinocytes, 1,25-dihydroxy-D has been shown to promote the repair of DNA damage induced by ultra-violet radiation (UVR), reduce apoptosis and increase survival after UVR and increase p53.44

The epidermis is unique in that under physiological conditions it is capable of producing vitamin D, converting it to 1,25-dihydroxy-D in the same cell and also responding to the

1,25dihydro-D produced.

As noted, 1,25-dihydroxy-D enables the keratinocyte to mount the innate immune response and suppress the autoimmune mechanisms that contribute at least in part to psoriasis.

Additionally, 1,25-dihydroxy-D promotes the differentiation of keratinocytes and inhibits their proliferation. In the epidermis, proliferation occurs in the basal layer, and as the keratinocytes move out of the basal layer, differentiation is initiated.

As the keratinocyte moves from one layer of epidermis to the next, differentiation proceeds in a sequential fashion, ultimately resulting in a lipid-rich matrix that provides the barrier function.

1,25-dihydroxy-D is involved in all steps of this process in that it limits proliferation in the basal layer and induces in a sequential pattern the expression of genes whose products ultimately produce the permeability barrier.45

1.4 Assessment of vitamin D status and deficiency

1.4.1 Which metabolite to assay?

Although 1,25-dihydroxy-D is the biologically active form of the vitamin, it is not a measure of vitamin D status. It has a short half-life (4-6 hours) and its circulating levels are 1000-fold less than 25-hydroxy-D.

With vitamin D deficiency there a decrease in intestinal calcium absorption and a transient drop in ionised calcium. This results in the production and secretion of PTH.

PTH acts by increasing renal tubular reabsorption of calcium, mobilisation of calcium from the skeleton and increasing renal production of 1,25-dihydroxy-D.

Thus, even when there is systemic deficiency, levels of 1,25-dihydroxy-D are preserved. This means that its measured serum levels are not a reflection of deficiency.

25-hydroxy-D, the major circulating and storage form of the vitamin D is therefore used to determine vitamin D status. It has a half-life of about 2-3 weeks.46

1.4.2 Vitamin D assays

Developing the appropriate assay to measure 25-hydroxy-D has presented challenges over the last 40 years. Several factors contribute to the analytical difficulties.

25-hydroxy-D is hydrophobic and therefore unstable in water. Its lipophilic nature means it strongly associates with vitamin D binding protein (VDBP). Endogenous lipids co-extract from plasma and serum, affecting binding and chromatographic separation.

It exists in several molecular forms; direct natural sunlight degrades both 25-hydroxy-

D metabolites and internal standards employed in some assays.

A suitable reference standard material has not been available until very recently.

Many methods have been developed over the years, with immunoassay becoming the most popular method. Recently, questions have been raised regarding immunoassays, with problems of accuracy and specificity being demonstrated and the recognition of fluctuations of assay performance noted.

Many clinical laboratories have therefore been moved to liquid chromatography-mass spectrometry (LC-MS/MS) technology, with potentially greater specificity and accuracy of measurement.

The recent generation of international standard reference material (SRM), and the development of reference method procedures should contribute to an improvement in the accuracy and precision of results generated by all methods in use currently and those developed in the future.47

In critically ill patients the value of a single level of 25-hydroxy-D48 and the effect of haemodilution, which may be seen during the resuscitation phase has been questioned49. These considerations and their impact on defining vitamin D status in critical illness are discussed in more detail in the final chapter of this thesis.

1.4.3 Defining vitamin D deficiency

Several criteria to define the optimal serum 25-hydroxy-D concentration have been described.

These include the level at which maximal suppression of PTH occurs, the level at which adequate renal production of 1,25-dihydroxy-D to ensure adequate intestinal calcium absorption occurs, and optimal level to prevent a defined clinical endpoint

(e.g., fracture).

The optimal serum 25-hydroxy-D concentration for skeletal health is controversial. The

Institute of Medicine (IOM) systematic review (https://www.nap.edu/read/13050) recommends concentration between 50 to 100 nmol/L (20 and 40 ng/mL), whereas other experts favour maintaining levels between 75 to 125 nmol/L (30 and 50 ng/mL).

Thus, the range of common agreement is 75 to 100 nmol/L (30 to 40 ng/mL).

Experts agree that levels lower than 50 nmol/L are suboptimal for skeletal health. The optimal levels for extra-skeletal health have not been established. The IOM recommendations are based upon evidence related to bone health. Other experts (the

Endocrine Society, the National Osteoporosis Foundation, the International

Osteoporosis Foundation], the American Geriatric Society) suggest that a minimum level of 75nmol/L is necessary in older adults to minimise the risk of falls and fracture.

The systematic review by the IOM also concluded there are insufficient data to determine the safe upper limit (https://www.nap.edu/read/13050/).

However, there was some concern at serum concentrations above 125 nmol/L. These concerns were based upon the increase in fracture in patients treated with high-dose vitamin D50 and conflicting studies describing a potential increased risk for some cancers (e.g., pancreatic, prostate) and mortality with levels above 75 to 120 nmol/L.

Similarly, definitions of deficiency and insufficiency are not universal. Some experts define deficiency as <50 nmol/L (<20 ng/mL) and vitamin D insufficiency as a 25- hydroxy-D level of between 52 and 72 nmol/L (21 and 29 ng/mL). Others define deficiency as <30nmol/L and insufficiency as 30-50nmol/L51-53 (1ng/ml=2.5mmol/L).

This definition is applied to a broad population, but as discussed above genetic factors related to DBP and others may mean that this definition may not be universally applicable.

1.5 Correction of deficiency and dosing strategies

For the most part, skin exposure to sunlight is the main source of vitamin D. It is found naturally only in small quantities in a few foods, such as wild-caught fatty fish.

Certain groups of adults are at higher risk for deficiency.54 This includes-

• Older or disabled people in residential care

• Housebound geriatric patients admitted to hospital

• Dark-skinned people, particularly those with modest dressing practice.

• People with a disability or chronic disease (e.g. multiple sclerosis)

• Fair-skinned people and those at risk of skin cancer who avoid sun exposure.

• Obese people

• Those taking medications that accelerate vitamin D metabolism such as

phenytoin

• Those with malabsorption including inflammatory bowel disease and coeliac

disease

• People working in an enclosed environment, such as office workers.

These patients would be at particular risk when they require admission to the ICU with an acute illness.

1.5.1 Recommendations

The two commonly available forms of vitamin D supplements are cholecalciferol

(vitamin D3) and ergocalciferol (vitamin D2).

In a meta-analysis of seven randomised trials evaluating serum 25-hydroxy-D concentrations after supplementation with cholecalciferol versus ergocalciferol, cholecalciferol increased serum 25-hydroxy-D more efficiently than ergocalciferol

(mean difference in serum 25-hydroxy-D of 15.23 nmol/L)55 The greatest difference was seen in trials that used weekly or monthly rather than daily dosing.

When sun exposure is minimal, supplementation of at least 600 IU (15 μg) of cholecalciferol per day for people aged ≤ 70 years and 800 IU (20 μg) per day for those aged > 70 years is recommended. People in high-risk groups may require higher doses.54

Recommendations for treatment of moderate to severe vitamin D deficiency are-

• 3000–5000 IU (75–125 μg) per day for at least 6–12 weeks, with a check on 25-

hydroxy-D concentrations after 3 months, followed by ongoing treatment with a

lower dose of around 1000–2000 IU per day followed by 1000 IU per day and

adequate calcium intake

• An alternative is 50,000 IU vitamin D3 once per month for 3–6 months. Monthly

dosing is similarly effective and apparently safe but takes 3–5 months for

plateau 25-hydroxy-D levels to be reached.56

• Most patients will need ongoing treatment with a lower dose (e.g., 1000 IU per

day or equivalent)

A systematic review of 30 studies of large single dose oral vitamin D in adult ambulatory cohorts reported that single vitamin D3 doses of 300,000 IU and greater are most effective at improving vitamin D status. This dose also suppresses parathyroid hormone concentrations for up to 3 months. They recommended that vitamin D doses >500,000 IU should be used judiciously in order to minimise adverse events.57

Few studies have documented complications following high-dose vitamin D supplementation. Some report minor gastrointestinal upsets. Rossini et al58 showed an increase in several bone turnover markers) following 600,000 IU of vitamin D3. Other studies report mild transient hypercalcaemia, hypercalciuria and increased urinary magnesium the effects of which are not found to be clinically significant.57 Another adverse effect of more clinical significance has been reported in a randomised controlled trial of 2,256 community dwelling elderly women, where a dose of 500,000IU cholecalciferol administered annually increased the rate of falls and fractures.

Incidence rate ratio for falls in the vitamin D group was 1.15; (95% confidence interval

1.02-1.30; p=0.03) and that for fracture was 1.26 (95% CI, 1.00-1.59; p=0.047)50

Supplementation in Intensive Care Unit (ICU) patients

In patients admitted to the ICU with an acute illness, supplementation of deficient patients is more challenging. This is because it might be desirable to achieve target levels in this population as soon as possible in order for vitamin D to provide its purported benefits.

In addition, the optimal route for achieving serum bioavailability during critical illness

would ideally be the intravenous route. However, there is no intravenous preparation of vitamin D apart from that available within intravenous multi-vitamin preparations in low dose.

The oral forms used in ambulatory patients are not as effective in ICU patients. This is due to impaired enteral absorption from decreased motility and splanchnic perfusion both of which are features of critical illness.

Intramuscular forms are available, but again poor peripheral tissue perfusion in shocked patients may result in suboptimal absorption of the dose administered.

However, this route is likely to be more bioavailable than oral supplementation.59

There have been a few studies assessing the ability of oral vitamin D to correct deficiency in ICU patients.60,61.

These issues and studies are discussed in more detail in Chapter 4 where I specifically addressed the question of vitamin D supplementation in the ICU setting.

1.6 Clinical significance of vitamin D deficiency

As discussed above the myriad functions that vitamin D undertakes, means that it has also been implicated to varying degrees in a number of disease states.

Previously, these included predominantly chronic disease processes in ambulatory populations, but now there is an increasing interest around vitamin D deficiency in acute critical illness.

1.7 Manifestations of deficiency in ambulatory patients

1.7.1 Skeletal effects

The classic manifestation of deficiency is rickets in children due to the inadequate mineralisation of growing bone. Osteomalacia refers to the failure of the osteoid formed

by osteoblasts to become mineralised with calcium and phosphorus.

Although histologically osteomalacia is characteristic of rickets, the term is also used to describe the inadequately mineralised bone caused by vitamin D deficiency in adults, who no longer have growing bones. It is seen only with severe deficiency.

Bone pain is a characteristic feature of osteomalacia, which can be confused with arthritis or fibromyalgia. Proximal muscle weakness and gait instability are often present.

The biochemical features of osteomalacia are similar to those of rickets, with increased serum alkaline phosphatase and PTH values, and low calcium, phosphorus, and 25- hydroxy-D values.26

In addition to the treatment and prevention of vitamin D–deficiency rickets in children and osteomalacia in adults, vitamin D has been associated with other beneficial skeletal effects.

There is a positive association between vitamin D levels and bone mineral density

(BMD) that has been observed in cross sectional studies in adolescents and adults.62,63

On the basis of RCTs, the evidence for the benefit of vitamin D relates to prevention of falls and fractures.

In a meta-analysis of 12 RCTs, a reduced non-vertebral fracture risk was demonstrated for doses of vitamin D greater than 400 IU/d (relative risk, 0.80; 95% confidence level

0.72-0.89).64 There are numerous studies and meta-analyses in this area with resulting controversy about vitamin D dose, level and degree of effect. Similarly, a meta-analysis of 8 RCTs demonstrated that vitamin D reduced the risk of falls (RR, 0.78; 95% CI,

0.64-0.94), but only if the dose was 700 IU/d or greater and the 25-hydroxy-D concentration was at least 75nmol/L.65 However, an RCT of a 500,000 IU annual dose of vitamin D in women of advanced age increased the median 25-hydroxy-D concentration one month later but resulted in an increased risk of falls and fractures in

the group receiving this regimen.50 More recently, a randomised controlled trial from

Switzerland explored the hypothesis that higher monthly doses of vitamin D or a combination with calcifediol (the liver metabolite which is 2-3 times more potent) would increase 25-hydroxy-D levels and reduce the risk of functional decline in home-dwelling men and women aged over 70 years. Although higher monthly doses were effective in reaching the threshold of 75nmol/L of 25-hydroxy-D, there was no benefit on lower limb function and of concern, the risk of falls increased with the higher dose compared with the low-dose control group (24,000IU D3).66

1.7.2 Extra-skeletal effects

The evidence for the extra-skeletal benefits is not as strong as that for the skeletal benefits.

All-cause mortality

In a prospective observational study of adults older than 65 years (NHANESIII cohort), the risk of death was 45% lower in those with 25-hydroxy-D values greater than

100nmol/L compared with those with values less than 25nmol/L (hazard ratio 0.55;

95% CI, 0.34-0.88).67

A meta-analysis of 18 RCTs of vitamin D supplementation in postmenopausal women reported a 7% lower risk of death in those receiving vitamin D supplementation (RR,

0.93; 95% CI 0.87-0.99). Baseline vitamin D levels were measured in 8 of the studies included and 9 studies had post-supplementation levels measured.

The dose used for supplementation varied with a few studies using single dose intramuscular cholecalciferol.68

A more recent meta-analysis of 32 observational studies compared the lowest

(<25nmol/L) to the highest (> 75nmol/L) category of 25-hydroxy-D. The hazard ratio for all-cause mortality was 1.9 (95% confidence interval = 1.6- 2.2; P < 0.001). Serum 25- hydroxy-D concentrations less than or equal to 75nmol/L were associated with higher

all-cause mortality than concentrations greater than 75nmol/ (p<0.01).69

On the other hand, two studies investigating the association between higher levels of

25-hydroxy-D and mortality found both high and low levels to be associated with increased risk of overall mortality.70 71

In a retrospective, cohort study, the CopD Study, in a single laboratory centre in

Denmark 25-hydroxy-D levels were analysed in 247,574 subjects from the general practice sector.

This demonstrated a reverse J-shaped relation between the serum level of 25-hydroxy-

D and all-cause mortality, indicating not only a lower limit but also an upper limit for optimal levels. The lowest mortality risk was at levels between 50–60 nmol/L.72

None of these studies are capable of proving a causative relationship between low or high levels and mortality and confounding factors such as active, outdoor lifestyles and compliance with medical care and follow up may have influenced the results.

Cardiovascular Disease

There has been debate as to whether vitamin D deficiency increases risk for cardiovascular disease. Several studies have found associations between 25-hydroxy-

D concentrations and hypertension, coronary artery calcification as well as prevalent and incident heart disease.53

Prevalence of myocardial infarction was found to be inversely associated with 25- hydroxy-D concentrations.

The NHANES III data in adults demonstrated those with 25-hydroxy-D levels of less than 50nmol/L had an increased risk of hypertension, diabetes, obesity, and high triglyceride levels—all metabolic manifestations associated with increased cardiovascular mortality.73

The CopD Study team from Denmark also analysed cardiovascular mortality in their

cohort and again found both low and high levels were associated with cardiovascular disease, stroke, and acute myocardial mortality in a nonlinear, reverse J-shaped manner, with the highest risk at lower levels.74

Overall, the Institute of Medicine (IOM) has concluded there is insufficient evidence to support cardiovascular benefits of vitamin D supplementation. This was supported by a meta-analysis of 51 RCTs of vitamin D supplementation. Baseline levels were recorded in a majority but not all studies. Dose regimes used for supplementation varied.

Vitamin D was associated with non-significant effects on patient-centred outcomes of death (RR, 0.96; 95% confidence interval 0.93-1.00; p= 0.08], myocardial infarction

(RR, 1.02; 95% CI, 0.93-1.13; p= 0.64), and stroke (RR, 1.05; 95% CI, 0.88- 1.25; p=

0.59).

These analyses were associated with minimal heterogeneity. Additionally were no significant changes in the surrogate outcomes of lipid fractions, glucose, or diastolic or systolic blood pressure, although these analyses were associated with significant heterogeneity.75

Diabetes Mellitus

Vitamin D receptors are present in pancreatic β cells, and vitamin D may augment insulin secretion and insulin sensitivity.

A systematic review and meta-analysis of observational studies and clinical trials in adults with outcomes related to glucose homeostasis has synthesised the evidence in this area41. The observational studies showed a relatively consistent association between low vitamin D status and prevalent type II diabetes or metabolic syndrome.

There were also inverse associations with incident type II diabetes or metabolic syndrome for highest vs. lowest combined vitamin D and calcium intake.

Evidence from trials with vitamin D and/or calcium supplementation suggests that combined vitamin D and calcium supplementation may have a role in the prevention of

type II diabetes only in populations at high risk (i.e. glucose intolerance). The available evidence is limited because most observational studies were cross-sectional and did not adjust for important confounders while intervention studies did not have adequate follow up, included small sample sizes, used a variety of doses and preparations of vitamin D and calcium or conducted post-hoc analyses.

The authors concluded that vitamin D and calcium insufficiency might negatively influence glycaemic control and combined supplementation may be beneficial in optimising glucose metabolism.41

Cancer Risk

As discussed above, vitamin D is known to promote cellular differentiation, inhibit cellular proliferation, and reduce the growth of certain tumors in laboratory animals.

However, the majority of clinical studies examining 25-hydroxy-D levels prior to cancer diagnosis do not support the hypothesis of low vitamin D and increased cancer risk, except for possibly colorectal cancer.

Three individual intervention RCTs in cancer were negative for benefit76-78. These data were then pooled with other RCTs and published in the Cochrane database as a systematic review looking at multiple clinical outcomes from vitamin D supplementation.79

In the subgroup analysis on cancer outcomes, a significant trend to reduced mortality was found. In this analysis of 95,286 participants, the authors concluded that vitamin

D3 significantly decreased mortality in elderly people (~11% decrease in mortality and

~12% decrease in cancer-related mortality)

A recent article reviewing current data concluded that higher concentrations of 25- hydroxy-D than are necessary for bone health might be effective in decreasing cancer risk and progression. The article also suggested that vitamin D metabolites or analogues might also be helpful in the treatment of cancer, especially when added to

existing therapies.80

Infection Risk

The stimulatory effects of vitamin D on the innate immune system through the expression of cathelicidin (LL-37) might imply that deficiency increases infection risk.

In observational data from NHANES III, persons with 25-hydroxy-D values lower than

25nmol/L were more likely to have had a recent upper respiratory tract infection than those with higher values in all 4 seasons of the year. This association was even stronger in those with asthma or chronic obstructive pulmonary disease.81

In a recent systematic review of 25 eligible RCTs (11,321 patients aged 0-95 years) vitamin D supplementation reduced the risk of respiratory infections. (Adjusted odds ratio 0.88, 95% confidence interval 0.81 to 0.96)82

Other infections where vitamin D has been demonstrated to play a role include tuberculosis, HIV and fungal infections.83-85

Multiple sclerosis

The incidence of multiple sclerosis increases with increasing latitude, corresponding with reduced ultraviolet B sun exposure and lower serum levels of 25-hydroxy-D.

A prospective, nested case-control study among more than 7 million US military personnel demonstrated that the odds of having multiple sclerosis were lower in the group with the highest 25-hydroxy-D levels- Odds ratio for a 50nmol/L increase 0.59;

95% confidence interval, 0.36-0.97.

In categorical analyses using the lowest quintile (<63.3 nmol/L) as the reference, the

ORs for each subsequent quintile were 0.57, 0.57, 0.74, and 0.38 (P = 0.02 for trend across quintiles). Only the OR for the highest quintile, corresponding to 25-hydroxy-D levels higher than 99.1 nmol/L, was significantly different from 1.00 (OR, 0.38; 95% confidence interval, 0.19-0.75; P = 0.006).86

Other conditions

Other conditions that have been proposed in which vitamin D may play a role include schizophrenia and depression87,88 and asthma or wheezing illnesses.89

1.8 Potential implications for critically ill patients

Vitamin D deficiency is generally considered a relatively mild, chronic condition that may have an impact in certain ambulatory patient cohorts.

However, in 2009, Lee et al90 reported in a group of 42 Intensive Care Unit (ICU) patients referred to the Endocrinology service a high prevalence of hypovitaminosis D, that was associated with adverse outcomes. The mean (SD) level of 25-hydroxy-D in this selected population was 41(22) nmol/L with only 7% of patients having sufficient levels (>60nmol/L) and 17% having undetectable levels. The three patients who did not survive their ICU admission all had undetectable levels of 25-hydroxy-D.

Following this, we published an article91 proposing potential mechanisms by which vitamin D deficiency might contribute to or worsen multi-organ dysfunction in critically ill patients. The knowledge of the pleiotropic actions of vitamin D on individual organ systems, cellular function, immune and inflammatory pathways was used to build this hypothesis.

This table illustrates some potential manifestations in critically ill patients of known associations of vitamin D deficiency seen in the general population.

Known functions Known association in general Potential manifestation in population critical illness Cardiac Myocardial Infarction Cardiogenic shock Heart Failure

Neurocognitive Stroke, Alzheimer’s disease, Encephalopathy, delayed diabetic neuropathy, MS neurological recovery, critical illness polyneuropathy

Glucose metabolism Diabetes Dysglycaemia

Calcium Osteomalacia, Osteoporosis Hypocalcaemia homeostasis

Mucosal barrier Inflammatory Bowel Disease Mucositis, translocation of bowel flora

Pulmonary Chronic Obstructive Pulmonary Acute Respiratory Distress Disease Syndrome

Endothelial Atherosclerosis, Hypertension Microcirculatory disturbance, multi-organ dysfunction

Innate immunity Tuberculosis Nosocomial infections, sepsis

Adaptive immunity Autoimmune disease, cancer Systemic Inflammatory Response Syndrome

Muscle Myopathy, myalgia Critical illness Myopathy

One supporting observation to this hypothesis is that hypocalcaemia, even after adjustment for protein and pH (i.e. ionised calcium), is common in critically ill patients.

It is seen in 15–88% of adult ICU patients.92-94 Various mechanisms have been proposed to explain how this occurs, including intracellular sequestration of calcium and relative hypoparathyroidism. Risk factors proposed include sepsis, burns, pancreatitis and rhabdomyolysis. Severe hypocalcaemia is an uncommon finding in non- critically ill patients, due to the presence of multiple compensatory mechanisms to maintain calcium homeostasis.91

Parenteral calcium supplementation has not been shown to improve the outcome of intensive care patients, as noted in a Cochrane review.95 It is possible that this hypocalcaemia commonly seen during critical illness may be a manifestation of unrecognised underlying vitamin D deficiency.

Although vitamin D insufficiency might not produce clinical manifestations in otherwise well individuals, deficiency may prove detrimental when a patient is critically ill.

The circulating vitamin D pool therefore might function as an important reservoir for conversion to active metabolites at tissue level during stress.

We therefore hypothesised that vitamin D deficient/insufficient states may worsen existing organ dysfunctions in critically ill patients, leading to adverse clinical outcomes.

In other words, the observed association between increased mortality and vitamin D deficiency in the general population may be augmented during critical illnesses, resulting in excess morbidity and mortality. The diagram below illustrates how patients who develop their critical illness in a vitamin D sufficient state, metabolic, immune and organ function may be enhanced with sufficient up-regulation of the tissue PTH-vitamin

D axis (panel A). However, critical illness developing during a state of vitamin D deficiency may lead to a breakdown in various tissue homeostatic mechanism as illustrated (panel B) resulting in morbidity and mortality.91

Figure 3

Following this, similar findings were replicated internationally61,96 and this motivated our programme of vitamin D research in the critically ill, resulting in this piece of work.

Since then, several larger studies confirming the high prevalence of vitamin D deficiency in critical illness and its association with poor outcomes were conducted by our group and others internationally. These are described in more detail in Chapter 3.

In addition to hypocalcaemia, a broader contributory role of vitamin D deficiency to critical illness seems biologically plausible based on our understanding of the functions of vitamin D as described above.

Particularly relevant to the ICU context is the contribution of vitamin D within the immune system and to the inflammatory response.

Critical illness is an inflammatory state

The understanding and knowledge of metabolic response to critical illness has rapidly evolved during the last decade. The response of the body to the stress of critical illness involves a neuroendocrine and an inflammatory/immune component.97

Cells of the innate immune system typically produce the initial inflammatory response to critical illness, even in the absence of an inciting microorganism. These include neutrophils, monocytes and macrophages. The pro-inflammatory mediators made by these cells are responsible for many of the pathophysiologic features of critical illness, which contribute to the multi-system organ dysfunction. The mobilisation of the innate immune system is a rapid and potent but nonspecific response. The adaptive immune system on the other hand is highly selective in its targets and is endowed with memory but is slow in initial activation. Critical illness results in derangements of all components of the immune response, which due to its complexity makes it challenging to correct the relevant derangements.98

In the presence of infection caused by a microorganism, this inflammatory response may be even more pronounced resulting in sepsis and septic shock.

Concurrent with the pro-inflammatory response, is a compensatory anti-inflammatory response. This involves the formation of anti-inflammatory mediators and impairment of innate immune cell function. This includes reduction of monocyte human leukocyte antigen-DR expression and impairment of the ability of innate immune cells to produce tumour necrosis factor alpha when stimulated. In its most severe form this is referred to as immunoparalysis and is associated with markedly increased risks for nosocomial infection and death in the ICU.99

The inflammatory response may be ongoing and is seen with an ongoing pro- inflammatory phenotype. It is associated with poor muscle function and physical recovery even following ICU discharge, so called chronic critical illness.100

Vitamin D in infection and sepsis

One of the most promising extra-skeletal roles of vitamin D in critically ill patients is its effect on the immune system. This was first indicated by the discovery of vitamin D receptors in almost all cell types across the innate and adaptive immune systems.101

Following this, studies have indicated that vitamin D modulates the immune response to bacterial endotoxin both in vitro and in murine models of sepsis.102,103 Vitamin D was also found to be involved in the monocyte response to Candida albicans infection.104

In addition to its effects on the humoral immune response, vitamin D produces a local tissue response to inflammation by inducing the production of anti-microbial peptides

(AMPs). In 2006, the critical role of vitamin D in the production of the AMP cathelicidin in response to Mycobacterium Tuberculosis infection was revealed.105

The active fragment of cathelicidin is LL-37, which has been shown to be produced by phagocytic leukocytes, mucosal epithelium and keratinocytes and to be present in mucosal secretions and plasma106.

The immune function of LL-37 includes direct bactericidal activity as well as disruption of Pseudomonas aeruginosa biofilms, promotion of phagocytosis and reactive oxygen

species, and chemotaxis of other immune cells to sites of infection106. These properties have been demonstrated to have efficacy in in vitro studies of human airway and bladder pathogens107,108

Clinical research in the area has supported basic science research to some degree.

Such studies have a wide range of study designs, populations, and interventions and have understandably demonstrated mixed results.

For the most part, observational studies among adults have revealed an association between low vitamin D and the incidence of respiratory infections81,109, although clinical trials have not substantiated the strong effect sizes110,111.

The potential effects of vitamin D on multiple physiologic and organ systems make it difficult to isolate its relationship to sepsis in the critically ill.

A study by Jeng et al112 in critically ill patients with sepsis demonstrated a strong positive correlation between 25-hydroxy-D concentrations and levels LL-37 suggesting defence against infection in critically ill patients may be regulated by vitamin D status.

Much of the current clinical science regarding vitamin D and sepsis is nested within the larger studies of critically ill patients and has yielded mixed results. These studies have been discussed in more detail in Chapter 3.

Of note, although the IOM report defined a 25-hydroxy-D concentration of 50nmol/L for skeletal health, we still do not know what level may be optimal for immune function.113

To summarise the above discussion relevant to the role of vitamin D in ICU patients, critical illness is characterised by varying degrees of local and systemic inflammation, as well as metabolic and immune dysfunction.

These are functions over which vitamin D exerts important regulatory actions from basic science and clinical research in ambulatory patients. Also, failure to up-regulate

PTH- vitamin D axis due to circulating vitamin D insufficiency may contribute to metabolic and immune dysfunction and ultimately multi-organ failure observed in

critical illness.

In the presence of sufficient vitamin D levels, target tissues are possibly able to increase local formation of 1,25-dihydroxy-D to meet tissue demand when critical illness occurs.

So, while low vitamin D levels may not pose immediate health hazards in otherwise well individuals, deficient states may prove detrimental during critical illness, if the circulating vitamin D pool functions as an important reservoir for activation at times of stress.

Therefore, vitamin D deficient states may worsen existing organ dysfunctions in critically ill patients, contribute to complications such as nosocomial infections and delay recovery, ultimately leading to worse outcomes.91

A knowledge gap was identified in this area of critical illness around the true prevalence of vitamin D deficiency, the clinical significance if any, of this and a safe and effective way to supplement these patients.

This led to the programme of research presented in the following chapters.

The outline of the programme is detailed in the next chapter.

Chapter 2: Research hypothesis and thesis outline

2.1 Hypothesis

Vitamin D deficiency is highly prevalent in critically ill adults, which results in exacerbation of existing immune and metabolic dysfunction and leads to worse clinical outcomes. Supplementation is safe, effective and improves patient-centred outcomes.

2.2 Aim

This thesis is designed to address the knowledge gap that exists around the true prevalence of deficiency and the clinical significance of the extra-skeletal, pleiotropic effects of vitamin D in adult critical illness.

The overall aim is to use a mixed methods approach to assess the feasibility of designing a phase-3 randomised controlled trial to test the hypothesis that vitamin D supplementation improves patient-centred outcomes and mortality in critically ill adults.

2.3 Objectives

Objective 1:

To describe how often vitamin D status is measured and corrected in current clinical practice in Australia and New Zealand intensive care units.

Objective 2:

To determine the prevalence of vitamin D deficiency in adult critical illness and describe associations between vitamin D deficiency, disease severity and clinical outcomes.

To study the parathyroid hormone–vitamin D–calcium axis during critical illness.

(Chapter 3)

Objective 3:

To determine the effect of dosing with cholecalciferol (vitamin D3) on pharmacokinetic and pharmacodynamic endpoints in critically ill patients.

(Chapter 4)

Objective 4:

To study a subgroup of critically ill patients with high illness severity (patients receiving extracorporeal life support) in relation to prevalence of vitamin D deficiency and the ability to augment levels of 25-hydroxy-D with cholecalciferol supplementation.

(Chapter 5)

Objective 5:

To provide a summary of the current knowledge and highlight the uncertainty that exists. This will inform a discussion about specific considerations that will be required for a proposed randomised controlled trial of vitamin D supplementation in critical illness.

(Chapter 6)

2.4 Outline of the thesis

This is submitted as a thesis by publication under the University of New South Wales guidelines.

The first chapter is an introduction to the topic and a review of the literature.

The main body of the thesis contains 3 published original manuscripts and includes material from other publications I undertook during my candidature.

Each chapter includes a comprehensive review of the data relevant to the area, and

the implications of these in the design of future studies.

Much of this has become available from international groups during my candidature.

This work was done as the basis towards designing a phase III randomised trial on the effects of vitamin D supplementation in critically ill adults. This trial is outside the scope of the current thesis, but the relevant considerations and future directions have been outlined in the concluding chapter.

Chapter 3 commences with a description of a cross sectional study that was undertaken in Intensive Care Units (ICUs) in Australia and New Zealand on a single calendar day.

This was followed by a prospective observational study, which is the manuscript described in Chapter 3.

The manuscript described in chapter 4 is an interventional dosing study.

It was funded by competitive, peer-reviewed grants obtained from the Intensive Care

Foundation and St. Vincent’s Clinic.

It forms the major component of this thesis and informs the design of the future phase

3 randomised controlled trial of vitamin D supplementation.

This study showed that while overall adequate supplementation could be achieved with the single intramuscular dose administered, there may be specific patient subgroups where this might not be possible.

One such subgroup identified was those patients who were on extracorporeal membrane oxygenation (ECMO) support. From the observational study described in chapter 3, it also appeared that the subgroup of ECMO patients had more prevalent and severe deficiency.

The manuscript described in chapter 5 therefore, focuses on vitamin D status and supplementation in ECMO patients.

The concluding chapter of the thesis summarises systematically the knowledge acquired to date in regard to vitamin D and its supplementation in critical illness- the

“known knowns”. It also highlights the areas of uncertainty and consequently the challenges to be considered in the design of a future phase 3 trial- the “known unknowns”.

Future plans for complementary post-doctoral research, include studying what determines response to vitamin D supplementation - why some patients respond more than others.

This will be performed by using stored serum samples from a large, multi-centre study of patients with severe septic shock. The study will describe perturbations in storage, transport, activation and catabolism of the vitamin, which determine not only disease severity but also influence response to treatment and clinical outcome.

In parallel but related to this programme of research we have reported a significant survival benefit in patients receiving bisphosphonate therapy prior to their ICU admission. Bisphosphonates are anti-resorptive agents used clinically in the treatment of osteoporosis but are also found to have anti-inflammatory and other beneficial properties. Future directions will include safety and feasibility studies of these agents and studies combining them with vitamin D in critically ill patients.

Chapter 3: Vitamin D-parathyroid-calcium axis and clinical outcomes in critically ill patients- an observational study114

3.1 Background

Cross-sectional Study

Prior to commencing this study, we conducted an observational, multi-centre, single day point prevalence study to understand current clinical practice in relation to vitamin

D status assessment and supplementation of deficiency in Australian and New Zealand

ICUs. This was undertaken under the auspices of the point prevalence programme conducted by the Australia New Zealand Intensive Care Society Clinical Trials Group

(ANZICS-CTG) in collaboration with the George Institute for Global Health (TGI). There were 506 patients admitted to 38 adult ICUs on the point prevalence day in 2010.

These included a combination of tertiary referral centres, metropolitan, rural and regional centres encompassing a wide variety of medical and surgical patients. Ten patients (2%) had received oral vitamin D supplements (400-600 IU/day) prior to their

ICU admission. None of the patients had been assessed for vitamin D status with a 25- hydroxy-D level during their current ICU admission and no patient received vitamin D supplementation other than that available in standard enteral or parenteral nutrition solutions.

This suggested that the true prevalence was unknown and implication of vitamin D deficiency in critical illness was not a consideration at the time. Other endocrine axes during critical illness e.g. thyroid, adrenal have been studied and are assessed when clinically indicated. However, the PTH-vitamin D-calcium axis had not been described in ICU patients. This information was used as background to the prospective, multi- centre inception cohort observational study described here.

3.2 Specific aims 51

This study was undertaken to define the prevalence of vitamin D deficiency and insufficiency in critically ill patients and to understand how levels change over the course of their critical illness.

There was also an imperative to study the evolution of the PTH-vitamin D-calcium axis during critical illness prior to designing interventional trials, as this was unknown.

We further aimed to explore associations between low levels with severity of illness and clinical outcomes to assess the impact of hypovitaminosis D.

3.3 Methods

The study took place in 3-university referral hospital ICUs in Sydney, Australia (33 degrees South latitude) over an 8-month period to include warmer and cooler months of the year.

One hundred patients who were expected to stay in the ICU for at least 48 hours were enrolled in order to exclude patients with lower illness severity such as those admitted for routine observation following elective surgery. Blood samples to assess vitamin D levels (storage and active forms), PTH and ionised calcium were collected once the resuscitation phase was completed in order to avoid the effect of acute haemodilution.

Samples were also collected on day 3 and day 7 or ICU discharge, whichever came earlier.

Standard assays performed under strict quality control were undertaken in a single laboratory. Levels >50nmol/L were considered sufficient, 25-50nmol/L insufficient and

<25nmol/L deficient.

We attempted make an estimate of bioavailable, free-vitamin D concentration (given the high level of binding to DBP described previously). DBP is a globulin and we

“corrected” for binding by dividing 25-hydroxy-D levels by the globulin level. Therefore, patients with higher globulin and consequently lower free-vitamin D would also have a

lower “corrected” level. This is not a validated method and was done as exploratory analysis.

3.4 Key results

Of the 100 patients studied, prevalence of vitamin D sufficiency at baseline was low at

11%. Levels were undetectable in 8% (<15nmol/L), deficient in 24% and insufficient in

55%, using the definitions above. Only 3% had levels above 75nmol/L–a level considered as a cut-off for sufficiency by some sources.

Admission 25-hydroxy-D results strongly correlated with 1,25-dihydroxy-D but no other parameters of the axis.

Ionised hypocalcaemia was seen in 83% of patients on admission. Secondary hyperparathyroidism (PTH>7pmol/L) was seen in 38% of hypocalcaemic and 33% of vitamin D insufficient patients. These PTH responders had significantly higher severity of illness scores, but there was no increase in mortality.

While 25-hydroxy-D and 1,25-dihydroxy-D levels remained stable through the ICU stay,

PTH levels dropped and ionised calcium levels dropped. Although these were statistically significant the changes did not appear clinically significant.

Admission 25-hydroxy-D levels showed only a modest correlation with severity of illness scores (SAPS-2 and APACHE-2), which increased somewhat, albeit still modest when a globulin correction for free levels was performed. (R=-0.3, p=0.01 to R=-0.32, p<0.0001).

Patients who were vitamin D deficient had significantly fewer hospital-free days, odds ratio 3.15 (1.18-8.43). There was no correlation between 25-hydroxy-D levels and mortality and in a multivariate analysis; age and APACHE-2 scores were the only variables that were independent predictors of mortality.

3.5 Interpretation 53

3.5.1 Principal findings

This study was able to characterise the vitamin D, calcium and PTH status in critically ill adults. It confirmed a high prevalence of hypovitaminosis D. It showed that parathyroid response occurred only in a third of deficient/insufficient patients and this was associated with a higher illness severity, but not mortality. Vitamin D deficient patients had fewer hospital free days. These results collectively show that there are marked changes in the PTH-vitamin D-calcium axis during critical illness, which are associated with adverse clinical outcomes.

3.5.2 Comparison with previous and subsequent studies

At the time we were undertaking our study, there was wide international interest in this area with several studies being published during and after our publication. These observational studies performed in ICU patients to date are summarised in Table 1.

All studies cited in the table were in adult patients. There are similar studies emerging in paediatric ICU patients.

They universally confirm a high prevalence of deficiency in critically ill patients.

However, the observed relationship with clinical outcomes is variable.

A systematic review of observational studies performed by in 2014 by de Haan et al 115 which included our study, reported that levels of 25-hydroxy-D < 50 nmol/L were associated with increased rates of infection (risk ratio (RR) 1.49, 95% (confidence interval (CI) 1.12 to 1.99), P = 0.007), sepsis (RR 1.46, 95% (CI 1.27 to 1.68), P

<0.001), 30-day mortality (RR 1.42, 95% (CI 1.00 to 2.02), P = 0.05), and in-hospital mortality (RR 1.79, 95% (CI 1.49 to 2.16), P <0.001). In a subgroup analysis of adjusted data including vitamin D deficiency as a risk factor for 30-day mortality the pooled RR was 1.76 (95% CI 1.37 to 2.26, P <0.001).

Overall it concluded that vitamin D deficiency increases susceptibility for severe infections and mortality of the critically ill.

This provides a basis to pursue interventional trials of vitamin D supplementation during critical illness to assess its effects on clinical outcomes.

Table 1: Summary of observational studies in ICU patients describing prevalence of vitamin D deficiency and/or relationship with outcomes-

N- number of patients; Gp-group, Obs.- observational; Retro-retrospective; Pts.-patients; vs.- versus; Ass.- association; Adm.- admission; SAPS-2- Simplified Acute Physiology Score-II; Med-medical; Surg.-surgical; VAP- ventilator associated pneumonia; LOS-length of stay; AKI- acute kidney injury; BSI- blood stream infection; def./insuff.- Deficiency or insufficiency; ALI- acute lung injury, HAI- hospital acquired infections, P/F- PaO2/FiO2 ratio, vit-Vitamin; dysfn.- dysfunction.

Author Year Design N Pt. group Aim Findings Lee90 2009 Obs. 42 Pts. Prevalence High prevalence referred to of Correlates with endocrine deficiency hypocalcaemia, service Correlation SAPS score with outcome Jeng112 2009 Obs. 49 Sepsis vs. Prevalence 100% Non- of prevalence in sepsis vs. deficiency sepsis patients. controls & effect on Ass. of Vit D with LL37 LL-37 levels Lucidarme96 2010 Obs. 106 ICU pts. Incidence & 79% incidence admitted risk factors Spring adm, low Spring/ for albumin, high Summer deficiency SAPS-2 predict deficiency Mata- 2010 Obs. 33 ICU pts. Vit. D 62% blood Granados61 Vs. blood status donors, 97% donors ICU pts.

deficient McKinney116 2011 Retro 136 Veterans Association Sufficient pts. in ICU with low had shorter LOS

levels and and decreased outcome risk of death Cecchi117 2011 Obs. 170 Sepsis vs. Vit. D Lower in sepsis trauma levels & No outcome outcomes relationship

Braun118 2011 Retro 2439 Level 1 Prediction Low levels year prior of outcome predicted to ICU, based on mortality and med/surg. Vit D level. blood culture ICU positivity. Ginde119 2011 Obs. 81 Adult ED Vit D status Higher sepsis pts. with & sepsis severity & organ sepsis severity dysfunction if vit D insufficient Venkatraman 2012 Obs. 437 Medical Vit D levels Increased 120 ICU and mortality in mortality deficient patients Flynn121 2012 Obs. 66 Surgical Vit D levels Increased LOS ICU and infection, Organ outcome dysfunction if low levels Matthews122 2012 Obs. 192 Surgical Relationshi -Increased VAP ICU p with VAP incidence, LOS, costs if deficient Arnson123 2012 Obs. 120 Ventilated Correlation Deficiency pts. with common, longer mortality survival time in sufficient pts. Braun124 2012 Retro 1325 Med/ Ass. of low Significant Surg. ICU, Vit D at predictor of all- admission cause mortality with mortality Higgins125 2012 Obs. 197 ICU Burden of Decrease in patients deficiency level during ICU, Ass. with Longer time to outcomes discharge, trend towards more infections Morandi126 2012 Obs. 120 Medical Ass. of Not a ICU pts. deficiency determinant of with delirium risk delirium Braun127 2012 Obs. 209 Med/ Vit D Predictor of AKI Surg. ICU deficiency & mortality & AKI relation Lange128 2013 Retro 23603 Med/ Pre- Associated with Surg. ICU hospital Vit mortality & BSI D & mortality

Su129 2013 Obs. 206 50 Ass. with No significant controls, severity of association 51 ICU, illness, 105 sepsis infection risk & mortality Aygencel130 2013 Obs. 201 Med ICU Prevalence 69% insufficient, & ass with higher mortality mortality but not an independent risk factor Azim131 2013 Obs. 158 Med/Surg. Prevalence 80% deficient, ICU no ass with mortality Amrein132 2014 Retro 655 Med/ Prevalence 86.5% def./ Surg. ICU correlation insuff. Higher in with winter. mortality, Associated with sepsis higher mortality mortality & BSI Barnett133 2014 Obs. 478 Mixed ICU Ass. of Vit D not ass deficiency with ALI with ALI, mortality in mortality sepsis, low levels ass with mortality in trauma. Moromizato 2014 Obs. 3386 Med/ Ass. of low Predictor of 134 Surg. ICU Vit D with sepsis, insuff. sepsis pts. with sepsis had higher mortality. Quraishi135 2014 Obs. 100 Surgical Prevalence Vit D levels ICU & ass. with predict LOS, outcome readmission, mortality. Rech136 2014 Retro 121 Sepsis Prevalence 54% deficient, pts. & ass. with higher mortality, mortality deficiency independent ass. with mortality Alves137 2015 Obs. 51 26 septic Vit D conc. 98% prevalence shock, 25 & variations of deficiency. - controls Decrease in organ dysfn. with higher levels.

Brook138 2015 Retro 300 Surgical Vit D levels If <50nmol/L- 3 x ICU & risk of non-home discharge discharge destination Chen139 2015 Obs. 236 ICU pts. Correlation Low levels ass with Vit D with mortality, levels, PCT inversely related & mortality to PCT, higher mortality in PTH responders Dancer140 2015 Obs. 52 52 ARDS, Ass. with All pts. with 57 ARDS & Vit ARDS & most surgical 8 D surgical pts. surg with deficiency deficient D dosing Ralph141 2015 Obs. 129 Medical Vit D & Levels ICU mortality >250nmol/L ass with illness severity & mortality Thickett142 2015 Retro 1985 Med/Surg. Vit D & Low levels ass ICU respiratory with risk of failure respiratory failure Kempker143 2015 Obs. 314 Medical Pt factors 57% low Vit D ICU ass with ass with winter low Vit D, admission, ICU risk of HAI stay, high P/F ratio. Not ass with risk of HAI Ala-Kokko144 2016 Obs. 610 ICU pts. Ass. with Not ass with Vit D sepsis mortality levels sepsis mortality De Pascale 2016 Retro 107 ICU pts. Very low Higher sepsis 145 levels & mortality, ass. with independent infectious & predictor, higher clinical rate of infection, outcome lower eradication, longer duration MV & pressors Dickerson146 2016 Obs. 158 Trauma Prevalence 76% deficient pts. & variables Penetrating ass. with injuries, African- deficiency American race, & obesity were risk factors

Zitterman147 2016 Obs. 3340 Cardiac Ass. with Ass. with low surgical low levels 1,25-dihydroxy- & post-op D & infection infection Quraishi148 2016 Obs. 94 Surgical Ass. with Inverse ass. with ICU low levels duration of MV & duration of MV Greulich149 2017 Obs. 48 32 SIRS, Vit D, PTH, Low levels of 25- 16 ICU 1,25- hydroxy-D & pts, 16 dihydroxy- 1,25-dihydroxy- healthy D, LL-37 D, SIRS pts. levels have even lower levels. Ratzinger150 2017 Obs. 461 SIRS Predictive 1,25-dihydroxy- criteria capacity of D predicted levels bacteraemia, not sepsis or death Vipul151 2017 Obs. 88 Sepsis Ass of low 74% deficient, levels with greater hospital morbidity LOS in deficient pts.

3.5.3 Key strengths

The population we studied was a broad representation of critically ill adults and patients were recruited serially, thereby eliminating a selection bias.

We studied serial levels over the ICU stay representing the hyper acute, acute and more chronic/recovery phase of the critical illness providing information on the PTH- vitamin D- calcium axis, which may be a relevant contributor to critical illness and recovery.

3.5.4 Key limitations

The heterogeneity of the population meant that there were only small numbers of patients in individual subgroups of patients. This makes it impossible to draw conclusions about effects in particular diagnostic groups.

The overall mortality event rate was low, making it difficult to determine a mortality relationship.

3.5.5 Clinical implications and implications for future research

Despite the high prevalence of vitamin D deficiency identified and its association with adverse clinical outcomes, it is not possible to establish a causative link. Vitamin D deficiency may be a marker of more severe illness and therefore and “innocent bystander” in critical illness.

Further, patients with poor general health may present with low vitamin D levels due to their inability to spend time outdoors, which may confound the relationship between vitamin D and outcomes.

There are studies in ambulatory72 and critically ill141 populations demonstrating increased mortality with high vitamin D levels.

Therefore, the equipoise remained about supplementation, particularly in high-risk critically ill patients.

Prior to undertaking a study of supplementation, establishing the safety, pharmacokinetic profile and pharmacodynamic effects in critically ill patient was required. This was undertaken and is the topic of the next chapter.

3.6 Publication details

Authors:

Priya Nair, Paul Lee, Claire Reynolds, Nguyen Dinh Nguyen, John Myburgh, John A.

Eisman, Jacqueline R. Center

Title:

Significant perturbation of vitamin D– parathyroid–calcium axis and adverse clinical outcomes in critically ill patients.

Journal:

Intensive Care Medicine (Journal of the European Society of Intensive Care Medicine and the European Society of Paediatric and Neonatal Intensive Care)

Impact Factor:

10.125 (rates second among intensive care/critical care journals)

Citation Index:

3.7 My role

I developed the concept, study design and protocol for this trial. This was refined following a presentation of the protocol at the Annual Scientific Meeting of the

Australian and New Zealand Clinical Trials Group.

I had oversight of patient enrolment, sample collection and data collection. I then organised this within a database. With professional statistical support, I undertook the data analysis and interpretation.

I wrote the first draft of the manuscript, and prepared a revision based on reviewers’ comments.

All authors provided advice and input into preparing the manuscript for submission and responding to reviewers’ comments.

3.8 Declaration

I certify that this publication was a direct result of my research towards this PhD and that reproduction in this thesis does not breach copyright regulations.

Signed ……………………………………………......

Date ……………………………………………......

3.9 Manuscript

Over page

Chapter 4: Randomised trial comparing two doses of

Intramuscular Cholecalciferol in Critically Ill Adults 152

4.1 Background

As discussed in the previous chapter, vitamin D deficiency is common in critically ill patients. Most observational studies have demonstrated that low levels are associated with adverse clinical outcomes.

Whether this relationship with bad outcomes is causal or merely a reflection of poor overall health remains unclear. Consequently, the role of supplementing vitamin D in insufficient patients in order to achieve higher levels is not routinely undertaken in the

ICU.

There is a plausible case for supplementation, made in the introduction chapter, based on the knowledge of both the skeletal and pluripotent benefits in other organ systems of vitamin D.

However, prior to undertaking a placebo-controlled randomised trial, it is important to establish the safety, appropriate dose, route and understand the effects of vitamin D supplementation in critically ill adults.

This trial was performed to study the effects of two different doses of intramuscular vitamin D in ICU patients who had evidence of systemic inflammation (using the definition of the Systemic Inflammatory Response Syndrome (SIRS))153. Patients with the SIRS were chosen in order to study the effect of vitamin D supplementation on markers of inflammation (pharmacodynamic effect of vitamin D). As discussed in the introduction chapter, an inflammatory process is almost a universal feature of critical illness. Both doses selected were in common clinical use in ambulatory patients.

4.2 Specific aims

We aimed to study the effect of two doses of intramuscular cholecalciferol on serial serum 25-hydroxy-D levels (pharmacokinetic effect) and on pharmacodynamic endpoints: calcium, phosphate, parathyroid hormone, C-reactive protein, interleukin-6, and cathelicidin in critically ill adults who had characteristics of the SIRS response.

4.3 Methods

This was a prospective, open-labelled randomised study conducted in a single tertiary referral adult ICU. Serial patients who demonstrated at least 3 of 4 SIRS criteria within

24 hours of admission and were expected to stay in ICU for at least 48 hours were enrolled. Patients who were pregnant, had chronic kidney disease, were hypercalcaemic (or at risk of developing hypercalcaemia) or who had a coagulopathy

(intramuscular injection) were excluded. As this study aimed to study the pharmacokinetic and pharmacodynamic effects of intramuscular supplementation, a placebo group was not included.

Intramuscular cholecalciferol was administered (150,000 IU or 300,000IU) as a single dose after baseline blood test samples were collected. The intramuscular dose was chosen due to the difficulties with enteral absorption during critical illness and the lack of availability of an IV preparation.

The study was approved by the hospital Human Research Ethics Committee

(HREC/12/SVH/322), and informed consent was obtained from the patient or the person responsible or healthcare proxy. Apart from cholecalciferol administration, other aspects of care did not differ between the two groups and was determined by individual clinicians blinded to the study drug allocation.

Blood samples were collected at fixed time intervals for up to 14 days or discharge from ICU. Assays for pharmacokinetic and pharmacodynamic endpoints as detailed in

the manuscript were collected. Assays were performed in a single accredited laboratory.

4.4 Key results

Demographic characteristics were well matched between the 2 groups. These were patients of relatively high illness severity as evidenced by the organ supports required, their illness severity scores and mortality.

Overall, 56% were vitamin D deficient (25-hydroxy-D <50nmol/L) and 16% were severely deficient.

25-hydroxy-D levels increased significantly in both groups from baseline. The mean change from baseline in the two randomised groups was 11.2 nmol/L (4.3–18.1 nmol/L; p = 0.002) at day 3, 18.9 nmol/L (9.4–28.4 nmol/L; p < 0.001) at day 7, and 23.3 nmol/L

(95% CI, 11.5 to 35.1 nmol/L; p < 0.001) at day 14. There was no significant difference in the mean change from baseline between the two groups over the entire study period

(2.6 nmol/L; 95% CI, −6.3 to 11.5 nmol/L; p = 0.57)

There was no change in 1,25-dihydroxy-D levels in the group as a whole (p = 0.36 and p = 0.41 at day 7 and day 14, respectively), and there was no difference between the two dose groups (p = 0.23).

There was a nonsignificant decrease in PTH at day 7 (mean change, −2.8 U; 95% CI,

−6.0 to 0.36 U; p = 0.08) and a significant decrease from baseline to day 14 (mean change, −3.2 U; 95% CI, −6.0 to −0.39 U; p = 0.03), but the overall difference between the two groups over total follow-up was not significantly different (−1.7; 95% CI, −3.4 to

0.06; p = 0.06)

CRP and IL-6 decreased significantly over the study period, but there was no difference in the decrease between the groups. The mean change in CRP from baseline was −43.9 mg/L (95% CI, −98.8 to 10.4 mg/L; p = 0.11) at day

7 and −74.4 mg/L (−112.4 to −36.4 mg/L; p < 0.001) at day 14. The difference between groups over total follow-up was not significant (p = 0.53). The mean change in IL-6 was

−16.3 pg/mL (95% CI, −32.9 to 0.4 pg/mL; p = 0.06) at day 1; −36.3 pg/mL.

(95% CI, −52.9 to −19.6 pg/mL; p < 0.001) at day 3; −49.2 pg/mL (95% CI, −66.1 to

−32.2 pg/mL; p < 0.001) at day 7; and −52.0 pg/mL (95% CI, −78.0 to −26.0 pg/mL; p <

0.001) at day 14. Again, the difference between randomised groups over the study period was not significantly different (p = 0.86).

At baseline, 25-hydroxy-D correlated positively with LL-37 (Pearson ρ = 0.03

and negatively with IL-6 (Pearson ρ = −0.17) although these were not statistically significant

Although there was a negative but nonsignificant relationship between 25-hydroxy-D and LL-37 over total follow-up, greater 25-hydroxy-D increments in the early phase of

ICU admission (day 1–3) were associated with larger increases in LL-37. Spearman ρ for the change in 25-hydroxy-D versus the change in LL-37 to days 1 and 3 were 0.33

(p = 0.04) and 0.46 (p = 0.004), respectively.

No clinically significant adverse effects were observed.

4.5 Interpretation

4.5.1 Principal Findings

A single intramuscular injection of either dose of cholecalciferol corrected vitamin D deficiency in critically ill patients. Supplementation in these doses was safe. Vitamin D repletion was accompanied by a reduction in pro-inflammatory (IL-6) and induction of the antimicrobial peptide, LL-37 responses during the early phase of critical illness.

4.5.2 Comparison with other studies

Just after this study was undertaken, a group from Austria published a phase II placebo-controlled randomised controlled trial of vitamin D supplementation of deficient 74

patients from a single institution.154 This is largest and most robust study to date in this area. They studied 475 medical and surgical adult ICU patients with vitamin D deficiency (25-hydroxy-D levels <50nmol/L) who were expected to stay in ICU for at least 48 hours. They excluded patients with gastrointestinal dysfunction (as they used enteral dosing), pregnant patients and those at risk for developing hypercalcaemia.

The primary end point for this study was hospital length of stay, which was not found to be significantly different between the groups. However, patients who were severely deficient (25-hydroxy-D<30nmol/L) had a significantly reduced 6-month mortality when supplemented with cholecalciferol. The study was not powered to detect a mortality difference, but these findings were described by the authors as hypothesis generating.

No serious adverse events were seen from supplementation.

The enteral route of supplementation was used in these patients. Only half the patients treated with vitamin D3 achieved serum 25-hydroxy-D levels >75nmol/L. The authors proposed that this low percentage of vitamin D3 responders may have been related to critical illness–associated compromised gastrointestinal function and to renal and drug- related alterations of the hepatic cytochrome P450 (CYP450) system that is implicated in 25-hydroxylation of vitamin D3.

A few smaller studies of supplementation in adult critical illness have been undertaken internationally and are summarised in this table.

Author Year N Population Dose Findings van den Berghe 2003 22 Prolonged critical 200 or 500 Fall in IL-6, CRP. 155 illness IU Ch Small increase in 25-hydroxy-D Mata-Granados 2010 33 Adult ICU 60,000IU Levels increased 61 oral Ch on with oral Ch, but D0 & D4 or not with IV calcitriol 2mcg IV calcitriol on alt days.

Amrein60 2011 20 RCT, Adult ICU 540,000IU 8/10 pts corrected oral Ch or within 2 days placebo No adverse effects Leaf156 2014 67 RCT, severe 2mcg No effect on clinical sepsis or septic calcitriol or outcomes, no shock placebo adverse effects, higher LL-37 & IL10 mRNA expression Quraishi157 2015 30 RCT, adult septic Placebo, Rapid, safe ICU patients 200,000IU improvement of 25- or hydroxy-D, 400,000IU concomitant oral Ch increase in LL-37 Han158 2016 31 RCT, ventilated Placebo, Increase 25- adult ICU patients 50,000 or hydroxy-D at D7, 100,000IU decreased hospital oral Ch LOS. No diff in LL- daily x5 37 or other clinical outcomes Smith159 2016 30 RCT, ventilated Placebo, Higher dose had adult ICU patients 50,000 or increase in Hb & (Same patient 100,000IU reduced hepcidin group as above oral Ch levels study) daily x5 Alizadeh160 2016 RCT, surgical Placebo or Increase in 25- ICU, stress 600,000IU hydroxy-D, induced IM Ch improved fasting hyperglycaemia BGL at D7, positive effect on adiponectin, but not HOMA-IR or AD

N-number of patients included, Ch- Cholecalciferol, IL-6- Interleukin 6, CRP- C-reactive protein, RCT- randomised controlled trial, IL-10- Interleukin 10, LOS-length of stay, Hb-haemoglobin, BGL- Blood glucose level

In 2017, Putzu et al published a meta-analysis of RCTs of vitamin D supplementation in critical illness161. Our study was not included in this analysis, as it did not include a placebo group. They concluded that vitamin D supplementation might reduce mortality in critically ill patients and that enteral administration is effective in increasing Vitamin D blood levels compared to placebo, without major adverse events. No statistically significant differences in length of ICU and hospital stay were found.

4.5.3 Key strengths

This was the first study of parenteral supplementation in critical illness.

It included patients with high illness severity and therefore represents real time pharmacological challenges in this patient group.

It also suggested mechanisms for the potential biological benefit on augmentation of vitamin D levels.

4.5.4 Key limitations

Importantly, this study did not include a placebo group who did not receive vitamin D.

Hence, although CRP and IL-6 levels fell during the study period, this could be a result of clinical improvement over time.

However, the primary aim of this study was pharmacokinetic characterisation, and we provided evidence supporting the efficacy, reliability, and safety of intramuscular vitamin D supplementation.

We did not measure DBP, free vitamin D, or vitamin D receptor polymorphisms although the implications and clinical relevance of these parameters are not well understood.

4.5.5 Clinical Implications and implications for future research

This study along with the other trials for a large, multi-centre RCT to study the effects of vitamin D supplementation on mortality in critically ill patients. It provides evidence of efficacy and safety of parenteral (intramuscular dosing) and a biologically plausible mechanism for benefit.

In order to design such an RCT, identifying inclusion and exclusion criteria are appropriate is necessary to ensure the results obtained will be externally valid. We noted from this study and our observational study that the subgroup of patients receiving organ support with extracorporeal membrane oxygenation (ECMO) appeared to have extreme values in terms of deficiency and were more difficult to supplement.

Further analysis of these patients was therefore undertaken, which is the topic of the

next chapter and manuscript.

4.6 Publication details

Authors:

Priya Nair, Bala Venkatesh, Paul Lee, Stephen Kerr, Dominik Hoechter, Goce Dimeski,

Jeffrey Grice, John Myburgh, Jacqueline Center

Journal:

Critical Care Medicine

Impact Factor:

7.442 (rates third amongst critical care/intensive care journals)

Citation Index:

4.7 My role

I developed the concept, study design and protocol for this trial.

This was presented at the Annual Scientific Meeting of the Australian and New Zealand

Clinical Trials Group. I was successful in securing two peer reviewed grants for this study (ANZICS Research Foundation and St Vincents Clinic Grant). This was used for pharmacy costs and sample processing.

I had oversight of patient enrolment, consent, randomisation, and sample collection/processing/storage and data collection. I undertook the data analysis and interpretation with statistical support.

I wrote the first draft of the manuscript. I submitted the manuscript to the journal and

coordinated a revision based on peer reviewers’ comments.

4.8 Declaration

I certify that this publication was a direct result of my research towards this PhD and that reproduction in this thesis does not breach copyright regulations.

Signed ……………………………………………......

Date ……………………………………………......

4.9 Manuscript

Over page

Chapter 5: Vitamin D status and supplementation in adult patients receiving Extracorporeal Membrane Oxygenation

(ECMO)

5.1 Background

Patients in the ICU are a heterogeneous in terms of their severity of illness, requirement for organ support and existing co-morbidities. These factors also contribute to altered pharmacokinetics and pharmacodynamics of drugs administered due to alterations in protein binding, volume of distribution, organ dependent metabolism etc.

Extracorporeal membrane oxygenation is a method to support patients with either severe respiratory and/or cardiac failure, which is refractory to conventional methods of support such as mechanical ventilation and pharmacological therapies. This support system consists of an extracorporeal membrane, oxygenator and circuit tubing. Drug disposition may be further affected by this device due to an additional volume of distribution, sequestration within elements of the circuit and other factors. Additionally, these patients represent a subset with extreme severity of critical illness and frequently have other chronic health conditions and prolonged hospital stays.

5.2 Specific aims

The aim of this study was to understand the characteristics of this subset of ECMO patients with respect to levels of vitamin D and its metabolites and response to supplementation with intramuscular cholecalciferol.

5.3 Methods

This study describes the group of patients who received ECMO support from the two studies detailed in chapters 3 and 4 and compares them to the patients who did not receive ECMO. The first study was an observational study of serial levels of vitamin D metabolites over a period of time and the second studied the effect on vitamin D levels following administration of a single dose of cholecalciferol intramuscularly.

5.4 Key results

A total of 24 ECMO patients were studied: 12 from the observational study and 12 from the vitamin D supplementation study.

The ECMO patients were younger, had a longer ICU and hospital stay and had a higher mortality confirming a greater severity of illness.

The prevalence of vitamin D deficiency was significantly higher in the ECMO compared to the non-ECMO group (88% vs. 52%, p=0.006). The median baseline level of 25- hydroxy-D was also significantly lower.

In the patients who did not receive cholecalciferol supplementation, vitamin D levels did not change significantly over their ICU stay.

Of the 12 ECMO patients who received cholecalciferol supplementation, half achieved correction of deficiency (reached levels >50nmol/L) compared with 36 of 38 (95%) in the non-ECMO group (p=0.001).

There were no occurrences of adverse effects of cholecalciferol supplementation- hypervitaminosis D, hypercalcaemia or renal calculi in the ECMO patients who received supplementation with cholecalciferol.

5.5 Interpretation

5.5.1 Principal findings

Critically ill patients who require ECMO support are almost universally vitamin D deficient. The levels of the active form 1,25-dihydroxy-D in the serum which are however preserved. This is not unexpected as the circulating levels of this active form are in picomolar concentrations and are generally preserved. However, deficiency of the storage form, 25-hydroxy-D might result in reduced tissue level activation.

Consequently, the pleiotropic functions of vitamin D previously described may well be compromised when the patient is vitamin D deficient even though serum 1,25- dihydroxy-D levels are within reference range.

Administration of cholecalciferol intramuscularly with a single dose in common clinical use is not an effective supplementation strategy and achieved correction of deficiency in only 50% of ECMO patients. However, in non-ECMO patients, the same dose regime achieved correction in virtually all patients.

5.5.2 Comparison with other studies

Vitamin D kinetics even in the absence of ECMO are altered as described in a study by

Czarnik et al.162 This appears to occur in parallel to and mirror disease severity; extreme instability in the resuscitation phase, followed by a steady state and then stabilisation. In another study, cardiopulmonary bypass was used as a model of haemodilution to mimic the changes that occur during the resuscitative phase of critical illness. This demonstrated that haemodilution significantly lowered 25-hydroxy-D levels which took up to 24 hours to resolve.49

In regard to patients receiving ECMO, there are a few studies in the neonatal and paediatric population. A study investigating 1,25-dihydroxy-D and PTH levels in neonates on ECMO described aberrant calcitriol-endocrine regulation of calcium with resulting hypercalcaemia. This finding was not replicated in our adult population.

The other pharmacokinetic studies on ECMO in adult patients relate to sedatives and antibiotics.

An ex-vivo study in ECMO patients, describing the disposition of micro and macronutrients, found significant alterations in concentrations. This was seen specifically for isoleucine, an essential amino acid and the vitamins A and E, but not D.

The authors suggest that the lipophilicity of vitamins A and E might explain their loss, but were not able to explain the relatively stable levels of vitamin D. The effect of supplementation was not described.

5.5.3 Key strengths

This is the only study describing vitamin D and metabolite levels and effect of supplementation in adult patients in-vivo. Patients studied had a high illness severity and were on multiple organ supports. The data collection is prospective and multiple measurements were assessed at precise time intervals from baseline and vitamin D metabolites, PTH and calcium profiles are described.

5.5.4 Key limitations

The key weakness is the small sample size and heterogeneity of the population in terms of underlying condition, organ dysfunction and co-existing illnesses. As a result, of this small number, propensity matching with a critically ill population not receiving

ECMO was not performed.

5.5.5 Clinical implications and implications for future research

This study suggests that ECMO patients are universally vitamin D deficient and alternative strategies such as higher or repeated doses need to be considered for supplementation.

This may also be the case for other lipophilic drugs in ECMO patients, many of which may be lifesaving. Appropriate dosing would require careful consideration and therapeutic drug monitoring where possible.

Randomised trials of vitamin D supplementation may have to either exclude this cohort completely or analyse as a pre-defined subgroup.

5.6 Publication details

Authors:

Priya Nair, Balasubramaniam Venkatesh, Dominik J Hoechter, Hergen Buscher,

Stephen Kerr, Jacqueline R Center, John A Myburgh

Journal:

Anaesthesia and Intensive Care

Impact Factor:

1.695 (2016)

5.7 My role

In collaboration with my supervisors I developed the concept, study design and protocol for this trial.

I undertook the data analysis and interpretation.

I wrote the first draft of the manuscript. I submitted the manuscript to the journal and coordinated a revision based on peer reviewers’ comments.

All authors provided advice and input into preparing the manuscript for submission and responding to reviewers’ comments.

5.8 Declaration

I certify that this publication was a direct result of my research towards this PhD and that reproduction in this thesis does not breach copyright regulations.

Signed ……………………………………………......

Date ……………………………………………......

5.9 Manuscript

Over page

Chapter 6: Summary of current knowledge and future considerations

6.1 Introduction

While the knowledge base surrounding vitamin D in critical illness has widened over the period of my candidacy from studies that have been undertaken internationally and by our group, many questions remain unanswered and a large, multi-centre phase III randomised controlled trial looking at patient centred outcomes in critical illness is required. The design of such a study presents a number of challenges.

The following manuscript was written as a review article towards the end of my candidacy summarising our current understanding and outlining the knowledge gaps in this area and has been accepted for publication in the peer reviewed journal, Critical

Care. (ccforum.biomedcentral.com)

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