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Congenital Heart problems - vitamin D levels drop even lower after surgery, loading dose probably required - thesis 2015

The Role of Daily High Dose Vitamin D in the Prevention of Post-Operative Vitamin D Deficiency in Children with Congenital Heart Disease
James Dayre McNally
Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Master’s degree in School of Epidemiology
Epidemiology and Community Medicince Faculity of Medicine University of Ottawa
© James Dayre McNally, Ottawa, Canada, 2015

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Background: Vitamin D deficiency (VDD) occurs in the majority of children following Congenital Heart Disease (CHD) surgery, and lower levels have been associated with postoperative cardiovascular dysfunction. Mechanistic studies have revealed that the high prevalence of VDD is due to poor pre-operative status and a 40% intraoperative drop in vitamin D. Available literature suggests that usual care with daily low dose vitamin D is ineffective and alternative regimens will be required to prevent post-operative VDD. Objectives: (1) To systematically review the pediatric clinical trial literature of high dose vitamin D, and (2) Determine whether pre-operative daily high dose vitamin D, based on the Institute of Medicine (IOM) Tolerable Upper Intake Level, can prevent post-operative VDD.

  • (1) Systematic review - Medline, Embase and the Cochrane Central Register of Controlled trials were searched for clinical trials reporting 25OHD levels in children after high dose vitamin D (> 1000 IU). Descriptive and meta-analysis techniques were used to determine the study characteristics associated with 25OHD response and adverse events.
  • (2) Trial - Design and initiate a dose evaluation trial. Recruit sixty two children with CHD into a double blind RCT and randomly assign to receive pre-operative cholecalciferol as usual (< 1 yr: 400 IU, > 1 yr 600 IU) or high doses (< 1 yr: 1600 IU, > 1 yr: 2400 IU). Primary outcome is immediate post-operative 25OHD concentration.

Secondary outcomes include vitamin D related adverse events and measures of trial feasibility. Study data to be reviewed by the Data Safety Monitoring Board after the first 30 participants - this time point was also chosen for preparation of the thesis results (25OHD data not available).
Results: (1) Systematic review - There were 88 eligible publications identified. Only two of six studies administering high dose daily supplementation (1000-4000 IU) to VDD children achieved group 25OHD levels above 75 nmol/L within 1 month; compared with nine of ten studies using loading therapy (> 40000 IU). In meta-regression, baseline 25OHD, regimen type, dose, age, presence of illness, and time factors were associated with final 25OHD levels. Increased risk of hypercalcemia was calculated with doses > 400 000 IU and group 25OHD levels above 200 nmol/L.
There were no reported cases of nephrocalcinosis and no significant increase in hypercalciuria risk with high dose vitamin D. (2) Trial -During the 19 months of active recruitment, there were 68 eligible referrals, full study was discussed with 49, and 35 consented (accrual rate of 1.8 per month). Of the 35 participants, 4 were withdrawn and 1 was awaiting surgery. For the 30 participants who completed all study procedures, the median number of doses was 21 (IQR: 4, 40) with 45% and 16% receiving more than 30 and 60 doses, respectively. The study safety protocol successfully identified one patient with levels approaching the upper safety threshold (parents had started an alternative medicine with vitamin D). Intra-operative or post-operative hypercalciuria occurred in 12 (40%) study participants. No study participant had pre or post-operative hypercalcemia. Immediate postoperative blood was collected for 25OHD determination in 100% (n=30) of study participants (to be determined as part of future work). Protocol limitations identified were: inability to collect baseline 25OHD (n=12/35, 34.2%), poor drug compliance (n=9/30, 30%), and protocol deviations (n=6/30, 20%) and excessive coordinator time to achieve early recruitment.
Conclusion: It is possible to recruit children with CHD into an RCT of high dose vitamin D. Due to both non-compliance and the short duration between enrollment and surgery the majority of CHD remain at risk for VDD following surgery despite pre-operative supplementation with doses approximating the IOM Tolerable Upper Intake Level.
Based on our findings, an alternative dosing regimen utilizing loading therapy may be necessary to achieve therapeutic levels of vitamin D.

Table of contents in thesis

Chapter 1 - Thesis overview
Global Objectives
Specific Objectives for the thesis
Presentation of thesis work 3
Chapter 2 - Background
Population and problem 5
Overview of Vitamin D 5
Vitamin D axis
Evaluation of vitamin D status
Vitamin D deficiency in CHD patients 7
Factors contributing to post-operative VDD 9
Primary vitamin D deficiency
Secondary vitamin D deficiency
Role of vitamin D deficiency in CHD patients 11
Critical illness hypocalcemia
Cardiovascular dysfunction
Immune dysfunction
Vitamin D supplementation regimens 14
Daily low dose vitamin D supplementation
Intermittent high dose supplementation
Vitamin D toxicity 17
Signs and symptoms
Toxic threshold levels
Risk factors for vitamin D related toxicity
Future Directions 19
Absence of clinical trial evidence
Supplementation with active vitamin D hormone
Nutrition and other vitamins
Summary 21
Bridging paragraph between publications 22
Chapter 3 - Rapid Normalization of Vitamin D: Systematic Review
Introduction 24
Methods 25
Eligibility criteria
Identification of studies
Data collection and risk of bias
Data analysis and reporting
Results 28
Adverse event analysis
Discussion 34
Conclusion 39
Bridging paragraph between publications 39
Chapter 4 - Protocol for Dose Evaluation RCT
Background 41
Objective and Hypothesis 45
Study Objectives
Methods 46
Sponsorships, approvals, principals
Study design
Study population
Study drug
Rationale for study design and interventions
Anticipated peri-operative 25OHD levels
Subj ect recruitment
Randomization, blinding and stratification procedures
Diagnostic and clinical outcome measures
Study procedures
Case report form
Statistical analysis
Sample size justification
Comments on power for evaluating vitamin D related adverse outcomes
Statistical procedures
Data and safety monitoring 62
Data Safety Monitoring Board
Safety measures and clinically relevant research findings
Discussion 64
Chapter 5 - Progress to date with conduct of dose evaluation RCT
Study initiation and time period for thesis
Study recruitment
Study participant characteristics
Duration of study drug intake and compliance
Research related testing and biological sample collection
Safety procedures and adverse events
Study modification
Study set-up, recruitment and sample collection
Sample collection and performance of safety procedures
Study drug intake and project 25OHD levels
Protocol limitations, deviations and modifications
Chapter 6 - Conclusion

Table 2A
Post-operative cardiac studies evaluating vitamin D

Table 3A Table 3B Table 3C Table 3D Table 3E
Patient, dosing and study characteristics of high dose study arms
Assessment of Study Design and Methodological Quality
Single-Variable Meta-regression of Post-Study Drug 25(OH)D
Multivariate Meta-Regression Predicting Post-Study drug 25(OH)D
Predicted final group 25OHD for vitamin D loading therapy (IU)

Table 4A Table 4B Table 4C Table 4D
WHO Structured trial summary
Vitamin D supplementation strategy
Definitions of Hypercalcemia and hypercalciuria
Biochemical measurements to be performed on research specimens

Table 5A Table 5B Table 5C Table 5D
Recruitment issues, withdrawals and protocol deviations
Study participant characteristics at baseline
List of participant cardiac lesions by RACHS category
List of study protocol modifications and initiatives

List of Figures

Figure 2A Figure 2B Figure 2C
Schematic of the endocrine pathway
Factors contributing to vitamin D deficiency in CHD patients
Organ systems negatively impacted by vitamin D deficiency
Figure 3A Figure 3B Figure 3C Figure 3D
Flowchart of study selection
Short term 25(OH)D response to high dose daily vitamin D
Short term 25(OH)D response to vitamin D loading therapy
Forest plot of hypercalcemia rates by dosing regimen

Figure 4A Figure 4B
Study related procedures and measurements
Flow diagram of study safety measures

Figure 5A Figure 5B Figure 5C Figure 5D Figure 5E
Participant flow diagram (CONSORT)
Actual vs. target recruitment rate
Flow diagram of collected research samples
Flow diagram of safety procedures and adverse event
Change in duration of enrollment over recruitment period

Author Contributions

- Chapter 2: Background
J. Dayre McNally: Manuscript conceptualization and design, data collection (review of literature), drafted the manuscript and approved the final as submitted.
Kusum Menon: Made substantial contributions to manuscript design, critically reviewed and revised for important intellectual content, and approved the final version to be published.
Chapter 3: Systematic review and meta-analysis
*As published in the journal
J. Dayre McNally: Study conceptualized and design, prepared data collection, carried out the data analysis, drafted the initial manuscript, revised for important intellectual content, and approved the final manuscript as submitted
Klevis Iliriani, Supichaya Pojsupap and Katie O’Hearn: Assisted with designed of the data collection instrument, determined study eligibility and acquired data, revised the article critically for important intellectual content, and approved the final manuscript as submitted. Margaret Sampson: Conceptualization and design of the search strategy, acquisition of data, drafting and revising the article for important intellectual content, final approval of the version to be published.
Kusum Menon, Dean Fergusson, Lauralyn McIntyre: Study conception and design interpretation of data, critical revision of the manuscript for important intellectual content, and final approval of version to be published;
- Chapter 4: Protocol for Randomized Controlled Trial
J. Dayre McNally: Study conceptualization and design. Prepared the initial grant, REB documents including consent and data collection forms. Liased with co-investigators for assistance with study design in their areas of expertise. Prepared the study protocol for submission to journal.
Kusum Menon, Dean Fergusson, Lauralyn McIntyre, Hope Weiler: All have significant clinical trial experience in PICU or on vitamin D. Each made significant contributions with study conceptualization and/or design. Critically revised the study protocol for submission to Heart and Stroke and/or Trials journal.
Katie O’Hearn: Contributed significantly to the study design in the areas of data collection, recruitment and consent, standard operating procedures for data monitoring, adverse event reporting and the Data safety monitoring board. Has lead a number of protocol modifications and critically reviewed the manuscript prior to submission.
Gyaandeo Maharajh, Stephanic Redpath, Margaret Lawson, Pavel Geier: Contributed to design of the study related to their specific areas of subspeciality expertise. All critically revised the grant and/or protocol manuscript for important intellectual content. GM and SR assisted with study design of procedures related to patient recruitment, data and research sample collection. ML and PG helped design the study safety procedures and adverse event analysis. ML has served as the study safety officer.

Copyrighted Contents

Chapter 2:
Reproduced with permission from Journal “Translational Pediatrics”, volume 2, pages 99- llt, Copyright @ 2013. See supplemental information, appendix S1.1
Chapter 3:
Reproduced with permission from Journal “Pediatrics”, volume 135, pages e152-e166, Copyright @ 2015 by the AAP. License number 3618860897610. According to the AAP terms and conditions we made no deletions, alterations, or other changes to the information or statistical data (note - figures and tables were renumbered). See supplemental information, appendix S1.2.
Chapter 4:
The protocol for the randomized controlled trial has been published in the Journal “Trials” volume 16, page 402 (doi: 10.1186/s13063-015-0922-8). This article is published by BioMed and is open-access allowing reproduction of text, tables and figures.

Chapter one - Thesis Overview
Summary of populaton and problem
Congenital Heart Disease (CHD) affects 1 in every 100 newborns with approximately 1500 Canadian children requiring heart surgery per year. Following surgery, these patients are often severely ill, requiring concentrated periods of expert medical care, consuming high- intensity resources, and spending weeks to months in hospital. New management approaches that reduce illness severity and speed recovery would benefit patients, families and the health care system. Vitamin D, well known for its role in bone health, is now recognized as a pleiotropic hormone important for the proper functioning of multiple organs, including the heart and lungs. A growing number of epidemiological studies in cardiovascular and adult critically ill populations have suggested vitamin D deficiency to be a modifiable risk factor for morbidity and mortality. Similarly, vitamin D deficiency has the potential to worsen illness following CHD repair through electrolyte disturbance, cardiac dysfunction, skeletal muscle weakness, altered immunity and impaired immumodulation. Until recently there were no studies reporting the prevalence of vitamin D deficiency in CHD patients admitted to the paediatric intensive care unit (PICU) following repair. Further, the potential relationship of vitamin D deficiency post CHD surgery with clinical outcomes has not been investigated.

The CHEO PICU research team recently completed a multicentre study demonstrating that 70% of critically ill children, including a post-operative CHD group, are vitamin D deficient at PICU admission. We demonstrated that patients with lower levels of vitamin D on PICU admission were more likely to require cardiovascular support. Secondly, a prospective single-centre study at CHEO focusing specifically on CHD patients confirmed the high rates (85%) of post-operative vitamin D deficiency. Importantly, that study identified that most children with CHD have borderline normal pre-operative vitamin D levels and that initiation of cardiopulmonary bypass (CPB) leads to an acute and sustained 40% decline in blood concentration. Similar to results from adult cardiovascular and ICU studies, lower postoperative vitamin D concentrations were associated with greater cardiovascular dysfunction. Our study findings, combined with the accumulating adult critical care literature, suggest that prevention of post-operative vitamin D deficiency could improve clinical outcomes following CHD surgery. As an inexpensive medication generally regarded as safe, vitamin D supplementation has the potential to be an ideal intervention for improving outcomes following CHD surgery.

Vitamin D deficiency following surgery for congenital heart disease (CHD) contributes to critical illness pathophysiology and worsens clinical outcome.
For patients with CHD, a personalized vitamin D supplementation approach that maintains normal vitamin D levels throughout the pre and post-operative stages will decrease morbidity, mortality and health resource utilization associated with CHD surgery.

Global Objective
The global objective is to identify supplementation strategies that will safely prevent post-operative vitamin D deficiency by significantly elevating levels pre-operatively.

Specific Objectives for the Thesis
Perform a systematic review of pediatric clinical trials reporting vitamin D supplementation meeting and exceeding the Institute of Medicine (IOM) Tolerable Upper Intake Level.
Report on the vitamin D levels achieved with different high dose regimens
Report on the occurrence of vitamin D toxicity with high dose regimens, including a comparison with study arms administering usual care.
Develop and initiate a pilot RCT evaluating whether a daily high dose vitamin D supplementation strategy based on the IOM recommendations can prevent postcardiac surgery vitamin D deficiency, when compared with usual care supplementation.
Develop a protocol acceptable for submission to Heart and Stroke Foundation
Evaluate the feasibility and barriers to a larger outcome based RCT
Compare the safety and toxicity of high dose and usual care dosing regimens

1.4 Presentation of thesis work
The thesis is presented in manuscript style. Chapter 2 represents a manuscript published in the Translational Pediatrics (July 2013) entitled “Vitamin D deficiency in surgical congenital heart disease: prevalence and relevance” describing the population, problem, knowledge gaps and proposes future work. Chapter 3 represents a manuscript published in Pediatrics (January 2015) entitled “Rapid normalization of vitamin D levels: a meta-analysis” describing the results and interpretation of the systematic review and metaanalysis. Chapter 4 contains a manuscript under review by Trials entitled “Prevention of vitamin D deficiency in children following cardiac surgery: a study protocol for a randomized dose evaluation trial”. The protocol manuscript provides an abridged version of the protocol that was submitted to the Heart and Stroke Foundation of Canada and awarded $138000 (Grant in aid, 2013). Permission has been obtained from the journals and have been reproduced with minor modifications to format but without modification of the content. Chapter 5 presents study findings after the first 30 participants completing all study procedures. To conclude the thesis, Chapter 6 provides an update of relevant literature, summarizes the results of the systematic review and RCT, and proposes the next steps for the field and research program.

2.0 Background

Population and problem
Congenital heart disease (CHD) is a common condition with an estimated prevalence of 1 per 100 in the general population (1). A significant proportion of these children require one or more corrective surgeries over their lifetime, collectively leading to 15,000 procedures per year in North America (1). Post-operatively, these patients may suffer significant morbidities which include a pronounced systemic inflammatory response, coagulopathy, respiratory failure, electrolyte disturbances, arrhythmia, myocardial dysfunction, kidney failure, infection and endocrine imbalances (2-5). Interventions that target one or many of these pathophysiological states could prevent illness, speed recovery, and decrease chronic morbidity in this high risk pediatric population.
Emerging literature suggests vitamin D deficiency to be a highly prevalent problem in the immediate post-operative CHD population. This observation is immediately relevant to researchers and clinicians since vitamin D is well recognized to be a pleiotropic hormone important for the proper functioning of organs critical to post-operative illness and outcome. Considered inexpensive and safe, vitamin D supplementation could prove to be an ideal intervention for improving outcomes in children with significant CHD.

Overview of vitamin D
Vitamin D axis
A schematic of the endocrine pathway is provided in Figure 2A (appendix 2.1). The vitamin D axis is primarily understood in the context of total body and serum calcium homeostasis (6, 7). In response to low ionized calcium, the parathyroid glands increase parathyroid hormone (PTH) secretion. Increased serum PTH leads to activation of vitamin D through an inducible renal enzyme, converting 25 hydroxyvitamin D (25OHD) to 1,25 dihydroxyvitamin D (1,25OH2D). The activated vitamin D, or calcitriol, works to restore serum calcium through bone breakdown, gastro-intestinal absorption, and increased renal reabsorption. Body stores of 25OHD are built and maintained through skin photosynthesis and dietary intake of pre-vitamin D that is immediately hydroxylated in the liver to 25OHD. Inadequate vitamin D intake through sun exposure and supplementation represents the most common reasons for diminished vitamin D axis functioning. Alternatively, or in addition, decreased renal or parathyroid function can impair 1,25OH2D levels despite normal 25OHD levels.
Evaluation of vitamin D status
Circulating 25OHD is the accepted marker of vitamin D status, with three threshold ranges commonly cited in the literature. Generally, sufficiency is accepted as a value above 75-80 nmol/L, deficiency is defined as a value below 50 nmol/L, with severe deficiency occurring in the 25-30 nmol/L range (7-13). These thresholds are based not only on biochemical indicators of axis stress, but also represent the values below which bone health or calcemic symptoms develop. Briefly, when 25OHD concentrations fall into the 50 nmol/L range, maintenance of active hormone levels requires elevation of serum parathyroid hormone (PTH) and increased renal enzyme activity (14, 15). As 25OHD falls below the 25 to 30 nmol/L range, production of the active hormone (1,25OH2D) falls and otherwise healthy individuals can develop electrolyte disturbances and clinically evident disease (rickets, seizures, myocardial disease) (15-17). Although overt clinical disease is often not evident until values drop below 30 nmol/L, population based research has established improved bone health with values over 50 nmol/L (11). Further research confirming or refuting 50 nmol/L as the appropriate threshold for prevention of non-bone related disease is required.
The clinical and research assays available for 25OHD determination can be divided into two analytical approaches: Liquid Chromatography-Mass Spectrometry (LC-MS) and antibody based immunoassays (9, 18). Although considered equivalent by some, there is emerging evidence that LC-MS may be superior for infants and young children. Superiority of the LC-MS methods relates to the ability to resolve 25OHD from a number of vitamin D metabolites that occur at relevant concentrations in infants and young children, particularly 24,25 dihydroxyvitamin D and 25-hydroxy-3-epi-vitamin D3 (3-epi-25OHD) (19-22). Recent studies have shown that the antibodies used in most immunoassays cross react with one or more of these metabolites, and counting these toward the 25OHD total may be inappropriate as they appear to have reduced or absent clinical effects (23).
Vitamin D deficiency in CHD patients
Previously described roles for vitamin D in the maintenance of electrolyte homeostasis, cardiovascular health, inflammation and innate immunity have led multiple research groups to investigate and report on the prevalence of vitamin D deficiency among both critically ill children and post-operative CHD populations. Two studies on vitamin D in pediatric critical illness were published in 2012, one of which included a subgroup of 120 patients with CHD (24, 25). This study reported that approximately seven out of every ten critically ill children were vitamin D deficient, and subgroup analysis on the post-operative
CHD participants confirmed a 73% deficiency rate with a mean 25OHD level of 40 nmol/L

To identify potential centre specific effects, post-operative 25OHD levels in CHD patients were compared at the three Canadian cardiac surgery sites and did not identify any statistically significant differences (26). A third PICU cohort study was published later in 2012 that reported a 40% vitamin D deficiency rate in post-operative cardiac surgery patients (27). In 2013 two studies were published focusing specifically on the post-operative CHD population (26, 28). Graham et al. used blood remaining from a glucocorticoid study of 70 neonates with CHD to show that 84% had 25OHD levels below 50 nmol/L (28). The other study prospectively evaluated 58 CHD patients with a range of ages and heart defects found a mean post-operative value of 35 nmol/L and an 85% vitamin D deficiency rate (26) (findings tabulated and provided in appendix 2.2).

Inspection of the 4 observational studies that included post-operative CHD patients demonstrates that all report statistically significant associations between lower vitamin D levels and need for cardiovascular support (vasopressor and/or inotropes). Findings from the sole PICU study that did not include post-operative cardiac patients also demonstrated a link between lower vitamin D levels and cardiovascular function (higher levels in patients with increasing CV-SOFA score, Cardiovascular Sequential Organ Failure Assessment, scores)

In addition, four studies showed an association between vitamin D levels and at least one other clinically important outcome measure including Pediatric Risk of Mortality III scores (25, 29), hypocalcemia (24) or calcium supplementation (27), fluid requirements (24), and PICU length of stay (24). Illness severity associations shown for the McNally et al. cohort study represent those for the entire group, while those for the study by Rippel and colleagues represent the CHD subgroup.

Additional evidence supporting vitamin D deficiency as a modifiable risk factor for poor outcome following CHD surgery can be found in a growing number of observational studies in adult ICU populations (30-38). A detailed evaluation of the adult critical care literature is beyond the scope of this article and has been reviewed elsewhere (39, 40). Briefly, the first adult publication on the topic in 2009 described 42 ICU patients, reported an average 25OHD level of 40 nmol/L, and demonstrated greater illness severity scores with lower hormone levels (30). Following a number of supportive small observational studies Braun et al. confirmed the association between lower admission vitamin D and mortality in a large observational study involving thousands of adult patients (41). Separately, this same research was also able to demonstrate pre-illness 25OHD concentration as a predictor of subsequent ICU related mortality (33). Although vitamin D deficiency has been associated with cardiovascular disease (36, 37, 42), the potential relevance of vitamin D deficiency to outcomes following adult cardiac surgery remains less well defined (43, 44). The best study to date comes from large prospective observational study published this year by Zitterman and colleagues, wherein they demonstrated that compared to normal 25OHD levels (75-100 nmol/L) having a value below 50 nmol/L was significantly associated with Major Adverse Cardiovascular and Cerebrovascular Events, or MACCE (43) .

Factors contributing to post-operative vitamin D deficiency
There are multiple pre-operative, intraoperative and immediate post-operative factors that may contribute to post-operative vitamin D deficiency (Figure 2B, appendix 2.3).

Primary vitamin D deficiency
Children with CHD may be at increased risk for pre and post-operative vitamin D deficiency due to inadequate sun exposure and poor vitamin D intake that may be related to their underlying disease. Significant pre-surgical vitamin D deficiency rates were described in both pediatric studies that measured 25OHD levels pre-operatively (26, 28). This observation suggests either poor compliance with guidelines or that vitamin D requirements (metabolism) differ for CHD patients compared with healthy children. The question of compliance with current recommendations (9, 11, 12) for vitamin D intake and supplementation was addressed through a targeted vitamin D food frequency questionnaire in the prospective study performed by McNally et al. (26). This questionnaire demonstrated that up to 50% were not achieving a daily vitamin D intake of 400 IU. This non-compliance with Vitamin D recommendations occurred despite close supervision by a large group of health care providers and has also been reported in the general population (45, 46).

Secondary vitamin D deficiency
In addition to the mechanisms outlined above, blood vitamin D levels may be further reduced either intra-operatively or immediate post-operatively due to large circulating fluid shifts, blood loss, blood ultrafiltration, fluid administration and interstitial leak of vitamin D binding proteins due to inflammation (47-50). This occurrence, and timing, was explored by McNally et al. through the collection of serial blood samples through surgery and over the first two post-operative days (26). The major finding was a 40% intraoperative fall in serum 25OHD immediately following initiation of CPB. Similar blood levels before and after modified ultrafiltration (MUF) and insignificant 25OHD in the ultra-filtrate do not support a loss of Vitamin D through ultrafiltation. Other possibilities therefore include either a dilutional effect from the prime volume or absorption of 25OHD by components of the bypass circuit (e.g., tubing, oxygenator membrane). No further change in group mean 25OHD levels occurred after PICU admission, evaluated over the critical first two postoperative days.

It is worth noting that in the retrospective study by Graham and colleagues an intraoperative decline in 25OHD concentration was not observed (28). The lack of even a small decline is not consistent with the study by McNally et al., two recent adult CPB studies and a small neonatal case series describing calcitriol concentrations before and after initiation of Extracorporeal Membrane Oxygenation (26, 47, 51, 52). Zitterman et al. reported a significant drop in 25OHD level following adult heart transplantation, but the exact timing could not be determined as the first post-operative levels were measured on the sixth postoperative day (51). The study by Krishnan and colleagues, demonstrated up to a 40% drop in serum 25OHD in 19 adults following the CPB prime (47). A potential explanation for the contrast in findings between Graham et al. and the other four studies could be a result of differences in research blood collection, processing and storage. Blood collection approaches may be essential as 25OHD levels have been shown to be consistently 20% higher when measured from capillary blood compared to venous blood (53). Further, and as noted by Graham and colleagues, the pre-operative vitamin D level and choice of CPB prime fluids may also contribute to the differences in findings (28).

Role of vitamin D deficiency in CHD patients
The role of vitamin D as a modifiable risk factor for post-cardiac surgery outcome has biological plausibility due to the number of known organs and body pathways through which it could cause or worsen post-operative pathophysiology (Figure 2C, appendix 2.4).

Critical illness hypocalcemia
Hypocalcemia is a common problem following pediatric cardiac surgery (~30%), with a 2008 study demonstrating the need for calcium supplementation as a risk factor for morbidity and mortality (5). Calcium homeostasis is important for patient well-being as calcium initiates and propagates nerve conduction, muscle contraction, and contributes to intra-cellular signal transduction. In addition to the negative impacts of hypocalcemia on cardiovascular dysfunction, abnormal calcium homeostasis could impair gas exchange and influence ventilator requirements through nerve dysfunction and muscle weakness (54, 55). A number of pediatric studies have confirmed hypocalcemia to be risk factor for worse ICU outcomes and that critically ill children with hypocalcemia are more likely to have abnormalities of their vitamin D axis, including 25OHD deficiency (56-58). No studies have evaluated whether optimization of vitamin D status prevents or reduces the severity of critical illness hypocalcemia.

Cardiovascular dysfunction
Post-operative cardiovascular dysfunction is common following cardiac surgery with many children requiring the continuous infusion of one or more vasoactive medications to support blood pressure and maintain cardiac output (59). A role for vitamin D in pediatric cardiac health can be found in case reports and case series describing cardiomyopathy secondary to isolated severe vitamin D deficiency (60-65). A recent case series identified 16 children with treatment responsive cardiomyopathy secondary to isolated severe vitamin D deficiency (63). Vitamin D responsive subclinical cardiac dysfunction has also been described in children with rickets, with 50% of the cohort demonstrating ECG and echocardiogram abnormalities at presentation (66). In addition to the indirect actions of vitamin D through calcium, it is well known that vitamin D influences myocyte structure and function via nuclear vitamin D receptors that alter gene and protein expression (67, 68). More recent research has also suggested that vitamin D may mediate structural and functional myocyte changes through non-nuclear VDRs (29). As an example, myocyte contractility has been observed to be favorably altered within minutes following 1,25OH2D supplementation; an effect mediated through signal transduction pathways, enzymatic reactions and ion channels (69, 70). Further, vitamin D appears to play a role modulating peripheral vascular resistance directly through receptors on smooth muscle cells and indirectly through the renin- angiotensin-aldosterone system (32). Finally, numerous studies have recently suggested that vitamin D deficiency could serve as an effect modifier, augmenting the impact of other deficiency states (e.g., adrenal) and medications (corticosteroids) on cardiorespiratory function (26, 71, 72).

Immune dysfunction
Cardiac surgery uniformly leads to a post-operative systemic inflammatory response that can contribute significantly to low cardiac output and respiratory dysfunction (73, 74). There is good evidence that vitamin D metabolites play important immunomodulatory roles mediated through functional vitamin D receptors present on all major immune cell types. Specifically, vitamin D has been demonstrated to inhibit antigen-induced T-cell proliferation, antagonize the pro-inflammatory Th1 (T-helper) response, suppress macrophage release of pro-inflammatory cytokines, and alter gene expression of adhesion factors, decreasing adherence and chemotaxis of neutrophils (75-77). Vitamin D signaling is also known to play a role in innate immunity through the production of cathelicidins, which are important
endogenous antimicrobial peptides that, provide protection again multiple viral and bacterial pathogens (78-80).

Vitamin D supplementation regimens
The cumulative body of basic science and clinical literature suggests that optimization of vitamin D status could improve clinical outcomes in CHD patients. Available evidence suggests that both primary deficiency and operative procedures potentially contribute to high post-operative deficiency rates. Although rapid restoration of vitamin D levels immediately following surgery would represent an attractive option for anesthesiology and intensivists, the lack an intravenous formulation of cholecalciferol or 25-hydroxyvitamin D prevents consideration of such an approach. Instead, the practioner caring for children with CHD will need to pre-operatively raise and maintain levels utilizing one of two approaches proven for other pediatric populations. Given the pharmacokinetics of vitamin D, prevention of post-operative deficiency patients may necessitate an understanding of both approaches, with application personalized based on patients factors such as compliance with daily intake and time prior to surgery.
Considering the primary literature, expert opinion and concerns about post-surgical inflammation, hypoparathyroidism and renal dysfunction, it would be ideal to target a postoperative 25OHD level above 75 nmol/L, with the goal of avoiding values below 50 nmol/L. Given the potential for a significant (40%) intra-operative decline, pre-operative levels in the 100-150 nmol/L range may be required to achieve these goals.

Daily low dose vitamin D supplementation
The most commonly used approach for building and maintaining levels of vitamin D relies on the daily consumption of an age specific low dose of ergocalciferol or cholecalciferol (ranging from 400 to 4,000 IU). To date there have been no pediatric trials of any vitamin D regimen in the CHD population, necessitating extrapolation of recommendations from the most recent guidelines or position statements for healthy children (9, 11, 12). Recently, at the request of agencies of the US and Canadian governments, the Institute of Medicine (IOM) assembled a committee to provide recommendation for intake of vitamin D based upon a rigorous and comprehensive review of literature. In the final report, the IOM provided two age specific doses: (i) Recommended Daily Allowance or Adequate Intake and (ii) Tolerable Upper Intake Level (11). As described in the IOM report, the Recommended Daily Allowance and Adequate Intake doses are intended to maintain blood 25OHD concentrations at or slightly above the 25OHD threshold (50 nmol/L) known to foster bone health. In calculating the age specific upper intake level, the IOM goal was to provide a daily dose that would significant elevate levels well above the cut-off for vitamin D deficiency while safely avoiding potential toxicity.

The efficacy and safety of the two age specific IOM dosing regimens have been supported by the publication of two well done trials since the IOM report (19, 81). First, a Canadian study confirmed previous work demonstrating that 2 or more months of daily dosing is required to achieve a new steady state 25OHD level (19). Second, both studies showed that with good compliance, 400 IU per day for 3 months will generate a mean preoperative level of 80-90 nmol/L, with almost all participants elevated above 50 nmol/L. However, these studies also suggest that utilization of this dose in CHD patients could still leave 50% or more at risk for post-operative vitamin D deficiency. Both studies evaluated a 1,600 IU/day regimen, closely approximating the IOM 6 to 12 month old Tolerable Upper Intake Level. At this dose, the mean 25OHD concentrations achieved in the two studies were 157 and 180 nmol/L, with almost all generating 3 month levels above 90 nmol/L. Importantly, after consideration of unwanted metabolites, no child exceeded a 25OHD level above 250 nmol/L. Neither study demonstrated additional cases of hypercalcemia or hypercalciuria in the groups receiving doses above standard of care.

Intermittent high dose supplementation
Some circumstances may necessitate consideration of an alternative approach to the daily low dose vitamin D regimen. First, poor compliance with vitamin D supplementation occurs in a certain percentage of patients as previously demonstrated in the general population (45, 46). Second, in a small but consistent percentage of the CHD population, particularly neonates and young infants, the time between diagnosis and surgery will not allow for 2 to 3 months of low dose vitamin D intake.

The second approach to supplementation involves the delivery of a 2 to 3 month prescription of vitamin D either as a single dose or over a period of days (82-84). This regimen is commonly referred to as Stosstherapy (or megadose therapy) and generally involves the oral or intramuscular administration of 50,000 to 600,000 IU. Not surprisingly the route of administration contributes significantly to the rate and final 25OHD level achieved. When given orally, the loading dose is immediately absorbed into the circulation, undergoes rapid liver hydroxylation, and gives rise to a peak 25OHD within a few days (17, 85-87). In contrast, with intramuscular administration, the rise in 25OHD levels occurs over many weeks due to slow resorption from the muscle, and may be more variable (88).

There is significant experience with the high dose regimen in certain regions of the world and it has been recommended as part of the Australia and New Zealand position statement (89). However, it is important to point out that the available pediatric clinical trials have largely focused on healthy children (without or without vitamin D deficiency) and administered doses intended to prevent or treat vitamin D related bone disease. The safety of this dose regimen has received limited evaluation in children with cardiac dysfunction or acute illness particular at doses intended to achieve 25OHD levels in the 100 to 150 nmol/L range. As there is evidence to suggest that some populations at certain doses may develop hypercalcemia additional work in this area will be required to determine the high dose supplemention approach that safely maximizes 25OHD level in CHD patients (90).

Vitamin D toxicity
Signs and symptoms
Vitamin D toxicity is a characterized by hypercalcemia and/or hypercalciuria, with the classic symptoms (lethargy, abdominal pain, anorexia, constipation, polyuria and nocturia) directly attributable to these abnormalities. In many instances symptomatology related to hypercalcemia and/or hypercalciuria is minor. However, as documented in case reports and case series the longstanding persistence of minor biochemical abnormalities or progression to severe electrolyte disturbances can give rise to more serious problems including dehydration, renal dysfunction and eventual nephrocalcinosis.

Toxic threshold levels
Presently no 25OHD level has been universally accepted as the threshold above which risk develops, with authors generally citing values between 250 and 750 nmol/L.
Although there is no evidence that children develop biochemical abnormalities or symptoms with 25OHD values at or slightly above 250 nmol/L, recent pediatric clinical trials of high dose vitamin D have focused on this threshold (19, 81). Application of this threshold for dosing studies is appropriate given that levels above this are supraphyiosological (cannot be achieved with excessive sun-exposure or healthy diets) and there is no evidence of benefit for 25OHD doses above 200 nmol/L (91, 92).

Risk factors for vitamin D related toxicity
Despite public and clinician concern regarding vitamin D toxicity, it is a rare event that generally occurs in the context of genetic susceptibility or inappropriate intake of high doses of vitamin D. Concern about the safety of daily vitamin D supplementation above 400 IU/day dates back to the 1950’s when a rise in idiopathic infantile hypercalcemia (IIH) cases coincided with the population based implementation of increased daily vitamin D intakes to ~4,000 IU/day (93-96). This small epidemic led to a decrease in recommended daily intake to levels known to prevent rickets and hypocalcemic seizures (400 IU/day). It has recently been argued that many, perhaps all, cases of IIH were due to rare genetic conditions (<1:10,000) that increase susceptibility to vitamin D toxicity (97). Of these, patients with Williams syndrome can have heart defects as part of their constellation of symptoms and it would be prudent to avoid higher vitamin D intake in this subgroup (98).
There is a substantial body of low level evidence suggesting that high dose vitamin D giving rise to shorter term cumulative intake at or above 600,000 IU is excessive and can lead to hypercalcemia, hypercalciuria and eventual nephrocalcinosis. This anecdotal evidence is also supported by one prospective pediatric clinical trial that demonstrated significant hypercalcemia rates among healthy infants who received intermittent, often repeated, high dose therapy with 600,000 IU (90). In most instances symptoms and biochemical abnormalities resolved following diagnosis and discontinuation of the vitamin D source allowing gradual decline of blood 25OHD levels below toxic levels. In some circumstances the nephrocalcinosis persisted despite discontinuation of vitamin D. A review of the literature on nephrocalcinosis shows that most cases associated with vitamin D have occurred in children with a rare genetic disorder called vitamin D resistant rickets and may be related to concurrent phosphate intake (99-103). Again, a review of case series and case reports demonstrate that otherwise healthy children only develop nephrocalcinosis following intentionally or unintentionally cumulative Vitamin D intake above 600,000 (99, 100, 104, 105). Our review of pediatric interventional trials on vitamin D supplementation identified 2 studies evaluating daily high dose vitamin D approximating below the IOM upper intake level and 4 with megadoses (100,000 to 150,000 IU range); none identified increased urinary calcium excretion or hypercalciuria (19, 81, 83, 106-108). Given these findings it would be prudent to avoid oral vitamin D dosing at or near 600,000 IU.

Future directions
Absence of clinical trial evidence
As stated previously, there are no studies evaluating ergocalciferol (D2) or cholecalciferol (D3) dosing in CHD patients. Although sufficient evidence exists to support administration of vitamin D regimens 2 to 4 times above the current standard of care (400600 IU/day), extrapolation of these safety findings from healthy infants to the CHD population may not be appropriate. CHD patients have unique metabolic demands, organ dysfunctions, and known and unknown genetic abnormalities that may make them more or less susceptible to vitamin D toxicity (98, 109-111). Similar to the recent approach taken from other severely ill populations, including pediatric heart failure (112), pediatric acute lower respiratory tract infection (113, 114) and adult critical illness (86, 115) it would be prudent to test feasable dosing regimens as part of phase II clinical trials.

Supplementation with active vitamin D hormone
Although recognized as the best indicator of vitamin D status, post-operative 25OHD concentration may not accurately reflect vitamin D axis functioning and calcitriol levels in the immediate post-operative CHD patient. As shown in Figure, appendix 2.3, the postoperative CHD patient has many congenital, pre-operative and surgically acquired risk factors for low post-operative calcitriol for reasons beyond impaired 25OHD levels. Of these, we speculate that CPB may lead to a significant acute decline in blood calcitirol levels, as demonstrated in studies following adult cardiac transplant and initiation of neonatal ECMO (51, 52). Further, as described in other patient populations, pre-surgical or acquired dysfunction of the parathyroid and renal organs may limit or prevent conversion of 25OHD to calcitriol (7, 116). If future studies report a transient or persistent decline in calcitriol levels following CPB, administration of active vitamin D may also be required to achieve the goal of optimizing vitamin D status. Clinical evidence demonstrating cardiovascular benefit of calcitriol administration, in addition to 25OH, can be found in the end stage renal disease literature, where a vitamin D deficient state emerges due to reduced renal activation of vitamin D. In this population, the administration of an activated vitamin D analogue reduces the increase (50-10 fold) in cardiovascular disease (18, 117).

Nutrition and other vitamins
The impact of cardiac surgery and CPB on the other endocrine axis regulating cortisol, glucose, thyroid and vasopressin have been well described. However, the impact of cardiac surgery and cardiopulmonary bypass on other vitamins and blood metabolites remains less well defined. A better understanding of how these metabolites respond to CPB and post-surgical care would inform researchers regarding the need for a multivitamin supplement in patients undergoing CPB.

2.9 Summary
Multiple observational studies have identified significant rates of post cardiac surgery vitamin D deficiency in a high-risk (CHD) pediatric population following CPB. Data from these same studies demonstrate that lower post-operative vitamin D are associated with a more protracted clinical course. Available data strongly suggest that the current approach to vitamin D supplementation will not reliably prevent post-operative vitamin D deficiency. The current lack of clinical trials evaluating the efficacy or safety of any alternative high dose vitamin D regimen prevents evidenced based recommendations. Therefore, it is important to conduct a systematic review of available literature prior to designing a clinical trial to explore alternative vitamin D supplementation strategies that will safely and effectively optimize post-operative vitamin D status in most CHD patients.

2.10 Bridging paragraph between publications
Prior to modifying current practice or embarking upon phase III clinical trials of high dose vitamin D, a fundamental step is a systematic review identifying all clinical trials of high dose vitamin D in children. In addition to identifying any clinical trials in the CHD populations such a review will prove crucial to understanding the short and longer term 25OHD response to different dosing regimens, the study and population characteristics that influence response, and what dosing regimens may be associated with adverse events. Understanding the different dosing regimens is particularly relevant to the CHD population where there is significant heterogeneity in lesion type, severity, comorbidity, age of presentation, and timing of surgery. Due to this heterogeneity, peri-operative optimization of vitamin D status is unlikely to be accomplished using currently approved doses and dosing regimens. Instead, individuals providing care for these children may need to understand and be comfortable with two or more dosing regimens so that care can be personalized and vitamin D deficiency is prevented for the vast majority of CHD patients.

In addition to uncertainty surrounding the appropriate dosing regimen for cholecalciferol, some research groups have also questioned whether other vitamin D metabolites, specifically calcitriol, might represent both a better marker of vitamin D status and more appropriate supplement given the availability of an IV formulation. Although limited, available research in cardiac surgery and critically ill populations have not demonstrated consistent advantage to the addition of 1,25(OH)2D to 25OHD measurements for predicting clinical course (118-120). A recent pilot RCT of calcitriol supplementation in adult severe sepsis did not demonstrate differences between groups in plasma concentrations of cathelicidins or cytokine levels(121). Finally, there is convincing evidence that elevating 25OHD through cholecalciferol administration also increases 1,25(OH)2D levels(122). Altogether the current wisdom supports trials of cholecalciferol prior to considering a more expensive metabolite with greater risk of toxicity(123).
Chapter 3 presents the result of a systematic review of clinical trials reporting vitamin D levels after receipt of high dose vitamin D. As the results of this review had applicability beyond the CHD population the publication was prepared with a more general pediatric audience in mind. Of additional note, the database we generated of all pediatric clinical trials of vitamin D has also been used as a resource for two other studies. First, we published a systematic review of clinical trials that evaluated how high dose vitamin D modifies asthma related outcomes; analysis of data from 5 studies demonstrated that high dose vitamin D decreased asthma exacerbations(124). Second, we have prepared a scoping review of high dose vitamin D clinical trials and created an online searchable database for clinicians and researchers (manuscript in preparation).

3.0 Rapid normalization of vitamin D: systematic review and meta-analysis

3.1 Introduction
The evidence for vitamin D in calcium homeostasis, cardiovascular and respiratory health, inflammation and innate immunity have lead to questions about whether deficiency might represent a modifiable risk factor in the prevention or recovery from acute and critical illness. A large body of observational literature from adult Intensive Care Unit (ICU) and cardiovascular populations have documented high Vitamin D deficiency (VDD) rates and association between blood 25-hydroxyvitamin D (25OHD) and organ dysfunction, health resource utilization and mortality. More recently pediatric observational studies have supported these findings in similar populations including asthma, ICU, and the post-surgical CHD (24-28).

Normalization of vitamin D status has the potential to speed recovery and improve outcomes in multiple acutely unwell pediatric populations. Most of the guidelines and clinical practice surrounding vitamin D dosing involves daily intake under 1000 IU (9, 11). As these standard dosing strategies target healthy children and require months to restore normal levels they are not applicable to the acute and critical care settings. Although other regimens that involve the administration of higher doses have been reported, there remains concern about both inadequate dosing and excessive doses leading to toxicity (82, 125). In the adult ICU setting, pilot trials of loading dose therapy have been performed, with results from a large trial evaluating clinical benefit completed but unpublished (86, 115, 122). No pediatric ICU studies have been completed at this time.

To inform clinical practice and future trials we have performed a systematic review with the goal of identifying all published pediatric trials reporting on the administration of high dose vitamin D (>1000 IU). The objectives of this review were to: 1) assess the ability of different dosing regimens to normalize vitamin D status, 2) determine study characteristics that influence post-drug 25OHD levels, 3) determine what high dose regimens are associated with vitamin D related adverse events, and 4) use the findings to recommend a dosing regimen for clinical practice and future clinical trials in pediatric acute and critical care settings.

3.2 Methods
Study objectives and protocol were determined a priori (PROSPERO protocol registration number: CRD42013006677) and reported according to PRISMA guidelines (Supplemental Information, Appendix S3.24) (126).

Eligibility Criteria
Studies were eligible for inclusion in the systematic review if they met all of the following criteria: i) an uncontrolled, controlled or randomized controlled trial (RCT) conducted in neonates, infants, children or adolescents; ii) the study administered at least one dose of cholecalciferol (D3) or ergocalciferol (D2) equal to or in excess of 1000 IU; iii) the study evaluated the effects of drug administration on 25OHD status. Studies were excluded if the population that was exclusively premature or low birth weight, had a genetic problem related to vitamin D metabolism, or were pregnant. Further, studies were excluded if they prescribed ultraviolet exposure, gave vitamin D as part of a food without precisely controlling quantity, or administered vitamin D with another vitamin or drug (without a control arm).
Identification of Studies

Medline (1946 to 2014 Week 2), Embase (1974 to 2014 Week 3) and the Cochrane Central Register of Controlled Trials (December 2013) were searched using the Ovid interface. The MEDLINE search strategy was developed by a librarian experienced in systematic review searching (MS), and peer-reviewed by another librarian (LK), using the PRESS standard (127). The MEDLINE search was then adapted for the other databases. No date, language or study design limits were applied. We searched conference abstracts from 2010-2013 through Scopus. The search strategies are presented in Supplemental Information (Appendix S3.1-S3.6). The initial search was conducted on April 30, 2013 and updated on Jan 21, 2014. We also conducted a grey literature search by reviewing ongoing trials registered with Clinicaltrials.gov, the citations of all eligible articles, and 24 systematic reviews of vitamin D in children.

Unless otherwise noted, two of the study authors independently reviewed the citations sequentially through three sets of screening questions to determine eligibility (Supplemental Information, Appendix S3.7). Level 1 screening was performed using Mendeley (Mendeley Desktop, version 1.10.3) and those citations that could not be excluded were uploaded to DistillerSR™ (Evidence Partners Incorporated, Ottawa, ON, Canada) for level 2 and 3 screening. Full texts of all potentially eligible citations were reviewed by two authors (KI/KO and DM). Conflicts between reviewers were resolved through discussion, with a third author available to resolve disagreement (MS). The eligibility of articles not in English, French or Spanish was determined by a single author after written translation or with the assistance of a translator.

Data Collection and Risk of Bias
Data from eligible studies were extracted by one review author and verified by a second author by independent review of the article (DM, KI, KO, SP). Data were collected and managed using REDCap (Research Electronic Data Capture) hosted at the Children’s Hospital of Eastern Ontario (128). Vitamin D or calcium data values that were only published graphically were extracted from figures using DigitizeIt software (http://www.digitizeit.de/, Germany). During the data collection process, 18 authors were contacted to clarify or request additional study 25OHD data, of which five responded. Study quality was described using the Cochrane risk of bias assessment tool (129).

Data Analysis and Reporting
Summary statistics and data from eligible studies and independent arms were described as text, through tables and figures. Clinical heterogeneity between studies was assessed using information on population (age, disease status, baseline vitamin D), dosing regimen (dose, frequency, form, route) and measurement features (time, assay type). Regimens were considered intermittent if they gave a vitamin D dose in excess of 40000 IU as a single administration (divided over two days) or was repeated with a frequency equal to or in excess of 1 month. Methodological heterogeneity was evaluated using information collected on study type (single arm, RCT, controlled non-RCT) and the Cochrane risk of bias tool. For specific dosing regimens, 25OHD response was presented using figures (Sigma plot, version 12.3) and the success of each dosing regimen was defined as achieving a group 25OHD average > 75 nmol/L.

Given significant heterogeneity in post 25OHD levels, we performed random effects meta-regression to evaluate the contributions of specific study level population, dosing and methodological characteristics. This analysis included study arms reporting a group 25OHD level between 1 and 13 weeks of drug initiation and an accurate cumulative dose could be calculated. Assessment of heterogeneity and meta-analysis was performed using Comprehensive Meta-Analysis (version 2) with meta-regression performed using the PROC MIXED function in SAS (version 9.3). Analysis used group mean or median 25OHD levels, and within study variance using provided or calculated standard errors (130, 131). For age, if the median or mean was not provided we used the mid-point of the age range as an approximation (132). Initially, single variable random effects meta-regression was performed and potentially significant variables were then tested in a multivariable meta-regression analysis. A potential interaction was sought between cumulative dose and age, and an interaction term was included in the regression analysis to control for and evaluate how timing from single or divided loading doses to 25OHD measurement influences the level. No new variables were to be added to the multivariable model once the ratio of variables to eligible 25OHD measurements exceeded 10:1. The final multivariate model was used to calculate the predicted group 25OHD response to a loading dose (or stosstherapy) of drug among four age groups of vitamin D deficient (30 nmol/L) children.

3.3 Results
Results of search
Figure 3A (appendix 3.1) shows the flow of studies through the identification and review process. A total of 2453 unique records were identified for screening. Of the 367 full text citations that remained after initial screening, 256 articles describing clinical trials were identified. Of these, 88 full text publications (17, 19, 81, 83, 84, 90, 106-108, 112, 133-211) and 10 conference abstracts (Supplemental Information, Appendix 3S.8-3S.9) met all population, dosing and 25OHD outcome-related eligibility criteria. Flow of eligible articles and study arms is presented in Supplemental Information, Appendix S3.10. The 88 full articles reported on 96 eligible study populations and included 199 different arms. Of these 199, three were ineligible due to UV exposure (n=2) or administration of active vitamin D (n=1). Of the remaining arms, 62 involved the administration of no vitamin D (e.g. placebo) or a dose under 1000 IU. Of the 134 high dose arms, 22% (29) and 78% (105) were from uncontrolled and controlled studies, respectively.

Patient populations
Tables 3A (appendix 3.2) and 3B (appendix 3.3) present the relevant clinical and methodological characteristics of the eligible high dose arms. Of the eligible high dose study arms, 73% involved administration of vitamin D to healthy children (49%), children with rickets (16%) or pediatric populations with subclinical VDD (8%). Populations of children with “other” disease states (e.g. HIV, arthritis, seizures) accounted for 19% of the study arms (Supplemental Information, appendix 3S.11). As shown in Table 3A, studies included children from all age ranges, with neonates being evaluated in 15% (n=20) of high dose study arms and adolescents in 50% (n=67). Vitamin D dosing regimens evaluating intermittent loading therapy accounted for 46% (n=62) of the eligible study arms, with daily regimens representing 38% (n=51). A minority of the eligible high dose arms (14%, n=19) described a dosing regimen that varied dependent on factors including baseline 25OHD, weight or age. The number of participants in each arm ranged from 5 to 233, with a median size of 27 (IQR: 13, 40).

At least one measure of average post-drug group absolute 25OHD (or change) was available from all but one of the high dose study arms. Of the 134 high dose arms, 35% (n=48) and 76% (n=106) measured 25OHD within 1 and 3 months of study drug initiation. Tables in Supplemental Information (Appendix S3.12-S3.18) show relevant information on population, dosing regimen, and 25OHD response for each study arm reporting 25OHD within 3 months of study drug initiation (17, 19, 81, 84, 90, 106-108, 112, 133-141, 143-189, 191-194, 212, 213).

Evaluation of 25OHD response by dosing regimen
Six independent treatment arms were identified that evaluated response to daily vitamin D between 1000-4000 IU in a group of children who were vitamin D deficient and reported 25OHD levels within the first month (144, 151, 159, 180, 184, 188). As shown in Figure 3B (appendix 3.4) none of the arms achieved a group 25OHD above 75 nmol/L with the first measurement, and 2 (33%) achieved this target within the first month. A single weekly dosing regimen was identified that enrolled VDD children and performed blood work within 1 month; this study reported an increase in 25OHD from 22 to 143 nmol/L with 4 weekly doses of 60000 IU. Ten independent study arms were identified that evaluated oral loading doses with vitamin D deficient populations and measured 25OHD within a month (17, 151, 162, 165, 173, 176, 214, 215). As shown in Figure 3C (appendix 3.5), 9 (90%) achieved an average post-study drug group level above 75 nmol/l, with three arms exceeding 200 nmol/L(151, 162, 173). Five additional arms calculated 25OHD change following oral vitamin D loads and reported increases ranging from 45 to 73 nmol/L(163, 178, 182). All arms with more than one post study drug measurement demonstrated a decline between the first and subsequent measurements. Dosing regimens that reported multiple measurements during the first week after oral loading suggested that 25OHD peaks on day 3 and declines from day 3 to 7 by an average of 15% (Supplemental Information, appendix S3.19) (17, 176).

Evaluation of variable loading dose regimens
Seven independent arms were identified that evaluated 25OHD response in vitamin D deficient children using a variable intramuscular (IM) dosing strategy (10000 IU/kg) (84,
170, 171, 183, 216). The single 10000 IU/kg IM dosing regimen that reported 25OHD within one month of therapy achieved a mean group level above 75 nmol/L (216). No published studies or conference abstracts evaluating 25OHD response within a month of a variable oral load were identified. One of the conference abstracts, published by Frizzell et al., evaluated response to an age based loading regimen (<3 yr:150000 IU, 3-12 yr: 300000 IU, >12 yr:
IU) among 40 children; approximately 6 weeks after treatment the group average increased from 27 nmol/L to 93 nmol/L, with at least one participant exceeding 300 nmol/L.

Factors associated with post study drug 25OHD levels
Significant heterogeneity in post study drug 25OHD was evident with group average levels ranging from 30 to 399 nmol/L, and a calculated I2 value of 99. Single variable random effects meta-regression identified eight variables to be statistically significant, with two additional variables approaching significance (Table 3C, appendix 3.6). Multivariate random effects meta-regression performed using data available from 102 independent arms identified 7 variables independently statistically significant in either the main effects or through an interaction (Table 3D, appendix 3.7). The interaction term between age and cumulative dose determined that the 0.27 nmol/L increase in final group 25OHD per 1000 IU is reduced by
013 nmol/L for every one year rise in age. Similarly, the interaction term between dosing regimen and time demonstrated that the group mean 25OHD gradually decreases following a loading dose by 5.6 nmol/L per week (CI: 3.48, 7.7). Inclusion of the study type variable demonstrated that non-randomized controlled studies, but not uncontrolled studies, were associated with higher post drug 25OHD levels. After including the variable for study design, no other measure of study methodological quality from the Cochrane risk of bias tool was statistically significant. Exclusion of the obese or malabsorption studies did not significantly change any of the parameter estimates.

The final multivariate model was used to predict group 25OHD levels following 4 loading doses in 4 age groups of VDD diseased children (Table 3E, appendix 3.8).
Regression analysis was also performed to model post study drug 25OHD standard deviation. Standard deviation was best predicted by the equation (SD = 0.42*final25OHD, R2 = 0.81); no other variable significantly improved the model R2 value.

Thresholds for potentially toxic vitamin D levels
Of the 88 eligible studies, 9 defined thresholds above which 25OHD was toxic or potentially toxic (range:125 to 374 nmol/L) (114, 139, 144, 152, 164, 169, 175, 206, 214). The most common definition was 250 (n=6) and another 2 used definitions of 374 and 375 nmol/L.

Adverse event analysis
There were 39 study arms reporting on high dose (>1000 IU) vitamin D regimens that provided hypercalcemia data within 3 months of drug initiation. Information on relevant population, dosing and adverse events measurements are provided in Supplemental Information (appendix S3.20-S3.21). There were 23 study arms who received intermittent, weekly or daily high dose loading regimens. Significant heterogeneity in hypercalcemia rates was calculated (I2=61%, see Figure 3D, appendix 3.9). Random effects meta-analysis identified a statistically significant difference in hypercalcemia rates between accepted daily dosing (2.6%, CI 1.1-5.9) and intermittent, weekly and daily high dose loading regimens (7.6%, CI: 4.1-13.7%, p=0.041). Further analysis identified higher hypercalcemia rates for the arms at or above 400000 IU (23.8%, CI: 16.3-33.3%) when compared with doses at or below 300000 IU (4.2%, CI: 2.0-8.8%, p=0.0001). Subgroup analysis using 25OHD data demonstrated that hypercalcemia was more likely among studies with average group levels above 200 nmol/L, compared to those below 200 nmol/L (3.9% vs 19.6%, p=0.006). Pooled hypercalcemia rates were similar for groups below 100 nmol/L and between 100 and 200 nmol/L. Further subgroup analysis by age was not possible due to the limited number of loading regimens administering doses above 300 000 IU.

For hypercalciuria (29 study arms; Supplemental information, appendix S3.22-S3.23) 13 distinct groups were identified that provided regimens corresponding to intermittent, weekly or daily loading doses. Of these, 10 reported no episodes of hypercalciuria and metaanalysis determined a pooled rate of 2.7% (CI: 0.8-8.9%). Exclusion of the study by Shajari and colleagues, reporting hypercalciuria in 28 of 30, reduced the pooled rate to 1.5% (CI:5-4.5%). Of note, the Shajari study was an RCT and the daily dosing arms reported hypercalciuria in 23 (200 IU/day) and 25 (400 IU/day) of the 30 children (168). Finally, our review did not identify any reported cases of nephrocalcinosis in the clinical trials administrating intermittent, weekly or daily loading dose regimens.

3.4 Discussion
Evaluation of daily vitamin D administration demonstrated that a dosing strategy approximating the IOM Tolerable Upper Intake Level (1000-4000 IU) will not rapidly normalize vitamin D levels in deficient children. However, administration of a loading dose of > 40 000 IU can rapidly elevate 25OHD. Our analysis also identified baseline 25OHD, age, cumulative dose, regimen type, disease status, time from loading dose and study type as independent predictors of final 25OHDlevel. Adverse event analysis found no increased hypercalcemia or hypercalciuria risk with loading doses at or below 300 000 IU, while a significant increase in hypercalcemia risk was observed with doses at or above 400 000 IU.

This systematic review identified 88 full text publications reporting 25OHD levels following the prospective administration of high dose vitamin D to one or more groups of children. Daily administration and loading dose therapy each accounted for roughly 40% of the eligible study arms. The rarity of loading dose arms originating from North America may explain why vitamin D position statements from Canadian and American pediatric societies make no mention of this therapy (9, 113). Slightly more than 75% of the eligible study arms included healthy, VDD, or children with rickets or kidney disease. None of the studies were from an acute or critical care settings with the most relevant study being a pilot RCT suggesting long-term clinical benefit in stable outpatient congestive heart failure (112). Inspection of excluded studies did not identify any performed in the pediatric critical care setting, with the most relevant evaluating high-dose intake in pneumonia and severe asthma (217-219).

Examination of post study drug 25OHD levels from high-dose study arms demonstrated a wide range of final group levels. To remove some heterogeneity related to clinical and methodological factors, we evaluated the short-term response to daily vitamin D approximating the IOM Upper Tolerable Intake Level (1000-4000 IU/day)(11). Overall, the results strongly advise that this approach will not normalize levels (> 75 nmol/L) in a time frame appropriate to potentially benefit acute and critically ill populations (144, 151, 159, 180, 184, 188). These findings are important as they will inform future studies, and help interpret the results of published RCTs. For example, these findings might call into question the validity of the pediatric RCT by Choudhary et al. evaluating the effect of 5 days of daily 1000 IU on recovery from pneumonia (217).

Conversely, there was convincing evidence that single or divided dose loading therapy is an effective means of rapidly raising 25OHD levels. We also observed that numerous studies generated levels well in excess of the 75 nmol/L target. Multiple study arms administering loading doses of vitamin D achieved potentially toxic levels (groups average > 200 nmol/L) (151, 162, 165, 173). Three of these administered doses in excess of 200000 IU to neonates or infants, and the fourth evaluated 600000 IU in toddlers and preschool children (151, 162, 165, 173). In contrast, the administration of 50000 IU to a group of toddlers and preschool children did not achieve levels of 75 nmol/L in more than half (17). These results suggest that with appropriate dose selection, single or divided loading regimens have the ability to rapidly normalize vitamin D status and may explain the positive benefits observed in clinical trials evaluating a loading dose in children with pneumonia and severe asthma (217-219).

This study also sought to further explain heterogeneity in post drug 25OHD levels due to population, dosing, and methodological characteristics. Single and multivariable random effects meta-regression identified that baseline vitamin D status, cumulative dose, age, regimen type, healthy vs. diseased status, and study type were significantly associated with post 25OHD level. Most importantly, we identified a statistically significant interaction between cumulative dose and population age, demonstrating that the 25OHD response per dose declines as age increases. Although this observation is most likely related to the high correlation between age and weight, differences in developmental pharmacokinetics may contribute (220). Further, our regression analysis identified lower post-study drug 25OHD levels in study arms originating from diseased populations, when compared to healthy children. There are multiple potential explanations including differential compliance, malabsorption, increased losses (e.g. capillary leak), and altered hepatic or end organ metabolism (48, 221-224).

Collectively, these findings suggest that rapid normalization of vitamin D status may require consideration of age (or weight), baseline 25OHD, and disease status. Prediction of 25OHD levels using the multivariate model suggested 50000 IU as appropriate in young infants, while doses in the 300 000 to 600 000 IU range may be required in adolescents. As weight-based dosing represents the standard of care in the pediatric medicine, these findings might be approximated to 10000 IU/kg. Review of published variable high dose regimens identified 7 independent pediatric populations having 25OHD measurements following the administration of 10 000 IU/kg IM vitamin D. These studies suggest that IM dosing might rapidly normalize vitamin D status, although the lack of measurements within the first month and paucity of enteral studies prevents definitive conclusions. Regardless results from the IM studies are relevant as many acute and hospitalized patients suffer significant malabsorption and/or are not able to take food and medication enterally (88, 225). The need for pediatric studies evaluating 10000 IU/kg using the enteral route is reinforced by evidence from adult studies showing significant differences in short-term response between enteral and IM routes (91, 92).

This review also examined whether high dose loading regimens were associated with vitamin D related adverse events and toxicity. Vitamin D toxicity is a characterized by hypercalcemia and hypercalciuria with the classic symptoms (e.g. abdominal pain, anorexia, constipation, polyuria) directly attributable to these abnormalities. Presently, there is no accepted 25OHD threshold that identifies increased adverse event risk. The lack of certainty is emphasized by our finding that 90% of studies did not use or cite a specific threshold. For the few that did report, the most common value was 250 nmol/l (114, 139, 152, 164, 169,
175, 206, 214, 226). Review of these articles identified that the more recent trials did not select thresholds based on known toxicity, but the idea that supraphysiologic levels (not achievable with sun-exposure, healthy diets) are unlikely to be of benefit (81, 97, 98, 114, 141). Our analysis supports a 200 to 250 nmol/L threshold as dosing regimens with averages above 200 nmol/L, being associated with increased hypercalcemia risk.

To better inform selection of dosing regimens, we also sought to understand whether there was a cumulative loading dose associated with increased hypercalcemia and hypercalciuria. Our evaluation did not identify increased risk of hypercalcemia with loading doses at or below 300000 IU (4%) but did find a significantly higher risk for those at or above 400 000 IU. In addition, our review identified only 3 cases of hypercalciuria among the 878 study participants who received intermittent, weekly or daily loading regimens (after exclusion of Shajari (168). Further, none of the eligible clinical trials reported a case of nephrocalcinosis with loading dose therapy. Taken together, these findings are consistent with the nephrocalcinosis literature, where most cases potentially associated with vitamin D have occurred in children with rare genetic disorders (99-103, 105) or following the intake of doses exceeding 600 000 IU in healthy children (85, 102, 227). Based on these findings we would suggest age or weight based loading doses, not exceeding 400000 IU or 25OHD levels 200 nmol/L. Of note, the increased hypercalcemia risk demonstrated with doses at or above 400000 IU is largely driven by multiple studies on young children and only one study administering 1.8 million IU to older children. Consequentially, our findings should not be interpreted to state that doses in the 400 000 to 600 000 IU range are toxic in adolescents. In fact, multiple adult studies, including pilot trials in the critical care setting, have not identified significant adverse events with loading dosing to adults in this range (86, 115,

Although this systematic review summarizes a large body of literature and provides valuable information, a number of limitations must be acknowledged. First, accurate information on a number of potentially relevant characteristics was not available including race, ultraviolet exposure, diet, physical activity, compliance, and blood collection techniques (53, 226). Second, study size was often small, with the associated random error in the determination of group 25OHD levels potentially negatively influencing our ability to quantify associations. Third, there were relatively few studies compared to the number of potentially relevant characteristics and interactions. For example, due to the absence of appropriate 25OHD measurements following intramuscular administration, no conclusions can be made about the rapidity at which this regimen achieves peak 25OHD levels. Further, to accommodate the discrepancy between potentially relevant factors and study number we were forced to combine patient groups into broad categories (e.g. diseased vs. healthy). As regression results generated using patient and study level variables are not always consistent, our results and recommendations will need to be affirmed through future clinical studies. Finally, our adverse event analysis was limited by lack of reporting in close to half of the studies for measures of hypercalcemia and hypercalciuria. Further, the lack of studies with loading doses at or above 400 000 IU to older children and adolescents prevents a more definitive statement of risk and benefit.
This systematic review provides valuable information on the ability of different dosing regimens to rapidly restore vitamin D levels. Our study findings indicate that age or weight-based loading therapy of 10000 IU/kg (maximum 400 000 IU) would be most appropriate. Given the absence of studies administering this dose enterally, and no studies on critically ill children, this dose along with vitamin D related adverse events including hypercalcemia and hypercalciuria should be evaluated in prospective RCTs prior to widespread use.

Bridging paragraph between publications
As described in Chapter 2, most children with CHD receive either no supplementation or doses based on the Recommended Daily Allowance or Adequate Intake suggested for healthy children. These doses are only intended to raise or maintain vitamin
D to levels that prevent bone disease and do not address extra-skeletal actions of vitamin D. Multiple research bodies and agencies have acknowledged that there may be situations and populations, like that identified for CHD, where higher daily dosing may be required or beneficial. Our systematic review evaluated all clinical trials administering daily high dose vitamin D in the 1000 to 4000 IU range - comparable to the IOM recommended Upper Tolerable Intake Level. This review suggested that given adequate time it should be possible to raise vitamin D levels high enough with IOM approved dosing to prevent postoperative vitamin D deficiency. As discussed in full detail in Chapter 4, 75 to 80% of CHD surgeries occur after 2 months of age, suggesting that it may be possible to prevent vitamin D deficiency with pre-operative administration of the IOM recommended Daily Upper Tolerable Intake Level. One specific population that may be at risk for inadequate response despite daily supplementation with the IOM high dose are neonates and young children who required immediate surgery. Further, there may be other factors that prevent adequate drug intake and increase vitamin D levels in children with surgical CHD. Evaluation of the entire surgical CHD population would allow us evaluate the feasibility of this approach in older children, and provide further support for our idea that this approach will not be effective in children. As such, we have designed a clinical trial intended to determine whether preoperative daily supplementation with the IOM Tolerable Upper Intake level can significant reduce the number of children with CHD who are vitamin D deficient postoperatively.

4.0 Prevention of vitamin D deficiency in children following cardiac surgery:

Development of a study protocol for a dose evaluation Randomized Controlled trial

4.1 Background
Congenital heart disease (CHD) is a common condition with an estimated prevalence of 1 per 100 in the general population. A significant proportion of these pediatric patients require one or more corrective surgeries over their lifetime, collectively leading to 15 000 procedures per year in North America (1). Post-operatively, these patients suffer significant morbidities, which may include a pronounced systemic inflammatory response, multiple organ failure, electrolyte disturbances, arrhythmia, infection and endocrine imbalances (2-5). Interventions that prevent or modulate post-operative pathophysiology may prevent illness, speed recovery, and decrease chronic morbidity in this high risk pediatric population.

Vitamin D status is well recognized as important to calcium homeostasis and musculoskeletal health. The importance of Vitamin D, and its physiological effects are well understood in the context of hypocalcemia(63, 228). Briefly, as serum calcium falls, the parathyroid increases parathyroid hormone (PTH) secretion leading to activation of vitamin-D through an inducible renal enzyme. The inducible renal enzyme works to convert serum 25-hydroxyvitamin D 25OHD to 1,25 dihydroxyvitamin D 1,25OH2D and this actived metabolite circulates to the bone, gut and kidneys to restore homeostasis. It is now known that many cell types do not rely entirely on kidney production and have enzymes capable of converting 25OHD to its active form for both autocrine or paracrine use. Circulating 25OHD is well accepted as the best marker for evaluating vitamin D status in the majority of health settings, including the general ICU population (8, 229). The generally accepted thresholds for defining vitamin D sufficiency is 75 nmol/L, with deficiency defined as below 50 nmol/L, and severe deficiency at 25 to 30 nmol/L.

Increasingly, vitamin D is accepted as a pleiotropic hormone important for the functioning of organ systems central to critical illness pathophysiology, including electrolyte homeostasis, cardiovascular health, inflammation and innate immunity (30, 39, 230). This has lead to the hypothesis that deficiency might represent a modifiable risk factor for critical illness. A growing number of observational studies in adult cardiovascular and intensive care populations have investigated this hypothesis and these studies have reported high vitamin D deficiency rates and associations between hormone level and organ dysfunction, health resource utilization and mortality (30-38). Further, a single moderately sized, interventional study on critically ill adults (VITdAL-ICU) suggests that rapid repletion of vitamin D may improve outcomes (122). This trial randomized 475 critically ill adults to an initial enteral 540 000 IU cholecalciferol loading dose (followed by monthly 90000 IU) or placebo doses. In this study there was a non-significant absolute risk reduction in hospital mortality in the vitamin D arm (7.0%, p=0.10). However, in the predefined subgroup of patients with vitamin D levels below 30 nmol/L at baseline this absolute difference became larger and statistically significant (-17.5%, p=0.01). Although large pediatric observational studies have also documented high rates of vitamin D deficiency and associations between hormone levels and clinical course within the PICU (24, 25), a high dose interventional trial in the PICU has yet to be undertaken (231).

Patients with CHD requiring surgery have been investigated as subgroups within large PICU studies and as distinct populations (24, 28). Analysis of the CHD patients enrolled in a large multicentre Canadian PICU reported a 70% deficiency rate and associations between vitamin D level and clinical course (24). Further, Graham et al. confirmed these observations with a secondary analysis of post-operative blood reporting not only that 84% of neonates with CHD were vitamin D deficient post-operatively, but that lower levels were associated with incrased need for inotropic agents (28). In addition, in a prospective longitudinal study we calculated an 85% vitamin D deficiency rate in the CHD population immediately following surgery, as well as an association between deficiency and post operative fluid and catecholamine requirements (26). Mechanistic studies determined the high rate of vitamin D deficiency to be secondary to borderline normal pre-operative 25 hydroxyvitamin D (25OHD) levels and an acute 40% intraoperative decline due to cardiopulmonary bypass (CPB), consistent with that described in an adult CPB study (47). In summary, the available data suggest that most CHD patients are vitamin D deficient following cardiac surgery and that the immediate post-operative levels are associated with subsequent clinical course.

A role for vitamin D in critical illness has biological plausibility, as there are multiple mechanisms through which deficiency could cause secondary pathophysiology. Hypocalcemia is a common problem following CHD repair (30%) and calcium replacement is associated with morbidity and mortality (5). Adult and pediatric ICU studies have shown that critically ill patients with hypocalcemia are more likely to have abnormalities of their vitamin D axis, including low 25OHD, hypoparathyroidism and/or renal dysfunction (5658). A role for vitamin D in cardiac health can be found in case reports and case series describing cardiomyopathy secondary to isolated severe vitamin D deficiency (60-63). A recent RCT of vitamin D supplementation in outpatient pediatric congestive heart failure showed improved cardiac function with a higher daily dose of vitamin D (112). Additionally, cardiac surgery with cardiopulmonary bypass uniformly leads to a postoperative systemic inflammatory response syndrome (73, 74). There is good evidence that vitamin D metabolites play important immunomodulatory roles mediated through functional vitamin D receptors present on all major immune cell types (75-77). Vitamin D signaling is also known to play a role in innate immunity, such as in the production of cathelicidins (78-80). Cathelicidins, important endogenous antimicrobial peptides, provide protection against multiple viral and bacterial pathogens. Prevention of vitamin D deficiency could decrease hospital acquired infections among CHD patients through improved innate immunity.

The current body of knowledge suggests that optimization of vitamin D status prior to and following CHD repair could improve clinical outcomes through reduced inflammation, fewer nosocomial infections, improved cardiac function, and faster postoperative rehabilitation and physical functioning (30-34, 38, 41, 51, 232). However, before these findings can be translated into clinical practice, a number of unknowns must be addressed. For instance, there have been no interventional studies establishing that prevention of post-operative vitamin D deficiency improves clinical outcomes in CHD patients. In addition, attempts to perform a large RCT would be premature as a dosing regimen that prevents post-operative vitamin D deficiency has not yet been identified. Moreover, there have been no vitamin D dosing studies or guidelines developed specific to the CHD population; presently children with CHD receive the same advice regarding supplementation as healthy children (11). Although it is tempting to extrapolate recent safety data from high dose vitamin D studies on healthy children to the CHD population, this may be inappropriate. CHD patients have unique metabolic demands, organ dysfunctions, as well as known and unknown genetic abnormalities that potentially make them more or less susceptible to vitamin D toxicity (109-111, 233). To begin addressing these knowledge gaps, we have designed a pilot dose evaluation randomized controlled trial (RCT) with the goal of identifying a supplementation regimen that safely prevents postoperative vitamin D deficiency in children requiring cardiopulmonary bypass for CHD.

4.2 Objectives and hypotheses
In pediatric patients requiring surgery for CHD, pre-operative supplementation with a daily high dose vitamin D regimen, modelled on the Institute of Medicine (IOM) Tolerable Upper Intake Level (UL), will significantly reduce post-operative vitamin D deficiency, when compared with usual care (11).

Study Objectives
The primary objective is to perform a double blind RCT to determine whether the preoperative administration of daily high dose of vitamin D based on the UL from IOM, compared with usual care, results in a significant reduction in post-operative vitamin D deficiency in a pediatric population with CHD.

Secondary objectives -
Determine the barriers and feasibility of conducting a larger phase III RCT evaluating whether vitamin D supplementation improves clinical outcomes in children who require CHD surgery (blinding, recruitment, compliance).
Determine whether the pre-operative regimen of daily high dose vitamin D, compared with usual care, results in a greater number of vitamin D related adverse events (hypercalcemia, hypercalciuria).
Determine whether the pre-operative regimen of daily high dose vitamin D, compared with usual care, improves established markers of vitamin D axis functioning (active hormone levels, cardiac function).

4.3 Methods
Sponsorship, approvals, principals
The Heart and Stroke Foundation of Canada is the Sponsor for the trial (Supplemental Information, appendix S4.1-S4.4). The study will be conducted in accordance with the ethical principles guided by Tri-Council Policy and the Declaration of Helsinki. The protocol is approved by Health Canada and the Children’s Hospital of Eastern Ontario Research Ethics Board (REB reference: 13/03E). The trial will comply with the principles of Good Clinical Practice and will be carried out in accordance with applicable legislation and the Standard Operating Procedures of the CHEO Research Institute. The trial will be reported in line with the Consolidated Standards of Reporting Trials (CONSORT) 2010 guidelines (234) and the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist (235). Protocol amendments will be communicated as necessary to the study team, health care team and REB. A structured summary of the trial is provided in Table 4A (appendix 4.1).

Study design
The trial is a single centre, double blind, parallel, randomized, controlled dose evaluation trial comparing the efficacy and safety of two vitamin D dosing regimens in the prevention of post-operative vitamin D deficiency in children undergoing surgery for CHD.

Study population
Between 36 weeks gestational age and 18 years
CHD requiring surgery within the next 12 months
CHD requiring surgical correction with cardiopulmonary bypass (CBP)
Born less than 32 weeks gestational age
Disease preventing enteral feeds or drug administration prior to surgery
Confirmed or suspected Williams syndrome (a neurodevelopmental genetic disorder with symptoms that include cardiovascular problems and high blood calcium)
Proposed surgery to take place at another centre (outside of CHEO)

Justification of eligibility criteria
In our previous study, CHD patients who did not receive CPB had a minimal (<10%) intraoperative drop in 25OHD. Although prevention of vitamin D deficiency is important for these patients, they do not require pre-operative elevation into the upper normal physiological range. Very premature infants (<32 weeks) are at significantly increased risk for nephrocalcinosis (236, 237). CHD patients with Williams syndrome have a genetic susceptibility to hypercalcemia and current guidelines recommend against any vitamin D supplementation (97, 233).

Study Drug
Study drug distribution
Europharm (Quebec) will provide the study drug (vitamin D) in the required concentrations, prepared in indistinguishable vials for blinding purposes. The study drug will be analyzed as per Health Canada regulations. The CHEO Pharmacy will administer the study drug (based on randomization, participant age and whether the patient is breast or formula fed). Infants with CHD assigned to the usual care arm will be given either a placebo (0 IU/mL) solution if they are receiving vitamin D as part of formula, or they will be given a 400 IU/mL solution if they are breast-fed.
Proposed supplement doses (Interventions) to be tested and rationale
The doses for evaluation have been modelled on the two age specific intake levels recommended by the IOM (11) (Table 4B, appendix 4.2).
1. The High dose group is based on the age specific UL from the IOM. These doses were chosen to elevate 25OHD well above 50 nmol/L, while minimizing risk of vitamin D toxicity (e.g. hypercalcemia, hypercalciuria). Patients under 1 year of age will receive 1600 IU/day, while those over 1 year of age will receive 2400 IU/day. Note - infants under 6 months of age in the high dose group will receive 600 IU/day more than the UL from IOM, while those between 6 and 12 months will receive 100 IU/day more than the UL.
The Usual care group will receive Adequate Intake (AI) for infants and Recommended Dietary Allowance (RDA) for children over 1 year (< 1 year, 400 IU;>1 year 600 IU/day). These doses were chosen by IOM to achieve blood 25OHD levels above 50 nmol/L in the vast majority of the healthy population.

Rationale for study design and interventions
Rationale for inclusion of the usual care arm -
Given that children with CHD receive the same vitamin D supplementation advice as healthy children (by default) it is tempting to conclude that usual care dosing will not be adequate to prevent vitamin D deficiency. Unfortunately, this conclusion may be wrong for the following reasons: (i) only 50% of previous study participants indicated daily vitamin D intake at or above 400 IU (24), (ii) compliance with vitamin D supplementation may be poor without motivation (research studies, vitamin D related disease), (iii) there is often uncertainty about vitamin D intake by caregivers, (iv) recommendations for usual care recently increased to 600 IU for children above 1 year (11). Given potential safety concerns regarding high doses of vitamin D in diseased population, it would be appropriate to properly evaluate the efficacy of usual care under ideal circumstances.

In addition, it is important to assess the baseline risk for vitamin D related adverse events in CHD patients receiving usual dosing. Although studies on healthy children have not identified adverse events (e.g. hypercalcemia, hypercalciuria) with doses at and slightly above the UL from IOM (19, 81), diseased populations including those with CHD may be predisposed at lower 25OHD levels.
Rationale for pre-operative daily enteral approach -
Our previous prospective study demonstrated that post CHD surgery vitamin D deficiency occurs due to borderline normal pre-operative values and a consistent cardiopulmonary bypass induced intraoperative decline (26). As there is no intravenous form of either cholecalciferol or 25OHD maintenance of appropriate post-operative levels will require elevation of pre-operative levels into the high normal range using enteral supplementation. Two basic approaches for enteral restoration and maintenance of vitamin D stores have been described. First, representing usual care, is the daily consumption of a relatively low dose of cholecalciferol (400 to 4000 IU/day). The second option is a single or divided megadose of vitamin D (100,000 to 600,000 IU) given intermittently throughout the year (83, 238). As safety concerns regarding the megadose approach in children have not been adequately addressed we have chosen to first evaluate regimens based on daily consumption (90).

Anticipated duration of study drug and peri-operative 25OHD levels
Duration of study drug - Vitamin D supplementation is generally initiated within a week of birth, so the duration of preoperative therapy would be from birth (or diagnosis of CHD) to the time of surgery. Timing of surgery is very dependent on the type of CHD lesion. Consistent with the literature, our recent observational study of perioperative vitamin D status demonstrated 8 months as the median age at surgery, with 2.4 months being the 25th percentile. Based on these findings, we anticipate that it should be possible for 75 to 80% of patients to receive study drug for more than 2 months (the time required to achieve a new 25OHD steady state with high dose daily supplementation). Whether this duration of study drug can actually be achieved pre-operatively, and the 25OHD levels achieved, will be reported as part of the pilot study.

Peri-operative 25OHD levels - Given the 40% intra-operative decline, pre-operative levels above 90 nmol/L will be required to maintain post-operative levels above 50 nmol/L (the value at which sufficient substrate to synthesize the active metabolite is available). The ability of certain vitamin D intake levels to achieve this pre-operative value can be inferred from recently completed dosing studies on healthy children level (19, 81). These studies have shown that usual care dosing for 2 to 3 months will achieve pre-operative levels of 90 nmol/L in only 40-50%. In contrast, studies evaluating doses approximating our higher daily intake level (1600 IU/day) achieved mean 25OHD levels of 130 to 150 nmol/L; suggesting that 80% or more of CHD patients could achieve pre-operative levels of 90 nmol/L or above.

Subject recruitment
Potentially eligible study participants will be identified in the ambulatory clinics (cardiology, cardiovascular) or inpatient wards (including pediatric intensive care and neonatal intensive care unit) by a member of the health care team or study staff. A research coordinator will provide study information and obtain informed consent as well as applicable
assent from each participant (Supplemental Information, appendix 4S.5-4S.19). Study staff will provide support to participants and encourage adherence to intervention protocols.

Randomization, blinding and stratification procedures
We will use a computer-generated randomization sequence. Only the CHEO pharmacy will have access to the randomization sequence and will be responsible for participant randomization and allocation. Only the pharmacist will know the identity of the study drug administered to a specific patient. Given the expected recruitment (2 to 3 per month) and potential impact of season on 25OHD, randomization will be performed in permuted blocks (4 within each stratum). We will blind patients, families, investigators, hospital staff, and research personnel to treatment arm. Blinding was considered necessary to avoid: (i) families altering the outpatient dose, and (ii) a number of secondary outcome measures are potentially subjective (echocardiography, timing of extubation, need for fluids or catecholamine infusion). Within each age group the two interventions will be indistinguishable (vial, volume, colour, taste, consistency and smell). Participants will be stratified into whether or not they are expected to receive at least 8 weeks of study drug prior to surgery. This stratification should guarantee that an equal number of CHD patients who will not receive 8 weeks of oral dosing end up in both the high and low dose arms. We will further stratify by age (under or over 1 year of age).

We will not protocolize post-operative co-interventions as the study is single centre and CHEO has standardized approaches to the common post-operative complications and adverse events (e.g. hypocalcemia, junctional ectopic tachycardia, necrotizing enterocolitis
constipation, sedation, catecholamine administration, etc). As the study is blinded, protocolization of co-interventions is less relevant and differences should relate to random chance or drug effects.

Diagnostic and clinical outcome measures
Blood 25OHD -
The primary objective will be evaluated using immediate post-operative blood 25OHD concentrations (collected on the day of ICU admission) with a level lower than 50 nmol/L used to define deficiency (7, 13). This is a well-established cut-off based on: (i) knowledge that the parathyroid and renal organs need to compensate for 25OHD levels below 50 nmol/L, and (ii) clinical studies showing increased risk of bone, cardiovascular, immune and other disease entities once concentrations fall below this level. 25OHD will be determined using a LC-MS assay from a laboratory participating in the Vitamin D External Quality Assessment Scheme (DEQAS) (24, 239).

Vitamin D related adverse events -
We will report, by intervention, on the occurrence of clinically significant adverse events. However, a measurable difference in clinically significant adverse events between the high dose and usual care arms of the study is unlikely. Therefore to enhance our ability to evaluate for potential toxicity we will use two well accepted surrogate outcome measures:
• Hypercalcemia: Will be defined as an ionized calcium level above 1.40 mmol/L (or above 1.45 mmol/L for children under 8 weeks) (Table 4C, appendix 4.3). We will assess calcium in blood collected immediately before surgery and throughout the
post-operative course (measurements are standard of care and any single episode of hypercalcemia not related to parenteral administration of calcium will be considered an adverse event) (190).
• Hypercalcuria - We will identify hypercalcuria using calcium:creatinine ratios defined using age specific norms and thresholds (Table 4C, appendix 4.3) (190, 240, 241). Measurements will be performed on urine collected in the operating room immediately prior to surgery and on the first post-operative day.

Vitamin D axis function -
We will evaluate vitamin D axis function through changes in blood 1,25OH2D (24) levels from blood collected at specified times following surgery. Based on our previous work we anticipate a 40% intra-operative decline in 1,25OH2D levels (229) and some children will experience a transient decline in 1,25OH2D levels into the deficient range (<50 pmol/L). Given adequate 25OHD levels and an otherwise properly functioning vitamin D axis we would anticipate restoration (or maintenance) of active hormone levels into the normal range within 12 hours of surgery. Impaired vitamin D axis function will be defined as an inability to maintain active hormone levels in the normal range at any point from blood work collected after the first post-operative day.

Organ function and ICU outcome measures -
Post-operative cardiovascular and immune function will be measured and compared between the two groups. Cathelicidin levels, an endogenous antimicrobial peptide, will be used as a surrogate measure of innate immune function (78, 79, 242). Clinically relevant measures of cardiac organ function will include echocardiograms (e.g. ejection fraction), inotrope requirements (e.g. vasopressor need, maximum inotrope score) and fluid resuscitation (positive fluid balance in first 48 hours). Further we will also evaluate standard PICU clinical outcome measures including time to extubation, PICU and hospital length of stay.

Phase III Study Feasibility -
We will determine the feasibility of a subsequent multi-centre large interventional study through an evaluation of study consent and accrual rate, protocol deviations and violations, proportion of adequate allocation concealment and blinding, proportion of study drug compliance, and proportion of study drop out/withdrawals.

Study Procedures
A summary of study procedures, biological sample collection and metabolite measurements has been provided as a flow diagram (Figure 4A, appendix 4.4) and the biochemical measurements on research specimens are summarized in Table 4D (appendix 4.5).
1. Prior to initiation of study drug -
Urine - After consent is obtained and the study participant is waiting for the pharmacy to prepare the study drug we will gather a urine sample for determination of calcium:creatinine ratios. Where developmentally appropriate the participants will be asked to provide urine into a container. Urine bags will be placed on younger children. If the child is unable to provide a urine sample during the time it takes to fill the study drug prescription, the participant will be provided with a container or urine bag and asked to provide an outpatient urine sample.
Blood - Neonates and other study participants requiring surgery within 2 months of diagnosis and enrollment will have 0.5 - 1 mL of blood collected prior to (or within 2 days) of starting study supplement for determination of 25OHD. Where possible, unused or discard blood will be obtained from the laboratory (243). If unused blood is not available, research blood will be collected at the time of clinically indicated blood work, or not at all. These patients will not have blood collected for research purposes again until they are taken to the operating room.
* Note -We will request initiation samples but children will still be enrolled if these samples cannot be collected or the families do not want these procedures.

During period of study drug administration -
All outpatient participants will have blood collected at the time of standard pre-surgical blood work (two to three weeks prior to surgery) for both 25OHD and Ionized calcium.
These samples are collected to ensure that patients do not go for surgery with potentially toxic levels of vitamin D. The safety officer (pediatric endocrinologist) will review the ionized calcium level on the patients chart and follow up if required as per the safety measures outlined below (Figure 4B, appendix 4.6). For those participants who will receive study drug for more than 6 months we will also perform additional blood work to ensure that potentially toxic levels are not maintained for long periods prior to surgery. The timing of this blood work will not be specified and will occur as part of clinically indicated blood-work during regularly scheduled clinic appointments

Intraoperative biological samples and measurements-
Blood - All study participants will have 2 mL of blood collected in the operating room following anesthesia and intubation, but prior to skin incision and initiation of cardiopulmonary bypass. Pre-operative ionized calcium will be determined. Remaining sample will be processed to plasma, aliquoted and stored at -80oC for determination of 25OHD at the end of the study.
Urine - All study participants will have urine collected after insertion of the urinary catheter and the calcium:creatinine ratio will be determined. Study results will not appear on the patient hospital chart, but will be labeled with the study ID number and forwarded to the study investigator and safety officer for review.

Post-operative biological samples and other study measurements
Blood - All study participants will have 2 mL of blood collected following separation from cardiopulmonary bypass (at admission to PICU). Table 2 shows the biomarkers to be measured. Further study participants will have 2 mL of blood collected on post-operative days 1, 3, 5 and 10 in the PICU. Samples will be collected from arterial or central venous catheters at the time of clinically indicated blood work. If these catheters have been removed, blood will be collected at the time of clinically indicated venipuncture. If patients are discharged to the ward before the day 10 research sample is collected, a discharge sample will be collected at the time of discharge and no further research blood will be gathered. To limit the volume of research blood collected, neonates will only have 1 mL of blood collected post operatively on days 3, 5, and 10.
Urine - All study participants will have urine collected from the urinary catheter on the first post-operative day. Calcium and creatinine concentrations will be determined.
Echocardiography - A comprehensive exam will be performed immediately post-operatively (standard of care) and on the first post-operative day by a trained technician or pediatric cardiologist. Between group comparison will evaluate for differences in left-ventricular in (LV) end-diastolic diameter, LV end-systolic diameter, LV ejection fraction (112).

Case report form
All trial information will be stored in a secure electronic database to maintain confidentiality. Data entry methods are in place to promote data quality. In addition, protocol deviations, including discontinuation of study participation will be recorded. The case report form will be developed using REDCap (128). Research Electronic Data Capture is a secure web application for building and managing online surveys and databases. The following data will be entered electronically:
Questionnaire - On the day of surgery the research coordinator will collect the participant diaries and unused study supplement. The patient diaries will contain information on which days the patient was or was not given the study drug, why not, and whether there were difficulties. Information will also be collected on prescribed medications, nutrition, additional supplement use, and symptoms associated with vitamin D intoxication (e.g. constipation, abdominal discomfort).
Operative details - The research assistant will extract detailed operative information, including: cardiac lesion type, surgery performed, RACHS score (244), total fluid intake and output, blood product and fluid administration and loss, hypothermia, need for deep hypothermic circulatory arrest (duration), aortic cross clamp times, CPB circuit volumes, CPB circuit constituents, CPB time, occurrence of intraoperative hyper or hypocalcemia, administration of parenteral calcium, need for catecholamines following separation from CPB, occurrence of intra-operative arrhythmias.
PICU course - Clinically relevant information on clinical course and organ dysfunction will be collected, including: death, ECMO, PRISM illness severity (245), cardiovascular dysfunction (fluid bolus requirements, inotrope/catecholamine use, arrhythmia), renal dysfunction (urine output, creatinine measurements, need for dialysis), hypocalcemia and calcium administration, duration of mechanical ventilation and duration of PICU stay.

4.4 Statistical analysis
Sample size justification
Based on our observational studies and findings from recent dose evaluation studies on healthy children, we estimate that no more than 40% of the usual care arm will have postoperative 25OHD levels above 50 nmol/L. Based on the 25OHD levels achieved with 1600 IU/day in recent studies on healthy infants we anticipate that 80% of the high dose arm will have post-operative levels above 50 nmol/L. Therefore group sample sizes of 28 in both treatment arms will be required to achieve 80% power to detect an absolute difference between the group proportions of 0.40. The test statistic used is the two-sided Fisher's exact test and the significance level of the test was targeted at 0.05. Assuming a 10% drop out rate, approximately 62 patients (total) will need to be recruited.
Comments on power for evaluating vitamin D related adverse outcomes:

  • 1. Hypercalcemia - Our previous observational study (n=58) identified no cases of preoperative or immediate post-operative hypercalcemia (26). With a baseline rate in the usual care arm between 0 and 10% our sample size would be sufficient to show a statistically significant absolute difference between groups if the rate in the high dose arm exceeded 30%.
  • 2. Hypercalciuria - Information on baseline rates of hypercalciuria prior to or following cardiac surgery with usual care vitamin D intake is not available. The proposed sample size would be sufficient to demonstrate a 35% absolute difference in proportions with baseline pre or post-operative rates up to 20%.

Statistical procedures
The analyses will be conducted using SAS software (Copyright SAS Institute Inc., Cary, NC, USA) and a p-value less than 0.05 will be considered statistically significant.
Descriptive statistics - Treatment groups will be described and compared using: (i) means with standard deviations or medians with inter-quartile range values for continuous variables or (ii) frequencies with percentages for categorical variables. Statistically significant differences will be determined using Chi-square and Fisher’s exact tests for categorical variables, and t-tests or nonparametric tests (e.g. Wilcoxon) for continuous variables, as appropriate.

Primary outcome - The primary analytical approach will be to evaluate all randomized patients in an intention to treat analysis. Differences in the primary outcome measure, proportion with 25OHD < 50 nmol/L, between the treatment groups will be evaluated using the Fisher’s exact test. Logistic regression analysis will be used if important variables are unevenly distributed between groups. We anticipate minimal missing data, as over 95% of participants from the recently completed observational study had an immediate postoperative sample(26).
Secondary outcomes - Secondary analyses will be evaluated between groups based on data type. Outcome measures that are continuous will be evaluated using the t-test, Wilcoxon sign rank test (where appropriate) or through linear regression analysis if important variables are not evenly distributed between groups. Binary secondary outcome measures (e.g. hypercalcemia, hypercalciuria) will be compared between the two treatment groups using Fisher’s exact or Chi-square. For the analysis of outcomes measures that represent time to event (e.g. restoration of 1,25OH2D levels to normal range, time to extubation, PICU length of stay/discharge) we will apply the log rank test. If randomization does not lead to equal distribution of important variables (e.g. weight) the analysis will be expanded to multiple regression modeling (e.g. logistic, linear, Cox proportional hazard).

Subgroup analysis - The well-known pharmacology of enteral vitamin D dosing shows that up to 2 months of regular daily intake is required to build body stores and achieve steady state blood levels of vitamin D. Consequently, neonates or other infants enrolled into the study who receive surgery within two months of birth or CHD diagnosis will be analyzed separately. Within this subgroup analysis, the primary objective remains reporting of proportions (in the usual care and high dose groups) that are vitamin D deficient postoperatively. However, given that these participants will receive study drug for a very short period we anticipate that the proportion with 25OHD levels above 50 nmol/L will remain low in the high dose group. Our program goal at this stage is to identify a dosing regimen that prevents post-operative vitamin D deficiency in 75% of CHD patients. Given this goal, and an estimated prevalence of 25% we would need 12 neonates (or children who receive < 2 months) to generate a confidence interval that excludes 75%. Feasibility - Most neonates with CHD who require cardiac surgery within the first few weeks of life have serious cardiac lesions that can limit enteral nutrition and medication delivery. Anticipating that most of these patients will not significantly elevate 25OHD levels with daily enteral intake at IOM high dose this study will provide important information on the willingness of health care providers to provide enteral study drug. This information will allow us to consider alternative dosing regimens for future studies based on single or divided doses representing a months or more worth of daily dosing (e.g. 5000-10,000 IU/kg). Only those children completing the entire study protocol will be included in analyses.

4.5 Data and safety monitoring
Data Safety Monitoring Board
In order to assess possible changes in risk/benefit ratio to study subjects and to obtain independent oversight of study conduct, an external Data and Safety Monitoring Board (DSMB) will be established to oversee the progress of the study. The DSMB will be composed of representatives from statistics, nephrology and endocrinology. External DSMB study reviews will be conducted after half of the participants (n=30) have completed all study procedures. The DSMB will review and monitor the study procedures and potential risks with a focus primarily on safety. Serious Adverse Events (SAEs) will be reviewed by the DSMB members in order to determine whether additional safety measures should be initiated. There are no predefined criteria for stopping the study, although the DSMB may recommend changing study drug concentration or stopping the study based on SAE or 25OHD data. If there are significant deviations from major study assumptions the DSMB or study investigators may choose to evaluate 25OHD levels and stop the study early.

The principal investigator and his co-investigators will be responsible for maintaining and assessing subject safety in the study, monitoring the presence and severity of adverse events, and monitoring compliance with study drug use. Information on specific vitamin D related adverse events will be obtained by laboratory findings, including 25OHD, ionized calcium and urine calcium:creatinine levels.

Safety measures and clinically relevant research findings
A flow diagram depicts the safety measures in place and response to clinically relevant research findings for individual study participants (Figure 4B, appendix 4.6). A standard operating procedure has been developed for adherence to the following safety measures. To avoid vitamin D overdose and related toxicities we have selected a supplement level recently proven to be safe in healthy children and will target the period of high dose supplementation to 6 months, and no more than 12 months. Study participants with elevated blood calcium and/or vitamin D levels will be identified and contacted by the safety officer. For 25OHD, although 500 nmol/L is generally considered the definitive acute toxicity threshold, we have chosen to intervene with 25OHD levels above 200 nmol/L as this value is supraphysiological and exceeds our study goal. The following details the actions that will be taken with abnormal values:

  • For 25OHD above 200 nmol/L with evidence of hypercalcemia (vitamin D toxicity): discontinue study drug immediately, repeat the values (fasting), and refer to endocrinology.
  • For 25OHD above 200 nmol/L without hypercalcemia: study drug will be reduced by 50%
  • For 25OHD above 250 nmol/L, without hypercalcemia: study drug will be discontinued
  • For hypercalcemia with 25OHD under 200 nmol/L: repeat the bloodwork (fasting) and refer to endocrinology

Both post-operative hypo and hypercalcemia will be managed by the clinical team as required. Study participants with persistently elevated blood calcium levels (for more than two days, not explained by intravenous calcium administration) will be referred to endocrinology. As prolonged exposure to hypercalciuria (> 3 months) could theoretically cause nephrocalcinosis, we will have ultrasounds performed prior to hospital discharge on all patients with elevated immediate pre-operative urine calcium to creatinine ratios. Any study participant with nephrocalcinosis will be referred to the nephrology service for further assessment. Participants that have study drug discontinued or decreased will be retained in the study, have peri-operative biological samples collected as outlined, and will be included in the analysis using intention to treat methodology.

4.6 Discussion
Research by our group and others has documented not only that 4 out of every 5 CHD patients have inadequate blood levels of vitamin D following surgery, but an association between immediate post-operative hormone levels and clinical course (24, 26, 28). Altogether, these findings and similar results in adult critical care and CHD surgery populations, suggest that optimization of vitamin D status following CHD repair could lessen inflammation, reduce nosocomial infection and improve cardiac function (30-34, 38, 41, 51, 232). As an inexpensive medication (~$15/month) that is generally regarded as safe, vitamin D has the potential to be an ideal intervention for improving outcomes following CHD repair. As protocols and guidelines should be evidenced based, well-designed clinical trials are essential to adjust clinical practice. This trial aims to determine whether a preoperative proposed dosing strategy will be sufficient to elevate pre-operative 25OHD and prevent post-operative vitamin D deficiency. Further clinical study will be required to investigate how normalization of vitamin D levels impacts the clinical course of patients with CHD requiring cardiac surgery.
Trial Status
At the time of writing 62% of the target sample size had been enrolled and the anticipated study completion date is January 2016.

Chapter 5 - Progress to date with conduct of the dose evaluation RCT

The primary objective of this study is to determine whether pre-operative supplementation with daily high dose vitamin D approximating the Tolerable Upper Intake Level can prevent post-operative vitamin D deficiency in children with CHD. As 25OHD are not yet available for the patients (and group assignments are unknown) we will instead describe our experiences with study drug intake, compliance and protocol violations. In addition we will described the other, equally important, feasibility objectives (i.e. recruitment, adverse events, collection of research samples).

Study initiation and time period for thesis
The thesis proposal was submitted in 01-Aug-2012. The Graduate Studies Committee requested minor modifications (15-Oct-2012) which were resubmitted on 23-Nov-2012. Official approval for the thesis was received from the Graduate Studies Committee on 11- Jan-2013. CHEO Research Ethics Board and Health Canada applications were submitted on 12-Dec- 2012 and 18-Dec- 2012, respectively. The ‘No Objection letter’ was received from Health Canada on 24-Jan-2013 (Supplemental Information, appendix S5.1). Notice was received of CHEO REB approval on 25-Mar-2013 and annual renewal has been requested and granted twice, most recently on 04-Mar-2015 (Supplemental Information, appendix S5.2). Following these approvals, study drug was requested from Europharm and was received by the CHEO pharmacy on 09-May-2013. The start-up DSMB meeting was held on 17-May-2013 where the terms of reference (Supplemental Information, appendix S5.3-S5.6) and reporting documents (Supplemental Information, appendix S5.7-8) were reviewed and approved. The only significant adjustment to the document was the addition of post-operative 25OHD levels included to the interim analysis. With all study procedures and approvals in place the Cardiology and Cardiovascular service were approached about initiating screening and recruitment. Due to significant recent turn over in administrative and nursing staff they requested that active screening for study participants begin in September 2013 after clinical training was complete.

The date of 30-Apr-2015 was selected as the end time point to describe study results for the thesis as this was the date when the first 30 participants had completed all study related procedures and the DSMB report was to be prepared. The chair’s summary of the DSMB meeting on 16-Jun-2015 can be found in Supplemental Information (appendix 5S.9).

Study recruitment
Participant flow
Although official active study recruitment was planned for Sept-2013 the first study participant was recruited early on 22-July-2013. This family approached the cardiovascular team about the trial after reading a newspaper article describing the findings of our initial study (Supplemental Information, appendix S5.10-S5.11). From 01-Sep-2013 to 30-Apr- 2015 there were 19 months of active recruitment (excluding 2 weeks over each of the 2013 and 2014 Christmas seasons where research coordinators were unavailable).
A participant flow diagram is shown in Figure 5A (appendix 5.1). During the 19 months of active recruitment research staff were contacted about 86 children of whom 67 were determined to meet study eligibility criteria. Of the 67 eligible patients there were 18 instances where research staff did not discuss the study with the family and/or patient. For the 49 children where the study was presented in full by a member of the research staff, consent and/or assent was given by 35 participants. The consent rate was calculated as 52% (35/67) for all referred eligible patients, and 71% (35/49) for those approached by study staff.

Explanation of excluded patients, declined participation and withdrawals
The two most common reasons referred patients were not eligible was either that the patient had CHD but did not require surgery (n=15) or had a lesion type that necessitated more complex surgery at another centre (n=3).
For the instances where study participation was not discussed with eligible children and families, refusal by the most responsible physician to allow study participation (n=5) or family unwillingness to discuss research due to stress (n=3), lack of interest (n=2) or inability to make decision (n=1) accounted for half of the cases. In 6 cases, the study was not discussed with the family as the research staff was unavailable (n=2), there was insufficient time after referral for consent and initiation of study procedures (n=2) or study staff were unable to make contact with family (n=2). In one circumstance, the family agreed to discuss the study with research staff but became distressed at the beginning of the conversation stating that they were unaware of the need for surgery. Study procedures were reviewed and it was confirmed that the most responsible physician (cardiology) had informed study staff that the family was aware of the need for surgery.

The reason for refusal after presentation of full study protocol was available for 9 of 15 cases (Table 5A, Appendix 5.3). In three of the cases the family vocalized that the need for daily supplementation and compliance contributed to their decision not to participate. In one case the mother decided not to participate as she did not think the father would reliably give the medication and did not want to negatively impact the study. In two other instances the caregivers did not want the added responsibility of another medication to give or track.
One family requested almost immediate withdrawal after enrolment over concerns that study drug might be worsening gastroesophageal reflux. For the remaining three cases withdrawal from the study occurred when the surgical plan changed and the patients were referred to the Hospital Sick Children in Toronto for more complex procedures.

Concurrent with randomization the 35 patients were stratified into four groups by age and expected duration of study drug. At time of randomization, 24 (69%) were under one year of age and 7 (20%) were expected to receive study drug for more than two months at time of enrollment. Of the randomized patients, 4 were ultimately withdrawn and 1 has not yet had surgery.
Recruitment of 35 patients over a 19 month study period represents an average accrual rate of 1.84 patients per month. Figure 5B (appendix 5.2) compares actual to target recruitment rate. Actual and target recruitment rates were well matched over the first 6 months, with accrual declining to 1.6 patients per month over the final 12 months.

Study participant characteristics
Baseline patient information is presented in Table 5B (appendix 5.4). Our enrollment survey found that the vast majority (78%, n = 27) of families identified themselves as being Caucasian and that 40% (n=14) reported taking a vitamin D supplement. Addition of the patients who were formula fed increased the percentage receiving vitamin D supplementation to 71% (n=25). At time of enrollment, the median age of the population was 3.0 months, with 74% (n=26) being under 1 year of age. Three patients (9%) had confirmed genetic syndromes. Age was also determined using the day of surgery (median 5.5 months, IQR: 1.5, 37.4) with 23% (n=7) and 71% (n=26) having surgery before 1 and 12 months of age, respectively. The majority of the study participants were in RACHS category 2 (n=13) or 3 (n=11). The primary cardiac lesion is listed for the study participants in Table 5B (appendix 5.5)

Duration of study drug intake, compliance and protocol deviations
Duration of study and compliance
The median time between enrollment and surgery for the 30 study participants completing all study procedures was 27 days (IQR: 6, 56). The number of study participants who could have received study drug for more than 30 and 60 days between enrollment and surgery was 50% (n=15) and 26% (n=8), respectively. The study participant withdrawn by family did so on day 3, while the withdrawals due to change in surgical plan occurred on days 12, 78 and 208. When available, study drug intake was recalculated using information from pharmacy for inpatient participants (n=10) and study diary for outpatients (n=16). The recalculated median number of doses received was 21 days (IQR: 4, 40) with receipt of more than 30 and 60 doses occurring in 45% (n=14) and 16% (n=5), respectively. The median per dose compliance rate was 97% (IQR 72%, 100%). Further, 11 participants reported having missed more than 10% of the doses, with 9 of 30 missing more than 20%. Excluding the 10 inpatients where drug was not dispensed to family, study vials were returned by 13 of the remaining 20 participants who underwent surgery. There were no cases where the volume of study drug returned exceeded that reported in the diary by more than 2 doses.

Protocol deviations
Protocol deviations with regard to study drug administration occurred in 6 patients. (Table 5A, appendix 5.3). On three occasions pre-operatively the caregivers decided to modify or change the regimen in a manner that would have reduced the potential rise in vitamin D. Pre-operatively one other family decided to start an immune supplement with additional vitamin D (described in more detail in section 5.1.6). During the post-operative period two separate participants received a short course of study drug resulting in 6 extra doses (both cases). In one instance the family did not return the study drug as requested and continued to give it despite instructions, and in the second case a physician wrote an order to re-initiate daily study drug and it was provided by pharmacy without approval from study staff or their knowledge.

Research related testing and biological sample collection
Research blood was properly collected, processed and stored for 97% (n=29) of patients at time of PICU admission and 93% (n=28) on the first post-operative day. One of either the PICU admission or first post-operative day blood samples was available for 100% of the participants. The number of additional research related biological samples collected at the initiation, intra-operative and post-operative time points has been summarized in Figure 5C (appendix 5.6). With the exception of the optional collection of enrollment urine and blood samples, more than 90% of the required research samples were collected.

Safety procedures and adverse events
Due to the possibility of vitamin D related adverse events, blood and urine samples were collected at specific time points. Results are shown in Figure 5D (appendix 5.7).
Mid-treatment - Pre-operatively, three participants were identified as receiving study drug in excess of 6 months. For one of the three, the 25OHD concentration was reported at 199 nmol/L. An interview with the family determined that since enrollment they had started an over the counter immune supplement and had not declared it with any of the research related telephone calls or appointments. The label indicated it contained vitamin D and, if accurate, the patient was receiving an additional 400 IU per day. As the family wanted to both continue the immune supplement and stay in the study, the decision was made to unblind to increase confidence the appropriate study drug solution was being provided. This confirmed the participant was in the high dose arm and receiving the 1600 IU/mL formulation; the family was asked to stop taking vitamin D study drug (~2 weeks) to allow the 25OHD level to decline slightly and then reinitiate using the provided 1200 IU/mL solution.

Pre-surgical samples - As per protocol, ionized calcium and 25OHD concentrations were determined on 20 participants (100% of required cases). None of these patients had elevated 25OHD concentrations or met the study definition of hypercalcemia.

Intra-operative and Post-operative samples -
Intraoperative blood and urine samples were collected on all 30 study participants. No participant was documented to have hypercalcemia, while 7 had elevated urine calcium to creatinine ratios. As per protocol, 6 of the 7 participants had a kidney ultrasound and no cases of nephrocalcinosis were documented. The one abnormal urine value that did not have a kidney ultrasound was not received by the safety officer until after hospital discharge. The patient had no clinical evidence of kidney dysfunction or urine abnormalities with their postoperative blood or urine samples; upon review with nephrology it was considered unnecessary to arrange for an outpatient ultrasound.

A post-operative urine sample was collected on 93% (n=28) of participants. Both of the participants with missing samples had surgery late in the week and due to an unremarkable clinical course were discharged from hospital the beginning of the following week; as they do not live in the Ottawa area we have been unable to arrange follow-up to obtain a post-operative urine sample. Five patients met criteria for post-operative hypercalciuria and had their findings (blood work and intra-operative urine measurements) discussed with a nephrologist who requested repeat urine measurements in all cases. Four of the patients had a repeat urine collection, while the fifth patient refused to provide a repeat urine sample prior to hospital discharge (will be attempted at a future clinic appointment). Three of the four repeat samples showed resolution of hypercalcuuria and the wrong analysis was performed by the laboratory on the fourth (urine albumin to creatinine ratio); attempts will be made to collect urine at future clinic appointments.

Study modifications
Table 5D (appendix 5.8) describes the protocol changes or initiatives and intended purpose(s). A number of specific changes were intended to improve accrual rate, lengthen time from enrollment to surgery (to increase cumulative study drug intake), or increase patient safety. Two major efforts were made to increase recruitment rate. First, it was recognized that approximately 10-15% of CHD surgeries at CHEO are from patients who reside in Kingston. As these patients are followed by cardiology in Kingston and are presented by videoconference they are generally only seen once by the CHEO team approximately 2 weeks prior to surgery. Our research team liaised with the pediatric cardiologist in Kingston and obtained REB approval to recruit patients; unfortunately the Kingston clinic did not recruit any patient from their clinic over a 12 month period. Second, we attempted to initiate recruitment at a second site. One of the investigators (Dr. Dermot Doherty) on the observational study of vitamin D in CHD (26) moved to Ireland (Dublin) and works in an ICU that provides post-operative care to children following CHD surgery.

Dr. Doherty was a co-investigator on the Heart and Stroke Grant and was interested in having Ireland as a second site for the study. Despite evidence of high vitamin D deficiency rates in their PICU (246) and agreement from the PICU and endocrinology services we were unable to convince the cardiovascular surgery team to enroll patients under 1 year of age. As the majority of CHD patients are under 1 year of age we decided not to proceed. In addition there were multiple protocol modifications made at CHEO intended to allow for either earlier access to families or easier follow-up. In addition to increasing accrual rate these efforts were designed to increase the duration of time on study drug prior to surgery. Analysis of whether there had been an increase in duration of time from enrollment to surgery suggested minimal to no change for the more recent study participants (Figure 5E, appendix 5.9). For example, the average mean period of time between enrollment and surgery increased from 37 (SD 29) to 48 (SD 64) days, while the percentage of study participants who received study drug for more than 30 days decreased from 53% (8/15) to 47% (7/15).

5.2. Discussion
This section discusses our experience with the set-up and initiation of a phase II dose evaluation double blind randomized controlled trial comparing pre-operative daily high dose
vitamin D supplementation to usual care in children with surgical CHD requiring cardiopulmonary bypass. The primary objective is to determine whether the high dose regimen can significantly reduce the prevalence of post-operative vitamin D deficiency. As 25OHD concentrations have not yet been determined from stored biological samples it is not possible to comment on the extent to which post-operative vitamin D status differs between groups. Instead we will focus the discussion on feasibility issues(study set-up, patient recruitment, study drug intake, safety procedures) and vitamin D related adverse events.

Study set-up and recruitment
The thesis objectives of designing, obtaining approvals for, and initiating a Health Canada regulated clinical trial was successfully achieved. Once regulatory approvals were in place the pilot study also demonstrated that the families of children with surgical CHD were willing to participate in a RCT of high dose vitamin D. Although below what we have reported for observational studies of vitamin D in ill children (24, 26, 247), the 70% consent rate is very similar to what has been reported for other studies evaluating hormones in surgical CHD(248, 249). For example, in recent placebo controlled trial of insulin for the management of post-operative hyperglycemia Agus and colleagues reported a 69% consent rate(248). Similarly, in their RCT investigating the benefits of post-operative thyroid supplementation, Portman and colleagues reported that 68% of those screened and eligible agreed to participate(249). Although the consent rate for approached patients was encouraging and consistent with recent literature, the study accrual rate was below that anticipated at the beginning of the study (1.8 vs. 2.5 per month per site). While below our target, the per month accrual rate is similar to what has been reported in the aforementioned multicenter studies of thyroid and insulin(248, 249). Our expectation of a higher consent and accrual rate was based on widespread public interest in vitamin D and the fact that most children would already be receiving some form of supplementation. Evaluation of the participant flow diagram identified two main reasons the accrual rate was below target. First, and most importantly, instead of the estimated 100 eligible patient referrals for the 19 month period the study team received only 67. Discussion with our cardiovascular surgery team determined that there has been a decline in the number of children having surgery for CHD at CHEO over the past few years. Second, was our observation that the research team was unable to discuss study participation in 26.9% (n=18/67) of eligible cases. Patient and/or parent refusal to discuss research is well known, but occurred in only 9% (n=6). Instead, physician refusal and inappropriately timed referrals (i.e. late) represented the most common reasons an eligible patients was not approached about study participation. Some of these barriers may be modifiable, and as our institution gains further training and experience with CHD research on high dose vitamin D, the proportion of eligible patients who do not have study participation discussed may decrease.

Finally, we documented that 4 of the 35 patients enrolled were ultimately withdrawn from the study. For three of the cases, the withdrawal occurred because the surgical plan changed and the child was to receive their operation in Toronto. Although a problem for our single center study, this change in the care plan may not negatively impact a large Canadian multicentre study if data and biological samples were collected at all sites.

Sample collection and performance of safety procedures
Primary objective -
In addition to recruitment, the one study procedure most important to the success of the study is the ability to collect biological samples for determination of post-operative vitamin D status. As anticipated based on the CHD observational study(26), immediate postoperative blood was collected and properly processed for 96.6% of study participants. For the single patient without a PICU admission sample, blood was intentionally not collected as the patient had a prolonged period of resuscitation immediately following return to the PICU.
For this patient we will use their post-operative day 1 sample as the preceding observational study did not indicate a significant difference between 25OHD levels at PICU admission and post-operative day 1(26). Finally, although it will not be possible to comment on postoperative vitamin D status for all withdrawn patients we did collect a mid-treatment sample from one participant prior to withdrawal; using this sample we will be able to comment on steady state 25OHD levels after approximately 6 months of study drug intake.

Safety procedures and adverse events -
As part of this trial we were able to design, set-up, and implement a “real time” safety protocol intended to evaluate for, document and minimize adverse events related to elevated vitamin D. It is well accepted that vitamin D toxicity is related to elevated 25OHD levels and that months of daily intake at or above the daily Tolerable Upper Intake Level would be required before levels rose sufficiently to increase risk of toxicity (11, 91, 250, 251). Given expected variability in CHD lesion type and duration of study participation prior to surgery we designed a safety protocol personalized to patient duration of study drug intake.
Regardless of patient type our safety protocol and adverse event analysis included intra-operative (pre-surgery) and immediate post-operative evaluation for hypercalcemia and hypercalciuria. Intra-operative and post-operative blood calcium levels were available for 100% of study participants, and no cases of hypercalcemia occurred. The absence of perioperative hypercalcemia matches what was reported in our observational study of CHD and is consistent with the 2.6% rate calculated for daily dosing regimens in our systematic review (26, 231). Similarly, evaluation for peri-operative hypercalciuria was equally successful with 100% and 93% of participants having intraoperative and post-operative urine samples sent for calcium:creatinine ratio, respectively. Altogether 40% of study participants had hypercalciuria on either their intra-operative or post-operative sample. This hypercalciuria rate contrasts significantly with the pooled 2.5% rate observed in the systematic review(231). Although different from what is observed in other pediatric populations, this high rate of peri-operative hypercalciuria was not unexpected and was one of the primary reasons the dose evaluation study was performed as a controlled trial. In deciding upon the study design we speculated that due to the abnormal physiology, diuretic administration, and kidney dysfunction an unknown, but potentially significant, proportion could have hypercalciuria. Having a control arm is essential to demonstrating whether hypercalciuria was related to CHD surgery or vitamin D. The closest data for comparison comes from the recently completed RCT by Amrein and colleagues, where they compared hypercalciuria rates in critically ill adults (n=475) who received placebo or a 540000 IU cholecalciferol load(122). This well powered study demonstrated no difference between the two groups, with hypercalciuria occurring in 25% of all patients at each time points(122). Once enrolment is complete and all participants have completed study procedures it will be important to evaluate for potential differences in the occurrence of hypercalciuria between study arms (and vitamin D level). In June 2015, the DSMB did compare hypercalciuria and serious adverse event rates by study arm and reported back no safety concerns or reasons to discontinue the study. Regardless of whether there are differences in peri-operative hypercalcuria rates it is important to emphasize that no participant with elevated intraoperative urine calcium levels had nephrocalcinosis and that all isolated episodes of post-operative hypercalcuira appeared insignificant and/or transient in nature. The lack of nephrocalcinosis with high dose vitamin D is consistent with the absence of any reported episodes in the clinical trials identified as part of the systematic review(231). Further, our review of nephrocalcinosis case series and cohort literature suggested that those cases associated with high dose vitamin D involved either massive doses (>600000 IU) or children with genetic abnormalities of their vitamin D axis (99-103).

Study drug intake and projected25OHD levels
Despite the lack of analysis and presentation of 25OHD results, important information is available from data collected on time from enrolment to surgery and cumulative study drug intake. Our study goal was to raise vitamin D levels pre-operatively to above 90 nmol/L, such that they would remain above 50 nmol/L after the 40% intraoperative decline. This idea was supported from the results of two recent well done clinical trials demonstrated that daily dosing in the 1200 to 1600 IU range achieved group means between 100 and 150 nmol/L in healthy infants(19, 81). Further supporting the dose selection was a more recent publication by Lewis and colleagues on a group of healthy school age children(252). Given a baseline 25OHD concentration of 70 nmol/L the group of children receiving 2000 and 4000 nmol/L increased their 25OHD levels by approximately 40 and 80 nmol/L, respectively. Importantly, these studies clearly demonstrate that change is time dependent with at least 2 months required to achieve new steady state (19, 81, 252).
Based on previous observations that the majority of CHD surgeries occur at or after 3 months of age we projected that ~75% of the study participants could receive 2 or more months of daily supplementation prior to surgery(26). Instead, the average (median) number of doses prior to surgery was only 27 days, with just 25% achieving two months of intake. The negative impact this will have on pre-operative vitamin D status can be estimated from the three RCTs and our systematic review of all high dose pediatric trials(19, 81, 231, 252). Based on the evidence from the three aforementioned clinical trials, dosing for 20 to 30 days may only be sufficient to raise levels by 20 to 30 nmol/L. However, this may be an overestimate of 25OHD response given our systematic review observation that disease children have a blunted response (per dose) when compared to healthy children (231). Inserting the study data into the equation generated from our systematic review and metaanalysis (baseline 25OHD of 60 nmol/L; cumulative dose of 40000 IU) returns a group average pre-operative 25OHD concentration of 70 nmol/L and standard deviation of 35 nmol/L.
Based on the short duration of study drug intake we expect the 25OHD levels to show that the majority of study participants do not raise pre-operative levels enough to prevent post-operative vitamin D deficiency. Although we did foresee that pre-operative daily high dose supplementation would not be effective in certain surgical CHD subgroups, the results of our pilot study suggest that the subgroup may represent the majority instead of the minority.

Protocol limitations, deviations and modifications
In addition to being unable to obtained adequate cumulative drug intake prior to surgery our study demonstrated that attempts to utilize daily supplementation for a trial on CHD patients presents other scientific, economic and potential patient safety issues.
First, in contrast to the success with collection of immediate post-operative biological sample, baseline blood and urine were collected for only 43% of study participants in whom it was desired. During study design, the risk and benefits of requiring a baseline 25OHD and urine calcium were debated by the investigators, REB and DSMB committee. Although all agreed that this information would be beneficial it was ultimately decided to make these investigations optional instead of mandatory. The single most important factor that played into this decision was consistent feedback during our previous studies that parents would decline participation if additional venipuncture for blood work was required (26, 247). These concerns are validated in a recent study by Menon and Ward where additional blood work was one of the most common reasons given for declining participation (253). Although it would have been an option to make baseline 25OHD mandatory and wait until the next set of clinically indicated blood work for initiation of study drug this would have further reduced cumulative dosing. Ultimately, the lack of baseline 25OHD data may end up as a significant study limitation.
Second, although we were able to recruit at an accrual rate similar to other surgical CHD trials involving hormones there were significant challenges not immediately evident through inspection of the flow diagram(248, 249). Unlike most other trials of surgical CHD where the intervention is given intra-operatively or during PICU admission, our intervention was given pre-operatively and requires early initiation to achieve the greatest chance of success. Shortly following the initiation of recruitment it became apparent that most patients were not being referred early enough and that most patients would not consent at the time of the first research encounter. Armed with the knowledge that the duration of time between enrolment and surgery matters, significant efforts were made to lengthen the time from recruitment to surgery. These efforts included REB approval for study staff to speak with potentially eligible patients prior to appointments, follow-up up with patients by telephone or home visit after appointments, and delivery of study drug by mail. Interestingly, although the study coordinators were of the impression that these changes were of value, no improvement in accrual rate or average duration of drug intake was shown. Importantly, the significant coordinator effort required to identify and repeatedly meet with families has resulted in coordinator time expenditure well in excess of the 2 hours budgeted for the study.

Third, we observed issues surrounding the administration of study drug to participants(254, 255). Included among the issues were anticipated problems with study drug compliance. Although the majority of caregivers gave the medication as instructed, approximately 30% gave less than 80% of the ordered doses.. Understanding and neutralizing poor compliance is important as it can significantly impair trial power, regardless of whether the outcome is biochemical or clinical (256, 257). In addition to problems with reduced study drug intake we also documented problems with protocol deviations by both families and physicians that resulted in increased vitamin D intake. Our observations also support the consideration of an alternative dosing regimen that is less susceptible to deviation and modification by families and health care staff.

Chapter 6: Conclusions

Summary of literature and update
Recent studies have demonstrated that many children with CHD have post-operative vitamin D deficiency and that these lower levels may place them at risk for greater postoperative morbidity (24, 26-28). The limited work in this area is supported by a recent systematic review, wherein the authors report a statistically significant association between vitamin D status and at least one post-operative outcome in 26 of 31 surgical studies (258). Although undesirable, the high prevalence of vitamin D deficiency also presents an opportunity as optimization could represent a simple and inexpensive means to improve short and/or long term outcomes. The idea that vitamin D deficiency could be relevant to critically ill patients received a significant boost recently with the publication of the VITdAL-ICU study suggesting a clinically meaningful but statistically insignificant absolute mortality reduction of 7% (p=0.10) with rapid normalization of vitamin D status in critical ill adults(122). As literature in other populations emerges, physicians and health care workers providing care for critically ill children, including those with CHD, are beginning to worry about vitamin D deficiency and ask whether and how they can optimize status(259). As a novel area of research, work has been limited and our systematic review confirmed that absence of clinical trials in the CHD or general PICU populations(231). The body of work completed for this thesis represents the next steps to address the knowledge gaps surrounding how to prevent or treat vitamin D deficiency in surgical CHD patients.
Summary of thesis work and findings
The primary project for the thesis was the design, set-up and initiation of a pilot dose evaluation RCT to determine whether pre-operative supplementation with daily high dose vitamin D, when compared with usual care, significantly reduces the rate of post-operative deficiency. Although there were other dosing regimens to consider, it was decided to begin clinical trial work in this area with a daily dosing regimen as it had the potential to be effective in approximately 75% of the population, is the only method of vitamin D supplementation recommended by the Canadian Pediatric Society, and is the only dosing regimen approved by Health Canada. The systematic review confirmed our suspicion that daily supplementation is the favoured regimen, as only one trial evaluating intermittent loading dose therapy has been performed and published (twice) in North America(135, 136). The thesis reports on our experience after the first 30 study participants completed all surgical procedures. Although vitamin D levels were not available for discussion there were important and relevant results available on study drug intake, safety and feasibility outcomes.

The majority of our initial observations related to the design, set-up, initiation and recruitment into the RCT were positive. First, the high consent rate suggests that patients and families are concerned about post-operative vitamin D deficiency and see value in a study of high dose vitamin D. Second, comparison with the CHD literature demonstrates that we could recruit study participants at a rate similar to other pediatric trials of hormone therapy (248, 249). Third, we confirmed our ability to collect biological samples for measurement of post-operative 25OHD and vitamin D related adverse events. Fourth, the safety procedures and adverse event analysis proved valuable. Especially meaningful was the finding that 40% of study participants had at least one episode of peri-operative hypercalciuria analysis. This rate is considerably higher than the 2.5% rate calculated as part of the systematic review and meta-analysis for healthy and less sick pediatric populations (231), and affirms our original decision to include a control arm in the dose evaluation study. Without the usual care arm it would not be possible to appraise whether the high hypercalciuria rate was related to critical illness or high dose vitamin D.
Although there were many positives observed with set-up, initiation, recruitment and sample collection many of other findings raise considerable concern that an intervention based on the pre-operative daily administration of the IOM Tolerable Upper Intake Level may not be the best choice for an eventual phase III trial. The primary concern was that the majority of participants could not be enrolled with adequate time to administer the desired cumulative dose of vitamin D. Only 25% of participants (instead of 75%) achieved the desired 2 month cumulative intake of study drug. Additional concerns about the preoperative daily high dosing regimen included the significant coordinator time required for early identification and recruitment, the inability to routinely measure baseline 25OHD status, and multiple instances of family or health care provider deviation from study protocol. These problems emphasize the need to explore alternative dosing strategies as the current strategy not only impacts the scientific integrity of the work but may place the patients at risk.
Prior to the initiation the dose evaluation RCT we recognized a subgroup of CHD patients (neonates) who were unlikely to adequately elevate 25OHD levels with the daily high dose regimen. Originally we believed that this group would be the minority (<25%) and would largely be made of those neonates and young infants presenting with lesions that require surgery within a few weeks of birth (or presentation). Our systematic review confirmed our concerns and demonstrated that high dose daily supplementation may not be adequate for the CHD patient presenting within a month of surgery(231). Fortunately, the systematic review did convincingly establish that it is possible to raise 25OHD levels within 48 hours with the administration of a single large enteral dose of cholecalciferol. Importantly, the adverse event analysis performed as part of the systematic review did show a greater risk for hypercalcemia with high dose regimens exceeding the Tolerable Upper Intake Level. Importantly, a detailed analysis revealed that this increased risk only occurred in the setting young children who received doses equal to or exceeding 400000 IU and achieved group mean 25OHD levels exceeding 200 nmol/L.
Proposed next steps
In conclusion, the preliminary findings suggest that it is possible to set-up and recruit into a study of high dose vitamin D in CHD but that this regimen may not achieve our goal of mitigating post-operative vitamin D deficiency. Therefore, prior to proceeding with a phase III RCT it will be important to consider an alternative dosing regimen. Based on available data we anticipate the need for a second dose evaluation RCT investigating loading dose cholecalciferol immediately prior to surgery. For this study, inpatients would receive their dose sometime in the week leading up to surgery, while outpatients would receive their dose at the pre-surgical appointment. Timing the dose with the pre-surgical visit would allow for collection of baseline research blood. Further using the knowledge generated from our systematic review it would be possible to personalize each participants dose to account for weight/age and baseline 25OHD level. The recent development and marketing of point-of- care devices (e.g. Qualigen, Nanospeed) that can assess 25OHD levels within 10-15 minutes would further make it possible to measure and respond to vitamin D status within the same patient encounter. This approach should reduce research coordinator time associated with aggressive patient recruitment and follow-up, improve study drug compliance, reduce the potential for patient and caregiver protocol deviations, reduce the variability observed with the implementation of the intervention, allow for the routine collection of baseline blood, and lead to a more consistent 25OHD response with better separation of the study arms.


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Figures and tables - only 3 are on web page, scores more in PDF

Figure 2A. Overview of vitamin D parathyroid renal axis—Functioning of the axis is demonstrated in the context of calcium homeostasis.
Table 2A. Findings reported within the 4 published PICU observational studies that included CHD patients.
Figure 2A: Flowchart of study selection based on inclusion and exclusion criteria.
Table 3A. Patient, dosing and study characteristics of high dose study arms.

Figure 3B: Short term 25(OH)D response to high dose daily vitamin D intake.
Figure 3C: Short term 25(OH)D response to vitamin D loading therapy.
Table 3C: Single-Variable Meta-regression of Post-Study Drug 25(OH)D.
Table 3D: Multivariate Meta-Regression Predicting Post-Study drug 25(OH)D.
Figure 3D: Forest plot of hypercalcemia rates by dosing regimen.

Table 4B: Vitamin D supplementation strategy.
Table 4C: Safety thresholds for ionized calcium, total corrected calcium and elevated calcium-creatinine ratio.
Figure 4A. Study related procedures and measurements
Table 4D: Biochemical measurements on research specimens.
Figure 4B. Flow diagram of study safety measures
Figure 5A. Participant Flow Diagram (CONSORT)
Table 5B: Study participant characteristics at baseline.
Table 5C: List of participant cardiac lesions by RACHS category.
Figure 5D: Flow diagram of safety procedures and adverse events

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