Clinical trial NCT03096314
Conditions: Acute Respiratory Distress Syndrome Vitamin D Deficiency Critical Illness
Keywords: ARDS Vitamin D
Sponsor: Massachusetts General Hospital
Start date: 2017-04-27; Completion date: 2018-12-11
Enrollment: 1358 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Vitamin D deficiency is a common, potentially reversible contributor to morbidity and mortality among critically ill patients. We conducted a randomized, double-blind, placebo-controlled, phase 3 trial of early vitamin D3 supplementation in critically ill, vitamin D-deficient patients who were at high risk for death. Patients screened as vitamin D deficient (\<20 ng/mL) were randomized. Randomization occurred within 12 hours after the decision to admit the patient to an intensive care unit. Eligible patients received a single enteral dose of 540,000 IU of vitamin D3 or matched placebo. The primary end point was 90-day all-cause, all-location mortality.

Detailed description:
Primary Objective: To assess the efficacy and safety of early administration of vitamin D3 (cholecalciferol) in reducing mortality and morbidity for vitamin D deficient patients at high risk for Acute Respiratory Distress Syndrome (ARDS) and mortality.

Primary Hypothesis: Early administration of vitamin D3 (cholecalciferol) will improve all-cause, all-location mortality to day 90 in vitamin D deficient patients at high risk for ARDS and mortality.

Methods: Patients were recruited from the emergency departments (EDs), hospital wards, operating rooms, intensive care unites (ICUs) and other acute care areas of the participating PETAL Network Clinical Centers. Screening included a test for Vitamin D (25OHD) levels using either the hospital's clinical laboratory or an FDA-approved point-of-care device (FastPack IP, Qualigen Inc). Patients screened as vitamin D deficient (\<20 ng/mL) were randomized. Half of the randomized patients received an early administration of high-dose vitamin D3 and the other half received a placebo. Both active and placebo products were given orally or via naso/orogastric tube.

Rational: Vitamin D has pleiotropic roles in regulating immune function and maintaining epithelial surface integrity. Strong preclinical data support the protective role of vitamin D in regulating pulmonary inflammation and disruption of the alveolar-capillary membrane that are fundamental to ARDS pathogenesis.

Arms: arm 1: A single dose of 540,000 IU vitamin D3 will be administered within 2 hours of randomization time. arm 2: A single, liquid enteral placebo dose administered either orally or via naso/orogastric tube will be administered within 2 hours of randomization time.
Interventions: intervention 1: Vitamin D3 intervention 2: Placebo
Intervention descriptions: intervention 1: 540,000 IU vitamin D3 delivered as a single, liquid enteral dose administered either orally or via naso/orogastric tube intervention 2: A single, liquid enteral dose identical in appearance and consistency to cholecalciferol administered either orally or via naso/orogastric tube.

Protocol documents:
Prot_000.pdf: 
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VIOLET: Vitamin D to Improve Outcomes by Leveraging 
Early Treatment 
 
Protocol Version 2.1 (12 September 2017) 
 
 
Protocol Co-Chairs:  
Adit Ginde, MD, MPH 
Professor of Emergency Medicine 
University of Colorado School of Medicine  
 
 
 
Daniel Talmor MD, MPH 
 
 
Professor of Anesthesia 
 
 
Division of Critical Care 
 
 
Beth Israel Deaconess Medical Center Boston 
 
 
  
 
 
 
 
 
 
_________________________           _____________ 
SITE INVESTIGATOR SIGNATURE     
        DATE 
 
 


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Table of Contents 
1 
ABBREVIATIONS AND DEFINITIONS ...................................................................................................... 5 
2 
TRIAL SUMMARY ................................................................................................................................... 9 
2.1 
Title ............................................................................................................................................... 9 
2.2 
Objective ....................................................................................................................................... 9 
2.3 
Hypothesis ..................................................................................................................................... 9 
2.4 
Study Design.................................................................................................................................. 9 
2.5 
Inclusion Criteria ......................................................................................................................... 10 
2.6 
Exclusion Criteria ......................................................................................................................... 11 
2.7 
Randomization and Study Initiation Time Window .................................................................... 11 
2.8 
Primary endpoint ........................................................................................................................ 11 
2.9 
Secondary endpoints .................................................................................................................. 11 
2.10 
Sample Size/ Interim Monitoring ................................................................................................ 12 
3 
TRIAL DESCRIPTION ............................................................................................................................. 13 
3.1 
Background ................................................................................................................................. 13 
3.2 
Vitamin D-Potential Benefits and Mechanisms .......................................................................... 13 
3.3 
Recent Clinical Trials ................................................................................................................... 14 
3.4 
Summary of Rationale ................................................................................................................. 15 
3.5 
Objectives.................................................................................................................................... 16 
3.5.1 
Primary Objective ................................................................................................................ 16 
3.5.2 
Primary Hypothesis ............................................................................................................. 16 
3.6 
Endpoints .................................................................................................................................... 16 
3.6.1 
Primary Endpoint ................................................................................................................ 16 
3.6.2 
Secondary Endpoints .......................................................................................................... 16 
3.7 
Subgroups ................................................................................................................................... 19 
4 
STUDY POPULATION AND ENROLLMENT ............................................................................................ 19 
4.1 
Number/Source/Screening ......................................................................................................... 19 
4.2 
Inclusion Criteria ......................................................................................................................... 20 
4.2.1 
Intention to Admit to ICU .................................................................................................... 21 
4.2.2 
Pulmonary risk factors ........................................................................................................ 21 
4.2.3 
Extra-pulmonary risk factors ............................................................................................... 22 
4.2.4 
Screening Vitamin D Test .................................................................................................... 22 
4.3 
Exclusion Criteria ......................................................................................................................... 23 


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4.3.1 
Reasons for Exclusions ........................................................................................................ 23 
4.4 
Randomization and Study Initiation Time Window .................................................................... 24 
4.5 
Informed Consent ....................................................................................................................... 24 
4.6 
Randomization ............................................................................................................................ 24 
4.7 
Minorities and Women ............................................................................................................... 24 
5 
STUDY PROCEDURES ........................................................................................................................... 25 
5.1 
Intervention Group (Vitamin D) .................................................................................................. 25 
5.1.1 
Stopping Rules for Vitamin D Administration ..................................................................... 25 
5.2 
Randomized Control Group ........................................................................................................ 25 
5.3 
Common Strategies for Both Groups .......................................................................................... 25 
6 
DATA VARIABLES AND SPECIMENS ..................................................................................................... 26 
6.1 
Background Assessments ............................................................................................................ 26 
6.2 
Baseline Assessments ................................................................................................................. 26 
6.3 
Assessment during Study ............................................................................................................ 26 
6.3.1 Specimen Collection ....................................................................................................................... 27 
All randomized subjects ....................................................................................................................... 27 
6.4 
Assessments after Hospitalization .............................................................................................. 28 
7 
STATISTICAL CONSIDERATIONS ........................................................................................................... 28 
7.1 
Statistical Methods Primary Analyses ......................................................................................... 28 
7.2 
Statistical Methods for the Screened Vitamin D Deficient Cohort ............................................. 30 
8 
DATA COLLECTION AND SITE MONITORING ....................................................................................... 30 
8.1 
Data Collection ............................................................................................................................ 30 
8.2 
Site Monitoring ........................................................................................................................... 30 
8.3 
Vitamin D Screening Quality Assurance ...................................................................................... 30 
9 
RISK ASSESSMENT ............................................................................................................................... 31 
9.1 
Potential Risks to Subjects .......................................................................................................... 31 
9.2 
Minimization of risk .................................................................................................................... 32 
9.3 
Potential Benefits ........................................................................................................................ 32 
9.4 
Risks in Relation to anticipated benefit ...................................................................................... 32 
9.4.1 
Procedures .......................................................................................................................... 32 
9.4.2 
Intervention ........................................................................................................................ 33 
9.5 
Safety Monitoring ....................................................................................................................... 33 
10 
HUMAN SUBJECTS ........................................................................................................................... 33 


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10.1 
Selection of subjects ................................................................................................................... 33 
10.2 
Justification of including vulnerable subjects ............................................................................. 33 
10.3 
Informed consent ........................................................................................................................ 34 
10.4 
Continuing consent ..................................................................................................................... 34 
10.5 
Withdrawal of consent ................................................................................................................ 34 
10.6 
Identification of legally authorized representatives ................................................................... 34 
10.7 
Justification of surrogate consent ............................................................................................... 35 
10.8 
Additional safeguards for vulnerable subjects ........................................................................... 35 
10.9 
Confidentiality ............................................................................................................................. 36 
11 
ADVERSE EVENTS ............................................................................................................................ 36 
11.1 
Safety Monitoring ....................................................................................................................... 36 
11.2 
Serious Adverse Events ............................................................................................................... 37 
APPENDICES ................................................................................................................................................ 38 
A. 
Time-Events Schedule ..................................................................................................................... 38 
C. 
Adverse Event Reporting and Unanticipated Problems ................................................................. 40 
D 
Data and Safety Monitoring Board ................................................................................................. 42 
E 
PETAL Network Steering Committee .............................................................................................. 42 
F 
SOFA Scoring System ...................................................................................................................... 43 
G 
De-identified data elements for screened, non-enrolled ............................................................... 44 
H 
References ...................................................................................................................................... 45 
 
 
 


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1 ABBREVIATIONS AND DEFINITIONS 
1,25OH2D = 1,25-dihydroxyvitamin D 
25OHD = 25-hydroxvitamin D 
ABG = arterial blood gases 
AE = Adverse Event 
APACHE = Acute Physiology and Chronic Health Evaluation 
ARDS = Acute Respiratory Distress Syndrome 
BiPAP = Bilevel Positive Airway Pressure 
BMI = Body Mass Index 
BUN = Blood Urea Nitrogen 
CCC = Clinical Coordinating Center 
CHF = congestive heart failure 
CPAP = Continuous Positive Airway Pressure 
CT = computerized tomography scan 
CXR = chest x-ray 
 
DNA = deoxyribonucleic acid 
DSMB = Data Safety Monitoring Board 
ED = Emergency Department 
FACTT = Fluid and Catheter Treatment Trial 
FDA = Food and Drug Administration 
FiO2 = Fraction of Inspired Oxygen 
GCS = Glasgow Coma Scale 
IBW = Ideal Body Weight 
ICU = Intensive Care Unit 
ID = Identification 
IFN-γ = interferon-γ 
IL-1β = Interleukin 1β 
IL-2 = interleukin-2 
IL-6 = Interleukin 6 
IL-8 = Interleukin 8 
IL-17 = interleukin-17 
IND = Investigational New Drug 
IRB = Institutional Review Board 
IU = International Unit 
INR = International Normalized Ratio 
ITT = Intent to Treat 
LC/MS/MS = Liquid Chromatography-Tandem Mass Spectrometry (gold standard Vitamin D 
measurement) 
LAR=Legally Authorized Representative 
LIPS = Lung Injury Prediction Score 
LTAC = Long Term Acute Care Facility 
LTFU = Lost to Followup 
MAP = Mean arterial pressure 
mBW = Measured Body Weight 
mRNA = messenger ribonucleic acid 


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NBAC = National Bioethics Advisory Committee 
NDI = National Death Index 
NHLBI = National Heart Lung and Blood Institute 
NIH = National Institutes of Health 
NNT = Number Needed to Treat 
OHRP = Office of Human Research Protections 
OR = Operating Room 
PBW = predicted body weight 
PETAL = Prevention and Early Treatment of Acute Lung Injury  
P/F = PaO2/FiO2 ratio 
PaCO2 = Partial pressure of arterial carbon dioxide 
PaO2 = Partial pressure of arterial oxygen 
PB = Barometric Pressure 
PBW = Predicted Body Weight 
PEEP = Positive End-Expiratory Pressure 
PI = Principal Investigator 
PSV = Pressure Support Ventilation 
SACE = Survival Average Causal Effect  
S/F = SpO2/FiO2 ratio 
SOFA = Sequential Organ Failure Assessment 
SBP = Systolic Blood Pressure 
SBT = Spontaneous Breathing Trial 
SNF = skilled nursing facility 
SpO2 = Oxygen Saturation via pulse oximetry 
SSN = Social Security Number 
SUSAR = Serious and Unexpected Suspected Adverse Reactions 
SAEs = Adverse events that are serious and unexpected and have a reasonable possibility that 
the event was due to a study procedure  
TNF-α = tumor necrosis factor-α 
VDR = vitamin D receptor 
VFD = Ventilator-free days 
WBC = White Blood Cell 
 
Adverse Event: Any untoward medical occurrence associated with the use of a drug or a study 
procedure, whether or not considered drug related. 
 
Adverse reaction: An adverse reaction means any adverse event caused by a drug. An 
adverse reaction is a subset of all suspected adverse reactions where there is a reason to 
conclude that the drug caused the event. 
All-cause, all-location mortality: Primary outcome to be assessed by phone call at 90 days for 
patients discharged alive from the hospital 
Assisted breathing: Any level of ventilatory support at pressures higher than the unassisted 
breathing thresholds (defined below). Completing 48 hours of UAB is defined as the date 
(calendar day) that the subject reaches exactly 48 hours of UAB. Example: if subject meets 


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UAB at 1900 on 6/1/15 and does not return to assisted breathing, then the date of completing 
48 hours of UAB would be 6/3/15. 
Controlled Ventilation: Any mode with a backup rate that allows clinicians to either set tidal 
volume to a target or adjust pressures to target a tidal volume. Examples include volume assist 
control, pressure assist control, pressure regulated volume control. 
Eligible patient: All patients ≥ 18 years old with an intention to admit to ICU from emergency 
department, hospital ward, operating room (except uncomplicated, elective post-operative 
admissions to ICU for routine monitoring), or outside facility with one or more acute risk 
factors for ARDS or mortality and no exclusion criteria. The time of eligibility will be based on 
documented time of intention to admit to ICU (see below). 
Enrolled participants: All eligible patients who have completed the consenting process and 
are eligible to receive the vitamin D screening test 
Extubation: Removal of an orotracheal, nasotracheal tube, or unassisted breathing with a 
tracheostomy 
Home: Level of residence or health care facility where the patient was residing prior to hospital 
admission. 
Intention to Admit to Intensive Care Unit (ICU): Documentation of plan to admit to ICU from 
emergency department (ED), hospital ward, operating room, or referring hospital that sets time 
zero for the 12-hour enrollment window. 
Intention to Treat (ITT): All eligible and consented patients who undergo randomization will be 
included in the ITT cohort for the purposes of analyzing the primary and secondary study 
outcomes. 
Invasive Mechanical Ventilation: Assisted ventilation delivered by a nasotracheal, orotracheal, 
or tracheostomy tube 
Legal Representative: An individual, judicial, or other body authorized under applicable law to 
consent on behalf of a prospective patient to the patient's participation in the clinical study. 
Funding: National Institutes of Health (National Heart Lung and Blood Institute) 
Nonrandomized participants: The subset of eligible and enrolled patients with screening 
25OHD levels 20 ng/mL or greater. 
Screen/Failure: Patients who meeting eligibility criteria 1-3 AND one or more exclusions.  
Sponsor: The Clinical Coordinating Center at Massachusetts General Hospital 
Study Day: The day of randomization is study day zero. The next day is study day one etc. 
Study Drug: Randomly assigned vitamin D3 (cholecalciferol) or placebo 
Study hospital: Defined as the hospital where the patient was randomized and enrolled. 


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Study withdrawal: Defined as permanent withdrawal from study before completion of study 
activities. This does not include those participants who have completed the protocol procedures 
or stopped procedures because they have reached unassisted breathing. If a patient or 
surrogate requests withdrawal from the study the clinician should seek explicit permission to 
continue data collection. 
Suspected adverse reaction: Any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. Reasonable possibility means there is evidence to 
suggest a causal relationship between the drug and the adverse event. A suspected adverse 
reaction implies a lesser degree of certainty about causality that adverse reaction (21 CFR 
312.32(a)) 
Randomized participants: The subset of eligible and enrolled patients with screening 25OHD 
levels less than 20 ng/mL that are randomized to receive either study drug or placebo.  
UAB (Unassisted Breathing): Spontaneously breathing with face mask, nasal prong oxygen, 
room air, T-tube breathing, tracheostomy mask breathing, CPAP ≤ 5 without PS or IMV 
assistance, the use of noninvasive ventilation solely for sleep-disordered breathing, or use of a 
nasal high flow system.  
Valid SpO2: Defined as SpO2 <97%, FiO2 ≥40%, PEEP ≥5 cm H2O, and at least 10 minutes 
after any documented change to FiO2 that is not considered spurious by the study staff and 
treating clinician. 
 
 


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2 TRIAL SUMMARY 
2.1 Title 
Vitamin D to Improve Outcomes by Leveraging Early Treatment (VIOLET) 
2.2 Objective 
To assess the efficacy and safety of early administration of vitamin D3 (cholecalciferol) in 
reducing mortality and morbidity for vitamin D deficient patients at high risk for ARDS and 
mortality. 
2.3 Hypothesis 
Early administration of vitamin D3 (cholecalciferol) will improve all-cause, all-location mortality to 
day 90 in vitamin D deficient patients at high risk for ARDS and mortality. 
2.4 Study Design 
VIOLET is a randomized, double-blinded, placebo-controlled, phase III trial (up to maximum 
n=3000) of early vitamin D3 in vitamin D deficient patients at high risk for ARDS and mortality. 
We will screen all patients for whom there is an intention to admit to ICU for study eligibility and 
will approach patients meeting inclusion/exclusion criteria for study enrollment. Screening will 
require screening for vitamin D deficiency using an FDA approved testing method for 25OHD, 
either by the hospital’s clinical laboratory or using the FastPack IP device (Qualigen Inc., 
Carlsbad, CA). We will obtain written informed consent for the protocol prior to the vitamin D 
screening test.  
 
We will randomize enrolled participants who are vitamin D deficient (initial screening 25OHD 
levels <20 ng/mL) to receive either 540,000 IU vitamin D3 (cholecalciferol) or placebo as a 
single, liquid enteral dose, administered either orally or via naso/orogastric tube. Randomization 
must occur within 12 hours of ICU admission decision and study drug administered within 2 
hours of randomization. We will assess trial endpoints for randomized participants by chart 
review and will contact participants or proxies at day 90. 
At baseline for all randomized participants, we will measure serum calcium (in real time, if not 
clinically available) and plasma 25OHD levels (batched and measured initially in monthly 
increments using the gold-standard liquid chromatography-tandem mass spectrometry 
[LC/MS/MS] method). We will collect additional plasma and whole blood for DNA banking. For 
the first 300 randomized participants, we will measure day 3 serum calcium (in real time, if not 
clinically available) and baseline plasma IL-6. We will also collect plasma at day 3 in the first 
300 randomized participants to measure 25OHD by LC/MS/MS and IL-6 (both batched) and for 
cryopreservation in the biorepository.  
The primary efficacy analysis cohort (confirmed vitamin D deficient cohort) will be defined as 
randomized participants having LC/MS/MS levels less than 20 ng/mL. The primary efficacy 
analysis will only involve these participants.  The FastPack device had some discordance with 
LC/MS/MS at a 20 ng/mL cutoff, and the previous study only showed a treatment effect in 
patients with low vitamin D levels. 1Since LC/MS/MS values will take at least 24 hours to 
process, we need to treat participants on the basis of the screening test result (FastPack or 


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clinical laboratory test) available within three hours of patient consent. A secondary analysis 
cohort (screened vitamin D deficient cohort), which will also be used for the primary safety 
analysis, will include all randomized participants who receive vitamin D or placebo.  
Figure 1 Study Flow 
 
 
 
 
 
 
 
 
2.5 Inclusion Criteria 
1. Age ≥ 18 years 
2. Intention to admit to ICU from emergency department, hospital ward, operating room, or 
outside facility 
3. One or more of the following acute risk factors for ARDS and mortality contributing 
directly to the need for ICU admission: 
Pulmonary 
a) Pneumonia 
b) Aspiration 
c) Smoke Inhalation 
d) Lung contusion 
e) Mechanical ventilation for acute hypoxemic or hypercarbic respiratory failure 
Extra-Pulmonary 
f) Shock 
g) Sepsis 
h) Pancreatitis 
4. Vitamin D deficiency (screening 25OHD level <20 ng/mL) 
 


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2.6 Exclusion Criteria  
1. Inability to obtain informed consent 
2. Unable to randomize within 12 hours of ICU admission decision 
3. Unable to take study medication by mouth or enteral tube 
4. Baseline serum calcium >10.2 mg/dL (2.54 mmol/L) or ionized calcium >5.2 mg/dL (1.30 
mmol/L) 
5. Known kidney stone in past year or history of multiple (>1) prior kidney stone episodes 
6. Decision to withhold or withdraw life-sustaining treatment (patients are still eligible if they 
are committed to full support except cardiopulmonary resuscitation if a cardiac arrest 
occurs) 
7. Expect <48 hour survival 
8. If no other risk factors present, a) mechanical ventilation primarily for airway protection, 
pain/agitation control, or procedure; or b) elective surgical patients with routine 
postoperative mechanical ventilation; or c) anticipated mechanical ventilation duration 
<24 hours; or d) chronic/home mechanical ventilation for chronic lung or neuromuscular 
disease (non-invasive ventilation used solely for sleep-disordered breathing is not an 
exclusion). 
9. Prisoner  
10. Pregnancy 
11. Greater than 72 hours since hospital presentation (or > 3 calendar days since hospital 
presentation if transferred with no time stamp).  
2.7 Randomization and Study Initiation Time Window 
All participants must be enrolled and randomized within 12 hours of meeting inclusion criteria. 
Study medication must be administered within 2 hours of randomization. 
2.8 Primary endpoint 
The primary endpoint is all-cause, all-location mortality to day 90. The primary analysis will be 
based on randomized participants with baseline 25OHD levels <20 ng/mL, as measured by 
LC/MS/MS (confirmed vitamin D deficient cohort). As below, additional secondary analyses 
will be based on all randomized participants (screened vitamin D deficient cohort).  
2.9 Secondary endpoints  
Clinical Endpoints 
• 
Hospital length of stay among survivors to day 90 
• 
Healthcare facility length of stay among survivors to day 90 
• 
Alive and home (prior level of care) at day 90 
• 
Ventilator-free days to day 28 
• 
Time to mortality to day 90 
Physiological Endpoints 
• 
Development and severity of ARDS to day 7 
• 
Change in organ failure severity to day 7  
• 
25OHD levels to day 3 
• 
IL-6 levels to day 3 
Safety Endpoints 
• 
Calcium levels to day 14 


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• 
Kidney stones to day 90 
• 
Fall-related fractures to day 90 
 
The primary analyses will be in the confirmed vitamin D deficient cohort.  These analyses will 
include an assessment of the secondary endpoints defined above. In addition, we will estimate 
the treatment effects on the primary and secondary endpoints in the screened vitamin D 
deficient cohort. If the analysis in the confirmed vitamin D deficient cohort is significant then 
estimates in the screened vitamin D deficient cohort will measure the treatment effect in patients 
who would be given the treatment if early LC/MS/MS is not available (or until next generation 
screening tests more closely approximate LC/MS/MS results).  We will also estimate the 
treatment effects on the endpoints above as a continuous function of baseline 25OHD level by 
LC/MS/MS. This function will provide an estimate of the extent to which efficacy (and safety) 
depends on vitamin D level. 
2.10 Sample Size/ Interim Monitoring  
1. We base our assumptions on finding a 5% or greater absolute difference in mortality. 
With a 20% mortality rate in the control arm and a 15% mortality rate in the intervention 
arm, the maximum required total sample size is 3000 randomized participants with  
87.4% power based on the assumption that 80% of the randomized participants will 
have baseline 25OHD levels < 20 ng/mL, as measured by LC/MS/MS, and one sided α = 
0.025. 
2. The principal analysis will be based on randomized participants with baseline 25OHD 
levels <20 ng/mL, as measured by LC/MS/MS (confirmed vitamin D deficient cohort), 
following an intention-to-treat principle (analysis according to randomization 
assignment). We anticipate that approximately 80% of randomized participants will have 
baseline 25OHD levels <20 ng/mL by LC/MS/MS. The maximum size of the screened 
vitamin D deficient cohort will be 3000 patients and the futility stopping boundaries will 
consider the possibility that the sample size will not reach 2400 (80% of 3,000) in the 
confirmed vitamin D deficient cohort if too small a fraction of randomized patients would 
have baseline 25OHD levels <20 ng/mL by LC/MS/MS.  
3. An independent Data and Safety Monitoring Board (DSMB) will determine if the study 
should stop for superiority or futility of the intervention, or safety concerns. There will be 
three interim analyses throughout the trial (at subject enrollments 750, 1500, and 2250 
randomized participants).  
4. For the first 300 randomized participants, we will analyze   calcium levels at baseline and 
day 3 and 25OHD levels at day 3 using LC/MS/MS. This analysis will evaluate the 
effectiveness and safety of the vitamin D intervention in correcting vitamin D deficiency 
(clinical outcomes will not be evaluated at this time). Based on pre-specified rules and in 
consultation with the DSMB (who will have access to additional adverse event data), we 
may require changes to the vitamin D dose or extend the duration of 25OHD/calcium 
monitoring. 


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3 TRIAL DESCRIPTION  
3.1 Background 
Acute respiratory distress syndrome (ARDS) is a common and devastating complication of 
critical illness or injury. Since effective therapies to reduce morbidity and mortality from ARDS 
are limited, prevention of ARDS in high-risk patients has become a high priority for the acute 
and critical care communities and a major goal of the PETAL Network.2 Currently, there are no 
safe or effective therapies for ARDS prevention. Vitamin D has emerged as a promising 
intervention to prevent ARDS and improve associated morbidity in critically ill patients. 
Specifically, strong preclinical data support the protective role of vitamin D in regulating 
pulmonary inflammation and disruption of the alveolar-capillary membrane that are fundamental 
to ARDS pathogenesis. Observational data indicate that vitamin D deficiency is common in 
critically ill patients and a key ARDS risk factor. Phase II trial data demonstrate high potential for 
early vitamin D supplementation to safely and effectively correct vitamin D deficiency and 
improve clinical outcomes in critically ill patients. Accordingly, this phase III trial is warranted to 
definitively test the efficacy and safety of early vitamin D repletion to reduce mortality and 
morbidity for vitamin D deficient patients at high risk for ARDS and mortality. 
3.2 Vitamin D-Potential Benefits and Mechanisms 
In addition to its role in calcium and phosphorus homeostasis, vitamin D has pleiotropic roles 
regulating immune function and maintaining epithelial surface integrity. The major circulating 
form of vitamin D, 25-hydroxvitamin D (25OHD) is converted systemically and locally in the lung 
to the active hormone 1,25-dihydroxyvitamin D (1,25OH2D), which binds to the vitamin D 
receptor (VDR) found on most immune and epithelial cells. Pre-clinical data indicate that vitamin 
D is a potent anti-inflammatory agent3,4 that reduces neutrophil infiltration,5 and is essential for: 
a) epithelial cell growth/differentiation,6,7 b) pulmonary surfactant synthesis,8,9 and c) expression 
of endogenous antimicrobial peptides such as cathelicidin (LL-37) by epithelial cells and 
leukocytes.10,11 
• 
Inflammation: The role of vitamin D in regulation of persistent inflammation is also critical 
in mitigating secondary organ injury (including ARDS), often seen in lung injury. 
Specifically, 1,25OH2D inhibits synthesis of pro-inflammatory cytokines IL-1β, IL-2, IL-6, 
IL-17, TNF-α, and IFN-γ in vitro and in animal models of sepsis. However, unlike other 
more non-specific anti-inflammatory interventions (e.g., glucocorticoids), vitamin D 
dampens pro-inflammatory responses without negatively impacting pathogen 
clearance,12,13 which is critical to recovery from infection-related ARDS.  
• 
Neutrophil Infiltration: 1,25OH2D also suppresses IL-8 production,14-22 an endogenous 
chemotactic factor for neutrophils key in the pathogenesis of ARDS.23-29 In a hamster 
model of lipopolysaccharide-induced ARDS, 1,25OH2D inhibited neutrophil recruitment 
in the lung by 40%.5 In this study, vitamin D supplementation inhibited neutrophils when 
administered early, but not later, in the course of ARDS development. This supports 
initiating vitamin D therapy early in the hospital course (i.e., in the ED) to optimize 
prevention of ARDS. 
• 
Epithelial Cell Growth/Differentiation: 25OHD or 1,25OH2D increases airway epithelial 
cell growth and differentiation in a dose-dependent manner.6,7 These trophic roles for 


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vitamin D are important in the preserving pulmonary epithelial integrity that is 
compromised during ARDS pathogenesis. 
• 
Pulmonary Surfactant: 1,25OH2D stimulates surfactant protein-B mRNA and protein 
synthesis in alveolar type II cells.8,9 Pulmonary surfactant is a complex of phospholipids 
and proteins that maintains alveolar integrity by reducing surface tension at the alveolar 
air-liquid interface. Since disruption of pulmonary surfactant is a contributor to the 
pathophysiology of ARDS, the ability of vitamin D to enhance pulmonary surfactant hold 
promise as a mechanism to prevent ARDS. 
• 
Antimicrobial: Vitamin D upregulates the production of the antimicrobial peptide 
cathelicidin (LL-37), aiding host defenses by killing a wide variety of pathogens.4,10,30-33 
Antimicrobial activity of vitamin D leads to control of the source infection and limits the 
host responses that lead to secondary tissue damage, including ARDS.  
3.3 Recent Clinical Trials  
Vitamin D deficiency is common in critically ill patients, with approximately 40-50% meeting the 
Institute of Medicine definition for vitamin D deficiency (25OHD levels <20 ng/mL [50 nmol/L]).34 
Consistent with preclinical data, observational clinical studies also support that vitamin D 
deficiency is a common, potentially reversible risk factor for ARDS.34-45 Specifically, early vitamin 
D deficiency in the ED or at ICU admission is associated with higher hospital and ICU lengths of 
stay, lung and other organ injury, prolonged mechanical ventilation, and mortality. 
Based on the rationale outlined above, several recent phase II trials have evaluated the 
potential of acute vitamin D repletion to improve outcomes in medical and surgical ICU. In ICU 
patients with sepsis, Leaf et al found that active vitamin D (calcitriol)-treated patients had 
increased expression of the potent antimicrobial peptide LL-37 (p=0.04) and observed a lower 
mortality rate vs. placebo (17% vs. 23%; p=ns).46 Dancer et al demonstrated that a 200,000 IU 
pre-operative vitamin D3 dose decreased post-operative extravascular lung water and 
pulmonary vascular permeability indices in esophagectomy patients at high risk for ARDS.7 
Quraishi et al found that a 400,000 IU vitamin D3 dose in ICU patients with sepsis increased 
25OHD levels by an average 12 ng/mL and led to increased expression of LL-37, as well as 
decreased pro-inflammatory cytokines (IL-1 and IL-6).47 Han et al found that 500,000 IU 
25OHD
Circulating 
pro-hormone
↑ Epithelial cell 
growth/differentiation
Vitamin D 
Supplementation
1,25OH2D
Active form
Prevent lung 
injury and 
mortality
↓ Inflammation/ 
Neutrophil infiltration
↑ Pulmonary surfactant
↑ Antimicrobial peptide
Hepatic
conversion
Systemic (kidney) 
& local (lung)
conversion
VDR
binding
25OHD level
<20 ng/ml—deficient (50%)
20-29 ng/ml—low normal (30%)
≥30 ng/ml—optimal (20%)
(>100 ng/ml—toxicity)
Figure 2: Proposed 
mechanisms of vitamin D 
and prevent ARDS and 
improve clinical outcomes 


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(administered over 5 days) safely increased 25OHD levels in ventilated ICU patients and was 
associated a lower hospital length of stay48   
Amrein et al published two randomized controlled trials in a mixed medical-surgical population 
of ICU patients in Austria that directly support the dose and rationale for the VIOLET trial. An 
initial pilot trial (n=25) demonstrated that a single 540,000 IU vitamin D3 dose administered 
enterally to vitamin D deficient (25OHD <20 ng/mL) ICU patients increased 25OHD levels by 
day 1 and above the target concentration (25OHD 30 ng/mL) by day 2.49 In a larger follow-up 
trial using the same 540,000 IU dose in 475 ICU patients, 25OHD levels increased above the 
target concentration (30 ng/mL) prior to day 3 and was sustained through at least day 7. Like 
prior trials, there were no vitamin D-related adverse events, particularly no clinically significant 
episodes of hypercalcemia nor incident kidney stones. Compared to placebo, vitamin D-treated 
patients had a lower observed 28-day mortality (21.9% vs. 28.6%; p=0.14) and 6-month 
mortality (35.0% vs. 42.9%; p=0.09).1 While the trial was underpowered for mortality, these 
results justify the need for a larger phase III trial to evaluate the impact of acute vitamin D 
supplementation on clinical outcomes in at-risk patients.  
 
The VIOLET trial incorporates three important differences from previous efforts. First, while 
preclinical and observational data support early supplementation to improve physiology and 
outcomes, the recent Amrein trial randomized patients later into their illness, a median of 3 days 
into the ICU course.1 VIOLET will be the first study to test early vitamin D supplementation, prior 
to ICU admission, which we believe will optimize the potential for clinical benefit. Utilizing 
PETAL infrastructure that includes acute care specialists (emergency medicine and acute care 
surgery), early administration (within 12 hours) will likely increase the probability of modifying 
disease progression and enhance the potential clinical benefit of the vitamin D intervention. 
Second, prior data strongly support plausible mechanism and potential benefit in patients at 
higher risk for ARDS and mortality. Compared to the Amrein trial, which enrolled all ICU 
patients, we focus enrollment on patients at higher risk for ARDS and mortality to increase the 
potential to improve clinical outcomes. Third, due to known variability in vitamin D measurement 
techniques, VIOLET will base the primary analysis on the gold standard LC/MS/MS 
measurement (i.e., confirmed vitamin D deficient cohort with LC/MS/MS <20 ng/mL) to decrease 
the heterogeneity of treatment effect according to baseline risk and maximize trial power to 
detect an efficacy signal. Further, there is scientific interest in the efficacy and safety of the 
intervention on the screened vitamin D deficient cohort including patients with LC/MS/MS ≥20 
ng/mL, which will also be addressed in VIOLET. 
3.4 Summary of Rationale  
Based on these data, there are several reasons why the PETAL Network should conduct a trial 
of early vitamin D repletion to improve clinical outcomes in vitamin D deficient patients at high 
risk for ARDS and mortality: 
1. Vitamin D deficiency is common in critically ill patients, and high dose supplementation 
can rapidly correct this deficiency.  
2. Promising pre-clinical and epidemiological data strongly suggest potential clinical benefit 
and plausible mechanism for high-dose vitamin D supplementation to prevent ARDS and 
improve clinical outcomes. 


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3. Robust phase II trial data support the dosing regimen, safety, and potential clinical 
efficacy in ICU patients. 
4. The trial leverages the PETAL infrastructure for early intervention prior to ICU admission, 
which is both feasible and desirable to optimize potential for clinical benefit. 
5. The data collection requirements are modest to monitor safety and measure clinically 
relevant outcomes, which facilitate this large, simple, generalizable and cost-efficient trial 
that is consistent with NHLBI and PETAL Network goals. 
6. Vitamin D is inexpensive, easy to administer, and safe, supporting the high potential to 
rapidly disseminate and change clinical practice if the trial results are positive. 
 
3.5 Objectives 
3.5.1 Primary Objective  
To assess the efficacy and safety of early administration of vitamin D3 (cholecalciferol) in 
reducing mortality and morbidity for vitamin D deficient patients at high risk for ARDS and 
mortality.  
3.5.2 Primary Hypothesis 
Early administration of vitamin D3 (cholecalciferol) will improve all-cause, all-location mortality to 
day 90 in vitamin D deficient patients at high risk for ARDS and mortality.  
3.6 Endpoints 
3.6.1 Primary Endpoint  
The primary endpoint is all-cause, all-location mortality to day 90. We will ascertain vital status 
at day 90 by chart review (for participants that remain in the hospital at day 90 or died in hospital 
prior to day 90). For participants that were discharged alive from the hospital prior to day 90, we 
will call the patient or proxy (e.g., research contact list and next-of-kin) on/after day 90 to 
determine vital status. For those that we are unable to verify vital status at day 90, we will use 
evidence from the medical record (e.g., healthcare visits after day 90), review of obituaries, or 
phone calls to healthcare facilities to determine that participants are alive. Finally, for 
participants that have missing 90-day mortality, we will use the Centers for Disease Control and 
Prevention’s National Death Index (NDI) to determine vital status, using each patient’s social 
security number (SSN) for an exact NDI match. At the time of final analysis, we will use best 
available mortality data and if vital status remains unknown (anticipated for <5%), will impute 
90-day mortality based on last known location. 
3.6.2 Secondary Endpoints  
3.6.2.1 Clinical Endpoints 
1. Study hospital length of stay among survivors to day 90: Study hospital length of stay is 
defined as the number of days from enrollment (typically the day of hospital admission) to 
the day of study hospital discharge up to day 90. This endpoint will be analyzed only in 
survivors because hospital length of stay in those who die in the hospital is non-informative 
for this endpoint. In addition, we will use survivor average causal effect (SACE) methods to 
estimate hospital length of stay among participants that would have survived in both 


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treatment and control groups.50 Accordingly, these methods adjust for the impact of 
differential survival between treatment groups. 
2. Healthcare facility length of stay among survivors to day 90: Healthcare facility length of 
stay is the time spent in another hospital or healthcare facility (e.g. long-term acute care 
[LTAC] hospitals or acute rehabilitation/skilled nursing facility), for the subgroup of 
participants that were discharged to another healthcare facility after the initial hospitalization. 
This measure is defined as the number of days from initial hospital discharge to the first 
facility discharge to home (pre-hospitalization level of care) up to day 90. Healthcare facility 
LOS is zero for patients discharged to home (pre-hospitalization level of care) from the study 
hospital. This endpoint will be analyzed only in survivors using SACE methods because 
healthcare facility length of stay in those who die during the follow-up period is non-
informative for this endpoint. 
  
3. Alive and home at day 90: This endpoint is the proportion of participants who have 
survived and are present at home, defined as pre-hospitalization level of care, at day 90. 
 
4. Ventilator free days (VFD) to day 28: VFD depends on both duration of ventilation and 
mortality through study day 28. In participants who survive 28 days, VFD is defined as 28 
minus duration of ventilation. Duration of ventilation is counted from the first study day of 
assisted breathing through the last day of assisted breathing provided the last day is prior to 
day 28. Otherwise, it is counted from the first study day of assisted breathing through day 
28. For participants discharged with assisted ventilation (e.g., to LTAC facility) prior to day 
28, a phone call will be required to assess ventilator status at day 28. Participants 
discharged prior to day 28 (but not to home) on unassisted breathing will be assumed to 
remain on unassisted breathing through day 28. Isolated periods of ventilation briefer than 
24 hours for surgical procedures and ventilation solely for sleep disordered breathing do not 
count towards duration of ventilation. In participants who never require assisted breathing, 
duration of ventilation is zero. Participants who do not survive 28 days will be assigned zero 
VFD. VFD is undefined in participants with chronic/home mechanical ventilation (except 
solely for sleep disordered breathing) and they will be excluded from this analysis.  
 
5. Time to mortality to day 90: Time to mortality will use best available data on vital status 
and date of death and censor based on last known alive date for participants with missing 
vital status. 
3.6.2.2 Physiological Endpoints 
6. Development and severity of ARDS to day 7: We will determine the presence and 
severity of ARDS for each day of mechanical ventilation to day 7 using the following 
approach: 
a. For each ventilator day: if ABG available between 2:00 AM and 8:00 AM, measure P/F 
(PaO2, FiO2 and PEEP) for all ABGs during this time window daily to day 7. Or, for 
ventilator days that no ABG available between 2:00 AM and 8:00 AM,  determine lowest 
imputed P/F from measured S/F (SpO2, FiO2, and PEEP).  See Appendix B for P/F 
imputation table. 
b. For participants with P/F <300 or imputed P/F <300, FiO2 ≥40%, and PEEP ≥5 cm H2O, 
determine if hypoxemia is valid, acute, and not fully explained by CHF or fluid overload 
c. If yes to item (b), local investigators will: 


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i. 
Review the first CXR (or CT) performed on each ventilated day with valid P/F or 
imputed P/F <300 (to day 7). If no chest imaging studies are present that day, site 
investigators may review imaging one day before or after to determine if ARDS 
imaging criteria met. 
ii. 
Assess if the images are consistent with ARDS (bilateral opacities not fully explained 
by effusions, lobar/lung collapse, or nodules). If equivocal, the reviewing investigator 
will adjudicate with additional investigators. 
 
7. Change in organ failure severity to day 7: We will measure acute organ failure severity 
daily to day 7 for three key organ systems: acute hypoxemic respiratory failure, acute kidney 
injury, and cardiovascular failure (i.e., shock). We will measure acute hypoxemic respiratory 
failure using data from the ARDS assessment, except this outcome does not require chest 
imaging consistent with ARDS. We will measure acute kidney injury by highest daily 
creatinine values or new use of dialysis/ renal replacement therapy (chronic dialysis 
participants are excluded). We will measure cardiovascular failure by highest daily dose of 
vasopressors (epinephrine, norepinephrine, dopamine, phenylephrine, or vasopressin). 
 
8. Plasma 25OHD levels: In addition to the screening vitamin D test (for trial entry), we will 
measure gold-standard plasma 25OHD levels at baseline for all randomized participants 
and at day 3 in the first 300 randomized participants to confirm biomarker response to the 
intervention. The gold-standard test will be done in batch using LC/MS/MS methods. 
Individual results will not be available to investigators and coordinators to avoid unblinding. 
Based on pre-specified rules and in consultation with the DSMB (who will have access to 
additional adverse event data), we may require changes to the vitamin D dose or extend the 
duration of 25OHD/calcium monitoring. 
 
9. Plasma IL-6 levels: We will measure plasma IL-6 levels at baseline and day 3 in the first 
300 randomized participants in batch concurrent with 25OHD testing. IL-6 is a pro-
inflammatory cytokine associated with increased incidence and severity of ARDS severity 
and worse clinical outcomes. Vitamin D may improve outcomes in critically ill patients by 
reducing levels of IL-6 (either causal association or as a result of improved recovery). 
3.6.2.3 Safety Endpoints 
10. Calcium levels to day 14: Because the half-life of 25OHD is approximately 2 weeks, we will 
record clinically available serum or ionized calcium levels through day 14 for all randomized 
participants. In addition, we will measure baseline and day 3 in the first 300 randomized 
participants in real time, either as part of routine clinical care or as a research procedure if 
not available. Day 3 is the when 25OHD levels peak in ICU patients given 540,000 IU of 
vitamin D.1,49 While no prior study of acute vitamin D repletion, using similar high doses as 
our trial, has observed clinically important hypercalcemia, we will confirm these findings in 
the VIOLET trial. Because little change in calcium levels is anticipated, access to individual-
level calcium results will not unblind study staff. During the trial, the DSMB may seek 
additional subject measurements for safety assessment, though this is not anticipated. 
 
11. Kidney stones to day 90: While an association between hypervitaminosis D and kidney 
stones has been suggested, the relationship with high dose vitamin D supplementation and 
kidney stones remains controversial. We will assess for incident kidney stones by chart 


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review at the end of the hospitalization and by self-report at the 90 day phone call for those 
discharged from the hospital prior to day 90. 
 
12. Fall-related fractures to day 90: The association between acute vitamin D 
supplementation and falls/fractures remains unclear. Most data suggest that high dose 
vitamin D in healthy outpatients may improve muscle function, balance, and bone mineral 
density, and thus decrease fall-related fractures, but other data suggest that high dose 
vitamin D supplementation may actually increase the incidence of falls/fractures. Because of 
this uncertainty and limited data in hospitalized patients, we will assess for incident fall-
related fractures by chart review at the end of the hospitalization and by self-report at the 90 
day phone call for those discharged from the hospital prior to day 90.  
 
3.7 Subgroups 
The primary analysis will be based on randomized participants with baseline 25OHD levels <20 
ng/mL by LC/MS/MS (confirmed vitamin D deficient cohort). Secondary analysis will be based 
on the screened vitamin D deficient cohort (all randomized patients). A priori subgroups for 
analysis will include age, sex, race/ethnicity, residence (independent vs. long-term care facility), 
BMI, pre-hospitalization vitamin D supplementation, baseline 25OHD level (by the LC/MS/MS 
method), baseline renal function, ARDS risk factor (e.g., infectious vs non-infectious), LIPS 
score, pre-randomization mechanical ventilation status, pre-randomization presence of ARDS, 
and source of ICU admission.  
4 STUDY POPULATION AND ENROLLMENT 
4.1 Number/Source/Screening  
The trial will randomize a maximum of 3000 participants from the EDs, hospital wards, operating 
rooms, ICUs and other acute care areas of the PETAL Network Clinical Centers. The 12-hour 
enrollment window requires engagement of the Network’s infrastructure for early recruitment. 
We will screen and enroll all medical, trauma, or post-operative patients at each site prior to ICU 
admission largely using available clinical data to determine potential eligibility.  
Tactics will include: 
i. 
Emergency Department (ED) screening: We anticipate that the majority of participants for 
this trial will be screened and enrolled in the ED. Eligible patients must have ICU admission 
intended (defined as an ICU consult or bed request/order) and meet inclusion and exclusion 
criteria.  
This assessment will be in person, by telephone, or through electronic screening. For 
capable sites, we encourage electronic triggers for hypotension, mechanical ventilation, 
hyperlactemia, trauma team activation, and vasopressor order that will identify patients likely 
to meet trial inclusion criteria even before the ICU admission is triggered. We also will 
engage key leaders and the ED staff to enhance knowledge of and effort to enroll in the trial 
as early as possible. 


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ii. Hospital ward screening: Ward patients who are destined for the ICU will be eligible for 
study inclusion. The primary trigger for screening will be hospital rapid response team, ICU 
consult or bed order, and clinician referral. Because typically these patients arrive rapidly to 
the ICU, we anticipate that enrollment and randomization will occur primarily in the ICU.  
iii. Operating room screening: Emergent/unscheduled operating room patients destined for 
the ICU will be eligible for study inclusion. In addition, patients with elective operations will 
be eligible if unanticipated complications occur that meet inclusion criteria (e.g., hemorrhagic 
shock, unplanned prolonged mechanical ventilation). 
iv. ICU screening: We will screen patients admitted to ICUs upon arrival if still within the 
enrollment time window, including admissions from the ED, wards, operating room, and 
outside facilities. Because the 12-hour enrollment window begins when the ICU admission 
decision is made (defined in Section 4, Inclusion Criteria), we anticipate that most trial 
participants will have screening and enrollment procedures begun prior to ICU arrival.  
v. Communication and hand-off: We will facilitate hand-offs with use of a unified screening 
log (to be used for ED, inpatient, and ICU screening); inservice training by site PIs to ED, 
OR, and ICU staff; and coordination by research staff for enrolled patients. When patients 
are enrolled prior to ICU admission (as we commonly anticipate in this trial), ED and other 
pre-ICU research staff will communicate with the ICU research staff to report patient 
information and current status (screening, consent, randomization, administration of study 
medication). 
As a Network, we will develop and update best practices for communication hand-offs to 
encourage interdisciplinary communication among research staff and between research 
staff and clinical providers. These will include enrollment notes and scanned consent forms 
in the electronic medical record; study acronym with coordinator name/contact on the white 
board in the patient’s room; and communication about VIOLET enrollment upon transfer to 
new units. Implemented strategies will be tailored to each hospital’s unique processes. 
  
vi. Study staff available for consent: Each site will have dedicated study physicians and 
coordinators who are certified and trained in human subjects protection and understand the 
study protocol. 
4.2 Inclusion Criteria  
1. Age ≥ 18 years 
2. Intention to admit to ICU from emergency department, hospital ward, operating room, or 
outside facility 
3. One or more of the following acute risk factors for ARDS and mortality contributing 
directly to the need for ICU admission: 
Pulmonary 
a) Pneumonia 
b) Aspiration 
c) Smoke Inhalation 
d) Lung contusion 
e) Mechanical ventilation for acute hypoxemic or hypercarbic respiratory failure 


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Extra-Pulmonary 
f) Shock 
g) Sepsis 
h) Pancreatitis 
4. Vitamin D deficiency (screening 25OHD level <20 ng/mL) 
4.2.1 Intention to Admit to ICU 
Randomization must occur within 12 hours of ICU admission decision. The occurrence of one or 
more risk factor conditions should contribute directly to the need for ICU admission per treating 
physician. The criteria recognize that recruiting sites have different processes for ICU 
admission, and therefore provides some flexibility, while ensuring that participants across sites 
would be recruited at similar/consistent times. The first occurrence of any of the following criteria 
will serve as time zero to start the 12-hour enrollment window: 
1. ED, hospital ward, or operating room patients at study hospital 
Whichever comes first: 
a. Initiation of ICU consult, OR 
b. Written order for ICU bed, OR 
c. Physician documentation of plan for ICU admission, OR 
d. Discussion with treating physician of intention to admit to ICU, documented by 
study coordinator or investigator 
2. Patients transferred from outside facility to study hospital ICU 
a. Acceptance of ICU transfer 
3. Patients transferred from outside facility to study hospital ED, hospital ward, or operating 
room at study hospital. 
a. Use same criteria as in item 1 above.  
4.2.2 Pulmonary risk factors 
Pneumonia 
Known or suspected based on physician charting or discussion with treating physician 
OR, both 1 and 2 below:  
1. Chest radiographs showing infiltrates, consolidation, or cavitation that is not known to be 
pre-existing (on prior chest imaging)  
2. Clinical signs of pneumonia (new cough, sputum, fever, or WBC>12,000) 
 
Aspiration 
Witnessed or suggestive history of inhalation of food, liquid, or regurgitated gastric contents 
 
Smoke inhalation 
Witness or suggestive history of smoke inhalation 
 
Lung contusion 
Blunt or penetrating trauma to thorax that results in related infiltrates in chest x-ray or CT. 
 
Mechanical ventilation 
Patients receiving invasive or non-invasive mechanical ventilation for acute hypoxemic or 
hypercarbic respiratory failure per treating physician. Mechanical ventilation exposes patients to 
ventilator-associated lung injury and is a marker for higher illness severity in ICU patients. 


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Therefore, these patients are at high risk for mortality,51 and prior data support the potential of 
vitamin D to improve outcomes in this population.52  
4.2.3 Extra-pulmonary risk factors 
Shock 
Infusion of vasopressors (epinephrine, norepinephrine, dopamine (>5 µg/kg/min), 
phenylephrine, or vasopressin)  
OR MAP <65 mmHg or SBP <90 mmHg after at least 1 liter intravenous fluids or 1 unit of 
blood product 
Sepsis 
Both 1 and 2 below: 
1. Suspected or proven infection  
• 
Treating physician suspects bacterial or fungal infection (document suspected 
source(s)) 
• 
Or, cultures: ordered in past 48 hours or positive cultures from referring ED  
• 
Or orders for antimicrobial medication 
2. Organ dysfunction defined as an acute change in total SOFA score ≥2 points 
• 
The baseline SOFA score can be assumed to be zero in patients not known to 
have preexisting organ dysfunction 
Pancreatitis  
Defined as two of the three following features, not explained by another obvious cause (e.g., 
diabetic ketoacidosis): 
a. Symptoms consistent with acute pancreatitis (e.g., abdominal pain or vomiting) 
b. Serum Amylase and/or Lipase ≥ 3 times the upper limit of normal 
c. Characteristic findings of acute pancreatitis on CT scan 
4.2.4 Screening Vitamin D Test  
After initial study screening, eligible patients will be approached by research staff for written 
informed consent for the protocol, prior to secondary screening for vitamin D deficiency.  
Vitamin D screening will use an FDA-approved test, by either the hospital’s clinical laboratory 
or the FastPack IP device (Qualigen Inc., Carlsbad, CA). Results of the screening test will be 
interpreted as vitamin D deficient (25OHD <20 ng/mL) or not vitamin D deficient (25OHD ≥20 
ng/mL). We will randomize participants with a screening 25OHD level <20 ng/mL within 12 
hours of ICU admission decision and administer study drug within 2 hours of randomization. 
Figure 3 Study Flow: screening & enrollment 


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4.3 Exclusion Criteria  
1. Unable to obtain informed consent 
2. Unable to randomize within 12 hours of ICU admission decision 
3. Unable to take study medication by mouth or enteral tube 
4. Baseline serum calcium >10.2 mg/dL (2.54 mmol/L) or ionized calcium >5.2 mg/dL (1.30 
mmol/L) 
5. Known kidney stone in past year or history of multiple (>1) prior kidney stone episodes 
6. Decision to withhold or withdraw life-sustaining treatment (patients are still eligible if they 
are committed to full support except cardiopulmonary resuscitation if a cardiac arrest 
occurs) 
7. Expect <48 hour survival 
8. If no other risk factors present, a) mechanical ventilation primarily for airway protection, 
pain/agitation control, or procedure; or b) elective surgical patients with routine 
postoperative mechanical ventilation; or c) anticipated mechanical ventilation duration 
<24 hours; or d) chronic/home mechanical ventilation for chronic lung or neuromuscular 
disease (non-invasive ventilation used solely for sleep-disordered breathing is not an 
exclusion). 
9. Prisoner  
10. Pregnancy 
11. Greater than 72 hours since hospital presentation (or > 3 calendar days since hospital 
presentation if transferred with no time stamp).  
 
4.3.1 Reasons for Exclusions  
We exclude patients <18 years old because of limited data on the safety of high dose vitamin D 
during critical illness in children. The 12-hour enrollment window underscores the trial rationale 
and PETAL Network goals for early intervention to optimize potential for benefit (criterion 2). 
The study drug is administered enterally and thus we must exclude patients who are unable to 
take the single vitamin D dose by mouth or enteral tube (criterion 3). Because vitamin D 
increases gastrointestinal calcium absorption, we will exclude patients with hypercalcemia to 
avoid further increases to calcium levels (criterion 4). Calcium levels should be obtained in the 
majority of ICU-bound patients as part of the routine clinical care. Results will be typically 
available within 1 hour of hospital arrival. If not available clinically in the pre-consent screening 
period (up to 72 hours prior to consent) and the clinical team does not plan to order a calcium 
level, the study team will order a serum calcium level as a research test in consented patients. 
Whether vitamin D may cause hypercalciuria and related kidney stones is controversial, it is 
prudent and consistent with prior vitamin D trials to exclude patients with a recent history or 
multiple prior episodes of kidney stones (criterion 5). Criteria 6 and 7 exclude patients who may 
not survive to important study endpoints and have limited chance to see benefit from the 
intervention. Criterion 8 limits enrollment of patients that have lower relative rates of mortality 
related to mechanical ventilation or confounds the interpretation of important trial endpoints (eg, 
VFD). Prisoners will not be enrolled due to their protected status as a vulnerable population, and 
preservation of autonomy for these patients will be difficult in the setting of critical illness 
(criterion 9). Exclusion of pregnant women is required because the safety of the single high 
dose of vitamin D is unknown for pregnant women and their fetuses, as this dosing has never 


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been studied even in healthy pregnant women (criterion 10). We would anticipate very few 
pregnant women would meet the eligibility criteria for this trial and thus, this trial would have 
limited potential to gain knowledge about the safety and efficacy of the intervention in pregnant 
women. We will ensure that non-pregnant women of childbearing age are not biased against 
inclusion in the trial.  Criterion 11 limits enrollment to acute conditions and presentations. 
 
Patients who meet inclusion criteria 1-3 and no exclusion criteria will be eligible for participation 
in the trial. After completing the informed consent process, eligible participants will then be 
assessed for randomized study treatment with a screening test for vitamin D deficiency. 
Participants meeting inclusion criteria 4 (screening 25OHD level <20 ng/mL) will be randomized 
1:1 to study drug or placebo. 
4.4 Randomization and Study Initiation Time Window  
All participants with screening 25OHD <20 ng/mL must be randomized within 12 hours of ICU 
admission decision, regardless of patient location. Study medication must be administered 
within 2 hours of randomization.  
4.5 Informed Consent  
All patients meeting inclusion criteria 1-3 will be entered on a screening log. If the patient meets 
an exclusion criterion we will document the reason and collect a minimum data set to the extent 
allowed. We will not seek informed consent. We will seek informed consent from each patient 
not excluded (or from their legally authorized representative (LAR)) prior to Vitamin D screening. 
We will assess the patient’s decisional capacity prior to consent. In order to facilitate possible 
long term follow-up ancillary studies separate from this primary trial, we will request consent to 
obtain social security number and Medicare number for all participants who consent to 
participation (both vitamin D deficient and not vitamin D deficient by the screening vitamin D 
test).  
4.6 Randomization  
After informed consent and completion of vitamin D screening, we will randomize each 
participant that has screened in as vitamin D deficient (screening 25OHD level <20 ng/mL).   
Randomization procedures will be specified by the CCC. Designated research team members 
will have permission to randomize participants. At randomization, each subject will be assigned 
a unique randomization ID number that the site pharmacy will use to determine study arm 
assignment to vitamin D or placebo.  The site research staff, clinical staff, patient and LAR will 
remain blinded to assignment. The CCC will send a confirming email to the randomizer 
containing the randomization ID number. We will stratify randomization by enrolling hospital. 
4.7 Minorities and Women 
No patients will be excluded on the basis of race, ethnicity, or sex. NHLBI considered the 
composition of minorities and women in the target study population in selecting the PETAL 
Network Clinical Centers so that studies reflect the population of the US census. The PETAL 
Network CCC will monitor recruitment of minorities and women. If necessary, additional 
recruitment efforts will be made at specific centers to ensure that the aggregate patient sample 
contains representative race/ethnicity and sex subsets. 


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5 STUDY PROCEDURES 
Trials should be conducted in a setting reflective of good clinical practice that can be clearly 
described and reproduced in a clinical (non-trial) setting. NHLBI selected Clinical Centers 
already providing high quality standardized ED and ICU care. We will monitor the provision and 
results of key processes of care and implement strict protocols (with training, monitoring, and 
feedback) for the administration of vitamin D. Almost all PETAL centers have existing protocols 
and order sets for the routine management of ED and ICU patients 
5.1 Intervention Group (Vitamin D)  
Participants randomized to the intervention group will receive a single enteral (oral or per 
naso/orogastric tube) dose of 540,000 IU vitamin D3 (cholecalciferol). Study medication will be 
provided in liquid form by BioTech Pharmacal (Fayetteville, AR), which has successfully 
provided study medication for prior NIH-sponsored vitamin D clinical trials. Because of the high 
dose of vitamin D and intention to prevent and treat disease, we filed a FDA Investigational New 
Drug (IND) application for this protocol. 
The two primary forms of vitamin D supplementation are enteral pre-hormone vitamin D (e.g., 
cholecalciferol, ergocalciferol) and enteral or parenteral active hormone (e.g., calcitriol or VDR 
agonists [e.g., paricalcitol]). The primary advantages of nutritional vitamin D supplementation 
include greater experience with its use, dosing, and safety; ability to measure dose-response 
using clinically relevant 25OHD levels; and a longer (2 week) half-life. Though VDR agonists 
can be administered parenterally, there are limited safety data with its use and dosing, 
particularly in critically ill patients. Therefore, we have selected nutritional vitamin D 
supplementation for this trial. Single bolus doses of 50,000-750,000 IU vitamin D are safe in the 
general population, and doses up to 540,000 IU in studies of ICU patients have not triggered 
clinically significant hypercalcemia, hypercalciuria, or other known vitamin D-related adverse 
events. Specifically, in a previous clinical trial, a single dose of 540,000 IU to ICU patients was 
safe, rapid, and effective in repleting vitamin D deficiency (25OHD <20 ng/mL).1 We chose this 
relatively high dose because of its known excellent safety profile in ICU patients and to 
maximize the chance to improve clinical outcomes through effective vitamin D repletion. 
5.1.1 Stopping Rules for Vitamin D Administration  
Because the intervention is a single dose of vitamin D, stopping treatment is not relevant to 
individual trial participants. However, trial participants will be followed for endpoints through day 
90, and the overall ongoing safety of the trial and the vitamin D intervention will be closely 
monitored during the conduct of the trial.  
5.2 Randomized Control Group  
Participants randomized to the control group will receive a single liquid enteral dose of placebo 
that is matched to the vitamin D intervention to ensure blinding. 
5.3 Common Strategies for Both Groups  
This trial will not mandate aspects of routine clinical care because 1) the intention is to evaluate 
the efficacy and safety of high dose vitamin D in the context of routine clinical care; 2) there is a 
broad spectrum of conditions that we will enroll in this trial; and 3) randomization in this large 
trial will balance routine care and we anticipate minimal impact on observed differences.  


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We will encourage sites to inform providers of this trial broadly and to ensure communication 
between research and clinical teams of specific participants that are enrolled. This will allow 
clinical decisions to be made in the context of the trial, such as any additional vitamin D 
supplementation or testing, to ensure the safety of participants and limit crossover and 
unblinding. Information on these items will be collected passively from the medical record for 
descriptive summary and analysis.  
6 DATA VARIABLES AND SPECIMENS 
6.1 Background Assessments  
1. Inclusion/exclusion criteria 
2. Demographic and admission data (including age, sex, race/ethnicity, pre-hospitalization 
level of care) 
3. Pertinent medical history (including Charlson co-morbidity score, renal function, chronic 
ventilation) 
4. Height; measured body weight (MBW); calculated predicted body weight (PBW); body 
mass index (BMI) 
5. Ventilator status prior to randomization 
6. Location when inclusion criteria met 
• 
ED, ward, operation room, referring hospital 
7. ICU Admission Service 
• 
Medical 
• 
Surgical scheduled 
• 
Surgical unscheduled 
• 
Trauma 
• 
Other 
6.2 Baseline Assessments  
1. Home vitamin D and calcium supplementation (as medication or supplement including 
multivitamin) 
2. SOFA Score: Cardiovascular, renal, respiratory, hepatic, and hematology organ function, 
as clinically available 
3. Lung Injury Prediction Score (LIPS) 
4. Calcium level, if not performed in routine clinical care 
5. Pregnancy test for women of reproductive age, if not performed in routine clinical care 
6. Health-related quality of life by EuroQol (EQ-5D-5L) 
6.3 Assessment during Study  
We will collect the following in-hospital data by medical record review to assess trial efficacy and 
safety endpoints. Most data may be assessed by medical record review.  
1. Duration of ventilation to day 28 (may require call to LTAC facility if hospital discharge 
with assisted breathing prior to day 28). 
2. Organ failure assessments daily to day 7 (ARDS, creatinine, and vasopressor use), 
using clinically available data. 


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3. In-hospital vitamin D supplementation daily to day 14 (medication only, not related to 
dietary intake or nutritional supplementation). 
4. In-hospital calcium levels daily to day 14, (required on day 3 for first 300 randomized 
participants, otherwise if clinically available). 
5. Adverse events and serious adverse events to day 14 
6. Safety endpoints (i.e., kidney stones and fall related fractures) 
7. Vital status and ventilator status (among survivors) at hospital discharge 
8. Date of hospital discharge or date of death, as applicable 
9. Hospital discharge disposition. “Home” will be defined as pre-hospitalization level of 
care. 
6.3.1 Specimen Collection  
We will collect 15 mL of blood at baseline (within 2 hours of randomization) for all randomized 
participants and 15 mL of blood at study day 3 for approximately the first 300 randomized 
participants (300 participants with match pairs of baseline and day 3 samples). We will measure 
plasma 25OHD on baseline samples for all randomized participants and on study day 3 
samples for approximately the first 300 randomized participants.  Additionally, we will measure 
plasma IL-6 at baseline and on study day 3 for approximately the first 300 randomized 
participants. Some of the blood collected on randomized participants at baseline will also be 
used for banking of plasma and DNA for future research. 
Table 1 Specimen Collection Schedule 
Subjects 
Collection Type 
Draw Time Points 
All randomized subjects 
Plasma for 25OHD 
Baseline 
Plasma for banking 
Whole blood for DNA banking 
Approximately the first 300 
randomized subjects 
Plasma for IL-6 
Baseline & Day 3 
Plasma for banking 
Day 3 
Plasma for 25OHD 
Day 3 
The 25OHD and IL-6 samples will be shipped to a central lab for batched measurements.  The 
baseline 25OHD samples will be measured by the LC/MS/MS method and compared to the 
screening test results, initially in monthly increments for quality control purposes. The samples 
for future research (plasma and whole blood for DNA) will be shipped to the network central 
repository.  All samples will be identified by a coded number during shipment and storage in 
both the central repository and central lab. 
The primary purpose of this trial is to evaluate efficacy and safety of the proposed vitamin D 
intervention, and we have attempted to limit additional data and specimen collection that extend 
beyond this aim. Accordingly, we designed the limited biospecimen collection plan to confirm 
that the vitamin D dose successfully corrects vitamin D deficiency and changes an important 
biomarker in critical illness (IL-6); this requires a small subsample (n=300) to evaluate. In 
addition, we will be able to stratify results based on clinical variables and vitamin D levels to see 
if there are subgroups that may have more or less potential to benefit. Although additional 
explanatory and mechanistic data collection would be beyond the scope (and budget) of the 
present trial, we will store baseline plasma/DNA to facilitate additional (ancillary) studies that 
would be potentially useful in explaining negative or positive results.  


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6.4 Assessments after Hospitalization 
For participants discharged to home or post-acute care facilities (e.g., LTAC facilities, SNF, or 
acute rehabilitation facilities) prior to day 90, we will obtain study endpoints by a telephone call 
with the patient, LAR, or facility staff to assess: 
1. Vital status at day 90 and date of death (if applicable) 
2. Location at day 90 (home, LTAC/SNF/acute rehab, hospital) 
3. Date of discharge home (prior level of care) for participants discharged from the hospital 
to post-acute care facility 
4. Safety endpoints (kidney stones and fall-related fractures) 
5. Health-related quality of life by EuroQol (EQ-5D-5L) 
 
The EQ-5D-5L53 is a brief, validated, reliable, and widely used54 QOL instrument that 
determines whether respondents have problems in each of five domains: mobility, self-care, 
usual activities, pain/discomfort, and anxiety/depression. Responses can be used to calculate a 
health utility score55 associated with the given health state that ranges from -0.11 to 1.00 (higher 
scores are better; 1.00 is perfect health).  
At the 90-day follow-up, patients may still be impaired. While proxies differ from primary 
respondents on almost all instruments, including the EQ-5D-5L,56,57 we are hopeful that proxy 
respondents will not be differentially distributed among intervention and control, allowing us to 
minimize confounding related to proxy vs. individual responses. 
We will collect contact information for the patient and alternative contact information for up to 3 
individuals. For participants who we are unable to contact by phone, we will use evidence from 
the medical record (e.g., healthcare visits after day 90) to identify participants that are still alive. 
We will also verify duration of survival for participants lost to follow-up or noted to have died 
using the Centers for Disease Control and Prevention’s National Death Index (NDI) and other 
federal databases. We will use each patient’s social security number (SSN) for an exact NDI 
match. Although we do not plan to collect long-term outcomes as part of primary trial endpoints, 
we will record Medicare number to facilitate future ancillary studies that wish to link with 
Medicare data on utilization and functional status. 
7 STATISTICAL CONSIDERATIONS  
7.1 Statistical Methods Primary Analyses 
The primary endpoint is intention to treat all-cause, all-location 90 day mortality in the confirmed 
vitamin D deficient cohort (LC/MS/MS <20 ng/mL). We will conduct analyses on a modified 
intention to treat basis (all randomized participants who received either vitamin D formulation or 
placebo in the confirmed vitamin D deficient cohort). 
Sample size is based on a comparison of 
binomial proportions with an overall one-
sided alpha level of 0.025. With 20% mortality 
rate in the control group and 15% rate in the 
vitamin D group, three interim looks at the 
data, and 80% of randomized patients with 
Figure 4 Study Flow: analysis plan 


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confirmed vitamin D deficiency by the LC/MS/MS method, the study will have a power of 87.4%. 
The trial will stop when the screened vitamin D deficient cohort reaches a maximum 3000 
randomized participants.  
The presumed 20% mortality rate in the control group is based on several recently published 
clinical trials. In the original Amrein study, mortality prior to discharge home before day 90 was 
40% in the control group.1 Because these participants were enrolled a median 3 days after ICU 
admission (some lower risk participants were already discharged from the ICU) and European 
ICUs tend to admit sicker patients that U.S. ICUs (some lower risk ICU patients in the U.S. 
would be managed in a non-ICU setting in Austria), we anticipate a lower mortality in our trial. In 
the ProCESS trial, which recruited ED patients early in the course of septic shock, the 90 day 
mortality was 32%.58 While similar septic shock patients will be recruited in VIOLET, other 
eligible patients (in addition to septic shock) will likely have a modestly lower mortality. For 
example, in the LIPS-A trial, which enrolled ED patients across broader ARDS criteria, the 
subgroup of patients admitted to the ICU had 11% in-hospital mortality (vs. 19% in-hospital 
mortality in ProCESS). While 90 day mortality was not collected in the LIPS-A trial, we would 
anticipate a 90 day mortality rate of approximately 18%. However, LIPS-A excluded many 
potentially eligible patients because of aspirin use, which selected against an older, more 
chronically ill population. These patients would be included in VIOLET (no aspirin exclusion 
criteria), which would likely increase the event rate. 
We plan to have three interim analyses at 750, 1500, and 2250 randomized (screened vitamin D 
deficient) participants. In addition, there will be an initial evaluation specifically of 25OHD and 
calcium levels after the first 300 randomized participants to confirm that the intervention repletes 
vitamin D deficiency without hypervitaminosis D or clinically significant hypercalcemia, 
consistent with prior trials using the same dose in ICU patients. DSMB meetings will occur as 
close to these planned analyses as scheduling allows. The study will be stopped for efficacy 
based on an analysis of the confirmed vitamin D deficient (LC/MS<20 ng/mL) participants.  
There will be an efficacy stopping rule at each interim analysis based on a the Lan-DeMets 
alpha spending function of 0.025t^4, where t is the proportion of the sample size that has 
accrued.  
In addition, Berry Consultants developed the futility stopping rules for this study according to the 
following specification. The study will continue until there are 3000 patients randomized, unless 
it stops early for efficacy or futility. The futility stopping rules will be based on the death rates in 
each treatment and the proportion of randomized participants who have LC/MS/MS level <20 
ng/mL (ie, the inclusion rate). The rule will calculate the predictive probability that the trial will 
reach statistical significance, when 3000 total randomized participants are enrolled. The futility 
rules will not be binding on the DSMB, as they will have other information on the secondary 
analyses of the study as well as information from outside the study.  The following table gives 
the power of the study under different scenarios under the assumption that 80% of the 
randomized participants will have a LC/MS/MS level <20 ng/mL. The plot below shows 
individual simulated trials with colors indicating whether they were positive and whether they 
were stopped early for futility. 
 
 


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Futility Stopping Boundaries 
 
 
7.2 Statistical Methods for the Screened Vitamin D Deficient Cohort 
The effect of treatment will be estimated in the screened vitamin D deficient cohort.  
Furthermore we will use a quadratic smoothing spline to estimate the relationship between the 
treatment effect and the baseline 25OHD level.  
8 DATA COLLECTION AND SITE MONITORING  
8.1 Data Collection  
The research staff will collect and record data on paper collection sheets or in a custom-
designed computer database. Data will be transferred to the Clinical Coordinating Center on a 
prescribed basis through a web-based data collection program. There will be recommendations 
but no protocol-mandated aspects of routine clinical care.  
8.2 Site Monitoring  
Data quality will be reviewed remotely using front-end range and logic checks at the time of data 
entry and back-end monitoring of data using SAS reports. Additionally, Clinical Center on site 
visits will be performed on a regular basis by the Clinical Coordinating Center to ensure that all 
regulatory requirements are being met and to monitor data quality. Patient records and case 
report forms will be examined on a spot check basis to evaluate the accuracy of the data 
entered into the database and monitor for protocol compliance. 
8.3 Vitamin D Screening Quality Assurance 
We will utilize a quality assurance plan to monitor accuracy of Vitamin D screening using 
Qualigen FastPack.  


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9 RISK ASSESSMENT  
9.1 Potential Risks to Subjects 
As noted in Section 5, high dose enteral vitamin D appears to be safe in critically ill patients with 
no related adverse events reported in a recent large trial using the dose proposed for this 
study.1 As such, the potential risks below are theoretical and have not been observed in 
previous clinical trials. 
Hypercalcemia: The primary risk associated with use of vitamin D supplementation is 
hypercalcemia, which may cause gastrointestinal symptoms, dizziness, fatigue, and less 
commonly cardiac arrhythmia. This risk is largely theoretical, as trials using similar vitamin D 
doses as in this protocol, including in ICU patients, have not observed clinically significant 
hypercalcemia. For example, in the largest trial to date using 540,000 IU vitamin D3 as the 
intervention (Amrein et al, n=475),1 the maximum total serum calcium level observed in the 
vitamin D group during the first 28 days was 11.2 mg/dL with no clinically or statistically 
significant difference in mean total serum or ionized calcium levels. To enhance trial safety, we 
will exclude patients with baseline hypercalcemia or 25OHD levels ≥20 ng/mL. 
We will confirm the minimal effect of acute vitamin D repletion on calcium levels by collecting 
and analyzing clinically available calcium levels to day 14 (after which 25OHD levels decline). 
We will also measure day 3 calcium levels (after plateau of 25OHD levels) as protocol specified 
testing in the first 300 randomized participants, and in additional participants or timepoints if 
required by the DSMB or cIRB to monitor ongoing safety.  
Kidney stones: While an association between hypervitaminosis D and kidney stones has been 
suggested, the relationship with high dose vitamin D supplementation and kidney stones 
remains controversial. However, we will exclude patients with a personal history of recent (past 
year) or multiple prior kidney stones and will closely monitor participants for incident kidney 
stones during the hospitalization and follow-up period. 
Fall-related fractures: The association between acute vitamin D supplementation and 
falls/fractures remains unclear. Some data suggest that higher doses of daily vitamin D 
supplementation (1,000-4,000 IU per day) may improve muscle function and balance, and thus 
decrease falls, which is also highlighted by a recent American Geriatrics Society clinical 
guideline recommending higher doses of daily vitamin D supplementation for fall prevention.60 In 
addition, the National Institute on Aging has recently sponsored a large phase III clinical trial 
with this hypothesis for evaluating vitamin D and fall prevention (NCT02166333). However, one 
report published in JAMA suggested that a single dose of 500,000 IU vitamin D supplementation 
annually increased the incidence of falls and possibly fractures in healthy older women.61 In 
addition, two recent trials in older adults found that monthly vitamin D supplementation (60,000-
100,000 IU) increased the incidence of falls but not fractures during the 1 year follow-up62,63. 
The recent VITdAL-ICU trial showed no difference in falls with 540,000 IU vitamin D3 in critically 
ill patients (6 month fall rate: 17.7% vitamin D and 24.3% placebo; p=0.17) with no difference in 
the rate of fractures (two fractures per group at 6 months follow-up).1 Because there remains 
some uncertainty around this issue, we will track fall-related fractures during the hospitalization 
and at the 90 day phone call as a secondary endpoint that is reviewed by the DSMB and 
reported in the final manuscript. 


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Placebo for those with vitamin D deficiency: Although vitamin D levels are not routinely 
measured as part of clinical inpatient care, during the course of this protocol clinicians may 
detect previously unrecognized vitamin D deficiency. However, the time span for vitamin D 
repletion to receive known benefit (e.g., osteoporosis prevention) is on the time scale of years. 
Acute vitamin D repletion is not the current standard of care in hospitalized patients for any 
clinical outcome. Thus, it is proposed that there is clinical equipoise to randomize vitamin D 
deficient participants to the placebo group for the duration of the study. 
Phlebotomy: All participants will have blood drawn for research purposes. As almost all 
participants will have invasive lines placed for clinical purposes, risk of blood draws are 
essentially nil, as blood can be easily obtained from these lines. In the rare case an invasive line 
is not present, the risks of drawing blood are uncommon and include bleeding and bruising. 
Commonly, drawing blood is painful, and rarely, drawing blood can lead to infections at the site 
of the blood draw. 
9.2 Minimization of risk 
Federal regulations at 45 CFR 46.111(a)(1) require that risks to participants are minimized by 
using procedures which are consistent with sound research design. There are several elements 
of study design inherent in the present protocol that meets this human subject protection 
requirement. High dose vitamin D has been used safely in a number of populations, including 
ICU patients. We selected the 540,000 IU dose as the highest dose studied in ICU patients with 
reassuring safety profile. In addition, we will exclude patients with baseline screening 25OHD 
levels ≥20 ng/mL, hypercalcemia, or significant kidney stones to further mitigate potential for 
risk. We anticipate that approximately 20% of randomized participants will have baseline 
25OHD levels ≥20 ng/mL by LC/MS/MS measurement. Based on our pilot data, nearly all of 
these participants will have a baseline LC/MS/MS level <30 ng/mL, and in this range, we believe 
there is still potential for benefit and low risk for vitamin D-related adverse events. Based on 
pilot data, we anticipate that the screening 25OHD testing will prevent randomization of 
participants with baseline LC/MS/MS levels >40 ng/mL, which further minimizes risk for 
participants. The DSMB will review data as outlined above and will examine not only efficacy but 
safety (inclusive of mortality) and may recommend halting the study at any time. 
9.3 Potential Benefits  
Study participants may or may not receive any direct benefits from their participation in this 
study. Early vitamin D repletion may result in reduced incidence and severity of ARDS and a 
lower associated morbidity and mortality. 
9.4 Risks in Relation to anticipated benefit  
Federal regulations at 45 CFR 46.111 (a)(2) require that “the risks to subjects are reasonable in 
relation to anticipated benefits, if any, to subjects, and the importance of the knowledge that 
may reasonably be expected to result.” Based on the preceding assessment of risks and 
potential benefits, the risks to subjects are reasonable in relation to anticipated benefits.  
9.4.1 Procedures 
Blood draws: The risks associated with these common clinical practices are small, however the 
knowledge gained in furthering our understanding of the pathophysiology and potentially leading 
to better and targeted therapy may be substantial. 


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9.4.2 Intervention  
High dose vitamin D has been previously studied in ICU patients and there is substantial data to 
support safety and potential efficacy. 
9.5 Safety Monitoring  
An independent Data and Safety Monitoring Board will monitor trial progress to determine if the 
study should stop for safety, futility, or efficacy. The DSMB will also monitor trial quality and 
feasibility. The first analysis will occur after randomization of 300 patients and will include a 
review of plasma 25OHD levels and safety data for the first 150 vitamin D-treated patients. 
Based on dose-response data in ICU patients1,49, we anticipate that the peak level will occur by 
day 3. We anticipate that the mean 25OHD level among vitamin D-treated participants will be 
~35-40 ng/mL, with the nearly all being in the normal range (20-80 ng/mL). Acute vitamin D 
toxicity has been reported only with 25OHD levels >150 ng/mL, with hypervitaminosis D being 
defined as >120 ng/mL. If mean 25OHD levels are lower than 30 ng/mL and there is no 
evidence of hypercalcemia, hypervitaminosis D, or other safety concerns, then the Steering 
Committee may recommend increasing the vitamin D dose. If mean 25OHD levels are higher 
than 50 ng/mL, then the Steering Committee may recommend decreasing the vitamin D dose. 
The DSMB will then review recommendations to modify the current dose. The DSMB will have 
access to unblinded rates of adverse events, clinical and laboratory findings, and study 
endpoints. The DSMB may also recommend prolonged (beyond 300 randomized patients) or 
additional monitoring of 25OHD or calcium levels if more data are deemed necessary to monitor 
trial safety. Additional DSMB reviews will occur at interim analyses after randomization of 750 
and 1500 and 2250 randomized participants and include safety, efficacy, and feasibility data 
(see Section 7, Statistical Considerations). 
10 HUMAN SUBJECTS 
Each study participant or a LAR must sign and date an informed consent form. Institutional 
review board approval will be required before any subject is entered into the study. PETAL will 
use a central IRB. 
10.1 Selection of subjects 
Federal regulations at 45 CFR 46(a)(3) require the equitable selection of subjects. The EDs, 
operating rooms, hospital wards, and ICUs of PETAL sites will be screened to determine if any 
patient meets inclusion and exclusion criteria. Data that have been collected as part of the 
routine management of the subject will be reviewed to determine eligibility. If any patient meets 
criteria for study enrollment, then the attending physician will be asked for permission to 
approach the patient or his/her LAR for informed consent. The screening vitamin D test will be 
performed as part of the eligibility process after written consent obtained. Study exclusion 
criteria neither unjustly exclude classes of individuals from participation in the research nor 
unjustly include classes of individuals from participation in the research. Hence, the recruitment 
of subjects conforms to the principle of distributive justice. 
10.2 Justification of including vulnerable subjects  
The present research aims to investigate the safety and efficacy of vitamin D repletion to 
improve clinical outcomes in vitamin D deficient patients at high risk for ARDS and mortality. 
Due to the nature of this patient population, the majority of these patients will have impaired 


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decision-making capabilities. Moreover, those with intact decision-making capacities probably 
have milder disease than those with impaired capacity. Therefore, this study could not be 
conducted if limited to enrolling only those subjects with retained decision-making capacity. 
Hence, subjects recruited for this trial are not being unfairly burdened with involvement in this 
research simply because they are easily available. 
10.3 Informed consent  
Federal regulations 45 CFR 46.111(a)(5) require that informed consent will be sought from each 
prospective subject or the subject’s LAR. We anticipate many consents will be from the 
subject’s LAR, and thus the remainder of this section will focus on LARs. Study personnel 
obtaining informed consent are responsible for ensuring that the LAR understands the risks and 
benefits of participating in the study, answering any questions the LAR may have throughout the 
study and sharing any new information in a timely manner that may be relevant to the LAR’s 
willingness to permit the subject’s continued participation in the trial. The consenter will make 
every effort to minimize coercion. All study participants or their LARs will be informed of the 
objectives of the study and the potential risks. The informed consent document will be used to 
explain the risks and benefits of study participation to the LAR in simple terms before the patient 
is entered into the study, and to document that the LAR is satisfied with his or her understanding 
of the risks and benefits of participating in the study and desires to participate in the study. The 
investigator is responsible for ensuring that informed consent is given by each LAR. This 
includes obtaining the appropriate signatures and dates on the informed consent document prior 
to the performance of any protocol procedures including administration of study agent. 
10.4 Continuing consent  
Subjects for whom consent was initially obtained from a LAR, but who subsequently regain 
decision-making capacity while in hospital will be approached for consent for continuing 
participation, including continuance of data acquisition. The consent form signed by the LAR 
should reflect that such consent should be obtained. 
10.5 Withdrawal of consent  
Participants may withdraw or be withdrawn (by the LAR) from the trial at any time without 
prejudice. Data recorded up to the point of withdrawal will be included in the trial analysis, 
unless consent to use their data has also been withdrawn. If a patient or LAR wishes to 
withdraw from the trial after completion of trial treatment, permission to access medical records 
for trial data will be sought. Due to the single dose of study drug, it will not be possible for 
subjects or their LARs to withdraw consent to terminate the study drug. Withdrawal of consent 
prior to randomization will constitute a screen-failure. Withdrawal of consent after randomization 
will lead to subject discontinuation but site staff will request access to medical records for data 
related to the trial.  
10.6 Identification of legally authorized representatives 
Many of the patients approached for participation in this research protocol will have limitations of 
decision-making abilities due to their critical illness. Hence, most patients will not be able to 
provide informed consent. Accordingly, informed consent will be sought from the potential 
subject’s legally authorized representative (LAR).  


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Regarding proxy consent, the existing federal research regulations (‘the Common Rule’) states 
at 45 CFR 46.116 that “no investigator may involve a human being as a subject in 
research…unless the investigator has obtained the legally effective informed consent of the 
subject or the subject’s legally authorized representative”; and defines at 45 CFR 46 102 (c) a 
legally authorized representative (LAR) as “an individual or judicial or other body authorized 
under applicable law to consent on behalf of a prospective subject to the subject’s participation 
in the procedures(s) involved in the research.” The Office of Human Research Protections 
(OHRP) defined examples of “applicable law” as being state statutes, regulations, case law, or 
formal opinion of a State Attorney General that addresses the issue of surrogate consent to 
medical procedures. Such “applicable law” could then be considered as empowering the LAR to 
provide consent for subject participation in the research. Interpretation of “applicable law” may 
be state specific and will be addressed by the PETAL central IRB. 
According to a previous President’s Bioethics Committee (National Bioethics Advisory 
Committee (NBAC)), an investigator should accept a relative or friend of the potential subject 
who is recognized as an LAR for purposes of clinical decision making under the law of the state 
where the research takes place 64. Finally, OHRP has stated in their determination letters that a 
surrogate could serve as a LAR for research decision making if such an individual is authorized 
under applicable state law to provide consent for the “procedures” involved in the research 
study. 65 
10.7 Justification of surrogate consent  
According to the Belmont Report, respect for persons incorporates at least two ethical 
convictions; first, that individuals should be treated as autonomous agents, and second, that 
person with diminished autonomy are entitled to protection. One method that serves to protect 
subjects is restrictions on the participation of subjects in research that presents greater than 
minimal risks. Commentators and research ethics commissions have held the view that it is 
permissible to include incapable subjects in greater than minimal risk research as long as there 
is the potential for beneficial effects and that the research presents a balance of risks and 
expected direct benefits similar to that available in the clinical setting 66. Several U.S. task 
forces have deemed it is permissible to include incapable subjects in research. For example, the 
American College of Physicians’ document allows surrogates to consent to research involving 
incapable subjects only “if the net additional risks of participation are not substantially greater 
than the risks of standard treatment.” 67. Finally, NBAC stated that an IRB may approve a 
protocol that presents greater than minimal risk but offers the prospect of direct medical benefits 
to the subject, provided that “the potential subject’s LAR gives permission…” 64 
Consistent with the above ethical sensibilities regarding the participation of decisionally 
incapable subjects in research and the previous assessment of risks and benefits in the 
previous section, the present trial presents a balance of risks and potential direct benefits that is 
similar to that available in the clinical setting, with the exception of the additional blood draws. 
10.8 Additional safeguards for vulnerable subjects 
The present research will involve subjects who might be vulnerable to coercion or undue 
influence. As required in 45CFR46.111(b), we recommend that sites utilize additional 
safeguards to protect the rights and welfare of these subjects. Such safeguards might include, 
but are not limited to: a) assessment of the potential subject’s capacity to provide informed 


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consent, b) the availability of the LAR to monitor the subject’s subsequent participation and 
withdrawal from the study. The specific nature of the additional safeguards will be left to the 
discretion of the central IRB, in conjunction with the sites. 
10.9 Confidentiality  
Federal regulations at 45 CFR 46 111 (a) (7) requires that when appropriate, there are 
adequate provisions to protect the privacy of subjects and to maintain the confidentiality of data. 
To maintain confidentiality, all laboratory specimens, evaluation forms, and reports will be 
identified only by a coded number. The coded number will be generated by a computer, and 
only the study team will have access to the codes. All records will be kept in a locked, password 
protected computer. All computer entry and networking programs will be done with coded 
numbers only. All paper case report forms will be maintained inside a locked office. Study 
information will not be released without the written permission of the patient, except as 
necessary for monitoring by the National Heart, Lung, and Blood Institute, and the PETAL 
Clinical Coordinating Center or their designees. 
11 ADVERSE EVENTS  
11.1 Safety Monitoring  
Assuring patient safety is an essential component of this protocol. Each participating 
investigator has primary responsibility for the safety of the individual participants under 
his or her care. A clinical trial adverse event is defined as any untoward medical event 
associated with the use of a drug or study procedure in humans, whether or not it is 
considered related to a drug or study procedure. 
The Investigators will determine daily if any adverse events occur during the period from 
enrollment (signing of the informed consent) through study day 14 or hospital discharge, 
whichever occurs first. Investigators will determine if the event is serious or related to the 
study drug. The rationale for this time window is the 2 week half-life of 25OHD, which 
helps to define the period at risk from vitamin D. 
The following adverse events will be collected in the adverse event case report forms: 
• 
Serious adverse events 
• 
Non-serious adverse events that are considered by the investigator to be related 
to study drug or study procedures  
Investigators will assess if there is a reasonable possibility that the study drug or procedure 
caused the event. Investigators will also consider if the event is explained by the patient’s 
underlying medical conditions, anticipated clinical course, previous medical conditions, and 
concomitant medications.  
 
 


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11.2 Serious Adverse Events  
A serious adverse event is any adverse event that results in one of the following outcomes: 
• 
Death 
• 
A life-threatening experience (that is, immediate risk of dying) 
• 
Prolonged inpatient hospitalization 
As per http://www.fda.gov/Safety/MedWatch/HowToReport/ucm053087.htm: Report if 
admission to the hospital or prolongation of hospitalization was a result of the adverse 
event. Emergency room visits that do not result in admission to the hospital should be 
evaluated for one of the other serious outcomes (e.g., life-threatening; required 
intervention to prevent permanent impairment or damage; other serious medically 
important event). 
• 
Persistent or significant disability/incapacity 
As per http://www.fda.gov/Safety/MedWatch/HowToReport/ucm053087.htm: Report if 
the adverse event resulted in a substantial disruption of a person's ability to conduct 
normal life functions, i.e., the adverse event resulted in a significant, persistent or 
permanent change, impairment, damage or disruption in the patient's body 
function/structure, physical activities and/or quality of life. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious adverse events when, based upon appropriate 
medical judgment, they may jeopardize the patient and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. 
Serious adverse events will be collected during the first 14 study days or until hospital 
discharge, whichever occurs first, regardless of the investigator's opinion of causation. 
Thereafter, serious adverse events are not required to be reported unless the investigator feels 
the events were related to either study drug or a protocol procedure. 
Study site personnel must alert the CCC of any serious and study drug or study procedure 
related adverse event within 24 hours of investigator awareness of the event. Alerts issued via 
telephone are to be immediately followed with official notification on the adverse event case 
report form. See Appendix for reporting timelines for serious, unexpected, study related events 
(SAEs) and serious, unexpected suspected adverse reactions (SUSARs).  
 
 


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APPENDICES  
A. 
Time-Events Schedule 
Measurement/Event 
0 
1 
2 
3 
4 
5 
6 
7 
8-14 
HOSP 
D/C 
90 
Demographics, history and physical 
X 
 
 
 
 
 
 
 
 
 
 
HCG (females) 
X 
 
 
 
 
 
 
 
 
 
 
Screening 25OHD test 
X 
 
 
 
 
 
 
 
 
 
 
Risk factors for ARDS and mortality 
X 
 
 
 
 
 
 
 
 
 
 
Home meds (calcium and Vitamin D) 
X 
 
 
 
 
 
 
 
 
 
 
Charlson Co-morbidity score 
X 
 
 
 
 
 
 
 
 
 
 
SOFA 
X 
 
 
 
 
 
 
 
 
 
 
LIPS 
X 
 
 
 
 
 
 
 
 
 
 
Study drug administration 
X 
 
 
 
 
 
 
 
 
 
 
ARDS Assessment 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Vasopressors (yes/no) 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Highest creatinine 
 
A 
A 
A 
A 
A 
A 
A 
 
 
 
Serum albumin level  
A 
A 
A 
A 
A 
A 
A 
A 
A 
 
 
On-study Vitamin D medication 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Calcium level (safety labs)† 
X 
A 
A 
X 
A 
A 
A 
A 
A 
 
 
Ventilator status 
X 
 
 
 
 
 
 
 
 
X‡ 
 
Plasma for IL-6 (first 300 randomized 
participants) 
X 
 
 
X 
 
 
 
 
 
 
 
Plasma for banking† 
X 
 
 
X 
 
 
 
 
 
 
 
Whole blood for DNA banking 
X 
 
 
 
 
 
 
 
 
 
 
Plasma for 250HD level† 
X 
 
 
X 
 
 
 
 
 
 
 
EQ-5D-5L 
X 
 
 
 
 
 
 
 
 
 
X 
Length of stay (hospital and 
healthcare facility) 
 
 
 
 
 
 
 
 
 
X 
X 
Assessment of kidney stones and fall 
related fractures 
 
 
 
 
 
 
 
 
 
X 
X 
Vital status 
 
 
 
 
 
 
 
 
 
X 
X 
X: required 
A: if available 
†: Baseline required for all randomized participants; Day 3 required for first 300 randomized participants only 
‡: If patient discharged from hospital prior to day 28 with ventilator-assisted breathing, day 28 ventilator status 
should be obtained (e.g., phone call to LTAC) 
 
 
 
 


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B. 
Imputed P/F using SpO2 and FiO2 
The equivalence table below determines the estimated P/F ratio from the FiO2 and SpO2. This 
data was generated by investigators at the University of Utah, on a cohort of critically ill patients 
with pneumonia 68-74. 
 
For altitude adjustment, we will incorporate the practice from prior ARDS Network studies of 
using average ambient to sea level barometric pressure (for Utah, 0.86; for Denver, 0.84). 
Additional requirements for the use of the S/F ratio include: 
1. SpO2 between 80-96% 
2. SpO2 should be measured at least 10 minutes after any change in FiO2. 
3. PEEP ≥ 5 cm H2O 
4. An adequate pulse oximeter waveform tracing 
 
 


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C. 
Adverse Event Reporting and Unanticipated Problems 
 As noted in section 11, investigators will report all adverse events that are serious and study 
drug or study procedure related to the CCC within 24 hours. The CCC will then notify the NHLBI 
and cIRB. 
The Medical Monitor at the CCC will work collaboratively with the reporting investigator to 
determine if a serious adverse event has a reasonable possibility of having been caused by the 
study drug or procedure (see Appendix C2). The Medical Monitor will also determine if the event 
is unexpected. An adverse is considered “unexpected” if it is not listed in the investigator 
brochure or the study protocol (21 CFR 312.32(a)). If a determination is made that a serious 
adverse event has a reasonable possibility of having been caused by the drug, it will be 
classified as a suspected adverse reaction. If the suspected adverse reaction is unexpected, it 
will be classified as a serious unexpected suspected adverse reaction (SUSAR).  
The CCC will report all unexpected and study related deaths, SAEs, and SUSARs to the DSMB, 
NHLBI, and cIRB within 7 days after receipt of the report from a clinical site. A written report will 
be sent to the NHLBI, DSMB, FDA and the cIRB within 15 calendar days. All unexpected and 
study related deaths and life threatening SUSARS will be reported to the Food and Drug 
Administration (FDA) within 7 days; all other SUSARS will be reported to the FDA within 15 
days. The DSMB will also review all adverse events and clinical outcomes during scheduled 
interim analyses. If the DSMB determines that the overall rate of adverse events is higher in the 
vitamin D group than the control group the cIRB and the FDA will be notified within 15 days of 
this determination (the latter via an IND safety report (21 CFR 312.32(c)(1)(i)(A)). The CCC will 
distribute the written summary of the DSMB’s periodic review of adverse events to the cIRB in 
accordance with NIH guidelines (http://grants.nih.gov/grants/guide/notice-files/not99-107.html). 
Unanticipated Problems (UP)  
Investigators must also report Unanticipated Problems, regardless of severity, associated with 
the study drug or study procedures within 24 hours. An unanticipated problem is defined as 
follows: any incident, experience, or outcome that meets all of the following criteria: 
 Unexpected, in terms of nature, severity, or frequency, given the research procedures that 
are described in the protocol-related documents, such as the IRB-approved research 
protocol and informed consent document; and the characteristics of the subject population 
being studied; 
 Related or possibly related to participation in the research, in this guidance document, 
possibly related means there is a reasonable possibility that the incident, experience, or 
outcome may have been caused by the procedures involved in the research; 
 Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
Determining Relationship of adverse events to study drug or procedures 
Investigators will be asked to grade the strength of the relationship of an adverse event to 
vitamin D or study procedures as follows: 
 


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• 
Definitely Related: The event follows: a) A reasonable, temporal sequence from a study 
procedure; and b) Cannot be explained by the known characteristics of the patient’s 
clinical state or other therapies; and c) Evaluation of the patient’s clinical state indicates 
to the investigator that the experience is definitely related to study procedures. 
 
• 
Probably or Possibly Related: The event should be assessed following the same criteria 
for “Definitely Associated”. If in the investigator’s opinion at least one or more of the 
criteria are not present, then “probably” or “possibly” associated should be selected. 
 
• 
Probably Not Related: The event occurred while the patient was on the study but can 
reasonably be explained by the known characteristics of the patient’s clinical state or 
other therapies. 
 
• 
Definitely Not Related: The event is definitely produced by the patient’s clinical state or 
by other modes of therapy administered to the patient. 
. 
• 
Uncertain Relationship: The event does not meet any of the criteria previously outlined. 
 
Events that are definitely, probably, or possibly related or of unknown relationship will be 
considered “study related” for the purposes of expedited reporting to the CCC. As noted above, 
the Medical Monitor at the CCC will work collaboratively with the reporting investigator to make 
a final determination if an adverse event or reaction has a reasonable possibility of having been 
caused by the study drug or procedure as outlined in 21 CFR 312.32(a)(1). 
 
Clinical Outcomes that may be exempt from adverse event reporting  
Study-specific clinical outcomes are exempt from adverse event reporting unless the 
investigator deems the event to be related to the study drug or the conduct of study procedures 
(or of uncertain relationship). The following are examples of events that will be considered study 
specific clinical outcomes: 
• 
Death not related to the study drug or procedures.  
• 
Multiple organ failures  
• 
Hypercalcemia 
• 
Kidney stone 
• 
Falls resulting in fractures 
 
 


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D 
Data and Safety Monitoring Board 
The principal role of the DSMB is to assure the safety of participants in the VIOLET trial. They 
will regularly monitor data from this trial, review and assess the performance of its operations, 
and make recommendations to the NHLBI with respect to: 
• 
Review of adverse events 
• 
Interim results of the study for evidence of efficacy or adverse events 
• 
Possible early termination of the trial because of early attainment of study objectives, 
safety concerns, or inadequate performance 
• 
Possible modifications in the clinical trial protocol 
• 
Performance of individual centers 
Three interim analyses will be conducted at approximately 25%, 50%, and 75% of target 
enrollment accrual. 
 
The NHLBI PETAL Network DSMB is appointed by the Director, NHLBI and makes 
recommendations to the NHLBI Director. The DSMB reviews all protocols for safety following 
review by an independent NHLBI Protocol Review Committee. The DSMB will consist of 
members with expertise in acute lung injury, emergency medicine, biostatistics, ethics, and 
clinical trials. An NHLBI staff member not associated with PETAL will serve as Executive 
Secretary. Appointment of all members is contingent upon the absence of any conflicts of 
interest. All the members of the DSMB are voting members. The Principal Investigator and the 
Medical Monitor of the CCC will be responsible for the preparation of all DSMB and adverse 
event reports and may review unblinded data. The DSMB will develop a charter and review the 
protocol and sample consent form during its first meeting. Subsequent DSMB meetings will be 
scheduled in accordance with the DSMB Charter with the assistance of the CCC. When 
appropriate, conference calls may be held in place of face-to-face meetings. Recommendations 
to end, modify, or continue the trial will be prepared by the DSMB executive secretary for review 
by Director, NHLBI. Recommendations for major changes, such as stopping, will be reviewed by 
the NHLBI Director and communicated immediately. Other recommendations will be reviewed 
by the NHLBI director and distributed in writing to the CCC, which will distribute to the PETAL 
steering committee with instructions for reporting to local IRBs when appropriate.  
Details of the NHLBI policies regarding DSMBs can be found at the following URL: 
http://www.nhlbi.nih.gov/funding/policies/dsmb_inst.htm  
 
E 
PETAL Network Steering Committee 
The PETAL Network Steering Committee is comprised of the Principal Investigators and Co-
investigators of all the Clinical sites, the CCC, and the NHLBI Project Officer who represents the 
NHLBI. All sites have two votes and the CCC has one.  
 
 


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F 
SOFA Scoring System  
 
SOFA Score 
0 
1 
2 
3 
4 
RespirationA 
PaO2/FIO2 (mm Hg) or 
imputed P/F using 
SaO2/FIO2 
>400 
<400 
<300 
<200 
<100 
Coagulation 
Platelets 103/mm3 
>150 
<150 
<100 
<50 
<20 
Liver 
Bilirubin (mg/dL) 
<1.2 
1.2-1.9 
2.0-5.9 
6.0-11.9 
>12.0 
CardiovascularB 
Hypotension 
No 
hypotension 
MAP <70 
Dopamine </= 
5 or 
dobutamine 
(any) 
Dopamine >5 
or 
norepinephrine 
</= 0.1 
Dopamine >15 
or 
norepinephrine 
>0.1 
CNS 
Glasgow Coma Score 
15 
13-14 
10-12 
6-9 
<6 
Renal 
Creatinine (mg/dL) or 
urine output (mL/d) 
<1.2 
1.2-1.9 
2.0-3.4 
3.5-4.9 
or <500 
>5.0 
or <200 
A: Values for scores 3 and 4 are with respiratory support 
B: Adrenergic agents administered for at least one hour (doses given in µg/kg/min) 
 
 


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G 
De-identified data elements for screened, non-enrolled 
The following data elements will be collected on screened subjects who met the inclusion 
criteria but were not enrolled. 
 Month of the year that patient met screening criteria (1-12). 
 Gender 
 Ethnicity 
 Age (if age >89, 89 will be entered for age) 
 Screening vitamin D level (25OHD level) 
 Method used for vitamin D level (clinical lab or Qualigen FastPack IP) 
 Patient location (e.g. MICU, SICU, etc.)  
 Reason(s) patient excluded from study 
 If not excluded, not enrolled, why? 
 ARDS risk factors 
o If shock, etiology 
o If sepsis, site of infection 
 
  
 
 


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H 
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SAP_001.pdf: 
 
 
Vitamin D to Improve Outcomes by Leveraging Early 
Treatment 
 
ACRONYM: 
 
VIOLET 
 
PROTOCOL CO-CHAIRS: ADIT GINDE, MD MPH 
 
                             
       
     ________________________                    ___06/25/18___ 
       INVESTIGATOR SIGNATURE                                     DATE 
 
 
               
DANIEL TALMOR, MD MPH     
 
 
 
 
 
             ________________________                    ___06/25/18___ 
     INVESTIGATOR SIGNATURE                                     DATE 
 
STUDY STATISTICIAN 
DAVID SCHOENFELD, PHD 
           
                                                                                                   
      
                      __________________                                     __06/25/18___ 
 INVESTIGATOR SIGNATURE                                   DATE 
 
 


Contents 
1. Introduction .............................................................................................................................................. 3 
2. Trial Summary ........................................................................................................................................... 3 
2.1 Title ..................................................................................................................................................... 3 
2.2 Objective ............................................................................................................................................. 3 
2.3 Hypothesis ........................................................................................................................................... 3 
2.4 Study Design ........................................................................................................................................ 3 
2.5 Study Flow ........................................................................................................................................... 4 
2.5.1 Inclusion Criteria .............................................................................................................................. 4 
2.5.2 Exclusion Criteria .............................................................................................................................. 5 
2.6 Randomization and Study Initiation Time Window ............................................................................ 5 
2.7 Primary endpoint ................................................................................................................................ 5 
2.8 Secondary endpoint ............................................................................................................................ 5 
2.9 Sample Size/Interim Monitoring ......................................................................................................... 6 
2.10Endpoints ........................................................................................................................................... 7 
2.10.1 Primary Endpoint ....................................................................................................................... 7 
2.10.2 Secondary Endpoints ................................................................................................................. 7 
2.11 Subgroups ....................................................................................................................................... 10 
3. Main Manuscript and Statistical Methods .............................................................................................. 10 
3.1 Statistical Methods for Main Manuscript ......................................................................................... 11 
3.2 Primary Endpoint .............................................................................................................................. 12 
3.3 Secondary Endpoints ........................................................................................................................ 12 
3.3.1Clinical Endpoints ........................................................................................................................ 12 
3.3.2 Physiological Endpoints.............................................................................................................. 12 
3.3.3 Safety Endpoints ........................................................................................................................ 13 
3.3.4 Baseline Characteristics ................................................................................................................. 13 
3.3.5 Figures ............................................................................................................................................ 13 
4. Supplementary Materials Outline and Statistical Methods .................................................................... 13 
4.1Statistical Methods for Supplementary Materials ............................................................................. 15 
4.2 Subgroup Analyses of Primary Endpoint........................................................................................... 15 
4.3 Adverse Events .................................................................................................................................. 15 
References .................................................................................................................................................. 16 
 


1. Introduction 
The following document is the Statistical Analysis Plan for the VIOLET Study. It describes the 
study and the statistical methods used to analyze the primary and secondary outcomes and the 
supplemental material for the primary manuscript. 
 
2. Trial Summary 
 
2.1 Title  
Vitamin D to Improve Outcomes by Leveraging Early Treatment (VIOLET) 
 
2.2 Objective 
To assess the efficacy and safety of early administration of vitamin D3 (cholecalciferol) in 
reducing mortality and morbidity for vitamin D deficient patients at high risk for ARDS and 
mortality. 
 
2.3 Hypothesis 
Early administration of vitamin D3 (cholecalciferol) will improve all-cause, all-location mortality to 
day 90 in vitamin D deficient patients at high risk for ARDS and mortality 
 
2.4 Study Design 
VIOLET is a randomized, double-blinded, placebo-controlled, phase III trial (up to maximum 
n=3000) of early vitamin D3 in vitamin D deficient patients at high risk for ARDS and mortality. 
We will screen all patients for whom there is an intention to admit to ICU for study eligibility and 
will approach patients meeting inclusion/exclusion criteria for study enrollment. Screening will 
require screening for vitamin D deficiency using an FDA approved testing method for 25OHD, 
either by the hospital’s clinical laboratory or using the FastPack IP device (Qualigen Inc., 
Carlsbad, CA). We will obtain written informed consent for the protocol prior to the vitamin D 
screening test.  
 
We will randomize enrolled participants who are vitamin D deficient (initial screening 25OHD 
levels <20 ng/mL) to receive either 540,000 IU vitamin D3 (cholecalciferol) or placebo as a 
single, liquid enteral dose, administered either orally or via naso/orogastric tube. Randomization 
must occur within 12 hours of ICU admission decision and study drug administered within 2 
hours of randomization. We will assess trial endpoints for randomized participants by chart 
review and will contact participants or proxies at day 90. 
At baseline for all randomized participants, we will measure serum calcium (in real time, if not 
clinically available) and plasma 25OHD levels (batched and measured initially in monthly 
increments using the gold-standard liquid chromatography-tandem mass spectrometry 
[LC/MS/MS] method). We will collect additional plasma and whole blood for DNA banking. For 
the first 300 randomized participants, we will measure day 3 serum calcium (in real time, if not 
clinically available) and baseline plasma IL-6. We will also collect plasma at day 3 in the first 
300 randomized participants to measure 25OHD by LC/MS/MS and IL-6 (both batched) and for 
cryopreservation in the biorepository.  
 
The primary efficacy analysis cohort (confirmed vitamin D deficient cohort) will be defined as 
randomized participants having LC/MS/MS levels less than 20 ng/mL. The primary efficacy 
analysis will only involve these participants.  The FastPack device had some discordance with 


LC/MS/MS at a 20 ng/mL cutoff, and the previous study only showed a treatment effect in 
patients with low vitamin D levels. 1Since LC/MS/MS values will take at least 24 hours to 
process, we need to treat participants on the basis of the screening test result (FastPack or 
clinical laboratory test) available within three hours of patient consent. A secondary analysis 
cohort (screened vitamin D deficient cohort), which will also be used for the primary safety 
analysis, will include all randomized participants who receive vitamin D or placebo.  
 
2.5 Study Flow 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.5.1 Inclusion Criteria 
1. Age ≥ 18 years 
2. Intention to admit to ICU from emergency department, hospital ward, operating 
room, or outside facility 
3. One or more of the following acute risk factors for ARDS and mortality 
contributing directly to the need for ICU admission: 
Pulmonary 
a) Pneumonia 
b) Aspiration 
c) Smoke Inhalation 
d) Lung contusion 
e) Mechanical ventilation for acute hypoxemic or hypercarbic respiratory failure 


Extra-Pulmonary 
f) Shock 
g) Sepsis 
h) Pancreatitis 
 
4. Vitamin D deficiency (screening 25OHD level <20 ng/mL) 
 
2.5.2 Exclusion Criteria 
 
1. Inability to obtain informed consent 
2. Unable to randomize within 12 hours of ICU admission decision 
3. Unable to take study medication by mouth or enteral tube 
4. Baseline serum calcium >10.2 mg/dL (2.54 mmol/L) or ionized calcium >5.2 mg/dL (1.30 
mmol/L) 
5. Known kidney stone in past year or history of multiple (>1) prior kidney stone episodes 
6. Decision to withhold or withdraw life-sustaining treatment (patients are still eligible if they 
are committed to full support except cardiopulmonary resuscitation if a cardiac arrest 
occurs) 
7. Expect <48 hour survival 
8. If no other risk factors present, a) mechanical ventilation primarily for airway protection, 
pain/agitation control, or procedure; or b) elective surgical patients with routine 
postoperative mechanical ventilation; or c) anticipated mechanical ventilation duration 
<24 hours; or d) chronic/home mechanical ventilation for chronic lung or neuromuscular 
disease (non-invasive ventilation used solely for sleep-disordered breathing is not an 
exclusion). 
9. Prisoner  
10. Pregnancy 
11. Greater than 72 hours since hospital presentation (or > 3 calendar days since hospital 
presentation if transferred with no time stamp).  
2.6 Randomization and Study Initiation Time Window 
All participants must be enrolled and randomized within 12 hours of meeting inclusion criteria. 
Study medication must be administered within 2 hours of randomization. 
 
2.7 Primary endpoint 
The primary endpoint is all-cause, all-location mortality to day 90. The primary analysis will be 
based on randomized participants with baseline 25OHD levels <20 ng/mL, as measured by 
LC/MS/MS (confirmed vitamin D deficient cohort). As below, additional secondary analyses 
will be based on all randomized participants (screened vitamin D deficient cohort).  
 
2.8 Secondary endpoint 
Clinical Endpoints 
• 
Hospital length of stay among survivors to day 90 
• 
Healthcare facility length of stay among survivors to day 90 
• 
Alive and home (prior level of care) at day 90 
• 
Ventilator-free days to day 28 


• 
Time to mortality to day 90 
Physiological Endpoints 
• 
Development and severity of ARDS to day 7 
• 
Change in organ failure severity to day 7  
• 
25OHD levels to day 3 
• 
IL-6 levels to day 3 
Safety Endpoints 
• 
Calcium levels to day 14 
• 
Kidney stones to day 90 
• 
Fall-related fractures to day 90 
 
The primary analyses will be in the confirmed vitamin D deficient cohort.  These analyses will 
include an assessment of the secondary endpoints defined above. In addition, we will estimate 
the treatment effects on the primary and secondary endpoints in the screened vitamin D 
deficient cohort. If the analysis in the confirmed vitamin D deficient cohort is significant then 
estimates in the screened vitamin D deficient cohort will measure the treatment effect in patients 
who would be given the treatment if early LC/MS/MS is not available (or until next generation 
screening tests more closely approximate LC/MS/MS results).  We will also estimate the 
treatment effects on the endpoints above as a continuous function of baseline 25OHD level by 
LC/MS/MS. This function will provide an estimate of the extent to which efficacy (and safety) 
depends on vitamin D level. 
 
2.9 Sample Size/Interim Monitoring 
 
1. We base our assumptions on finding a 5% or greater absolute difference in mortality. 
With a 20% mortality rate in the control arm and a 15% mortality rate in the intervention 
arm, the maximum required total sample size is 3000 randomized participants with  
87.4% power based on the assumption that 80% of the randomized participants will 
have baseline 25OHD levels < 20 ng/mL, as measured by LC/MS/MS, and one sided α = 
0.025. 
2. The principal analysis will be based on randomized participants with baseline 25OHD 
levels <20 ng/mL, as measured by LC/MS/MS (confirmed vitamin D deficient cohort), 
following an intention-to-treat principle (analysis according to randomization 
assignment). We anticipate that approximately 80% of randomized participants will have 
baseline 25OHD levels <20 ng/mL by LC/MS/MS. The maximum size of the screened 
vitamin D deficient cohort will be 3000 patients and the futility stopping boundaries will 
consider the possibility that the sample size will not reach 2400 (80% of 3,000) in the 
confirmed vitamin D deficient cohort if too small a fraction of randomized patients would 
have baseline 25OHD levels <20 ng/mL by LC/MS/MS.  
3. An independent Data and Safety Monitoring Board (DSMB) will determine if the study 
should stop for superiority or futility of the intervention, or safety concerns. There will be 
three interim analyses throughout the trial (at subject enrollments 750, 1500, and 2250 
randomized participants).  
4. For the first 300 randomized participants, we will analyze   calcium levels at baseline and 
day 3 and 25OHD levels at day 3 using LC/MS/MS. This analysis will evaluate the 


effectiveness and safety of the vitamin D intervention in correcting vitamin D deficiency 
(clinical outcomes will not be evaluated at this time). Based on pre-specified rules and in 
consultation with the DSMB (who will have access to additional adverse event data), we 
may require changes to the vitamin D dose or extend the duration of 25OHD/calcium 
monitoring. 
2.10Endpoints 
2.10.1 Primary Endpoint  
The primary endpoint is all-cause, all-location mortality to day 90. We will ascertain vital status 
at day 90 by chart review (for participants that remain in the hospital at day 90 or died in hospital 
prior to day 90). For participants that were discharged alive from the hospital prior to day 90, we 
will call the patient or proxy (e.g., research contact list and next-of-kin) on/after day 90 to 
determine vital status. For those that we are unable to verify vital status at day 90, we will use 
evidence from the medical record (e.g., healthcare visits after day 90), review of obituaries, or 
phone calls to healthcare facilities to determine that participants are alive. Finally, for 
participants that have missing 90-day mortality, we will use the Centers for Disease Control and 
Prevention’s National Death Index (NDI) to determine vital status, using each patient’s social 
security number (SSN) for an exact NDI match. At the time of final analysis, we will use best 
available mortality data and if vital status remains unknown (anticipated for <5%), will impute 
90-day mortality based on last known location. 
 
2.10.2 Secondary Endpoints 
 
Clinical Endpoints 
1. Study hospital length of stay among survivors to day 90: Study hospital length of stay is 
defined as the number of days from enrollment (typically the day of hospital admission) to 
the day of study hospital discharge up to day 90. This endpoint will be analyzed only in 
survivors because hospital length of stay in those who die in the hospital is non-informative 
for this endpoint. In addition, we will use survivor average causal effect (SACE) methods to 
estimate hospital length of stay among participants that would have survived in both 
treatment and control groups.50 Accordingly, these methods adjust for the impact of 
differential survival between treatment groups. 
2. Healthcare facility length of stay among survivors to day 90: Healthcare facility 
length of stay is the time spent in another hospital or healthcare facility (e.g. long-
term acute care [LTAC] hospitals or acute rehabilitation/skilled nursing facility), for 
the subgroup of participants that were discharged to another healthcare facility after 
the initial hospitalization. This measure is defined as the number of days from initial 
hospital discharge to the first facility discharge to home (pre-hospitalization level of 
care) up to day 90. Healthcare facility LOS is zero for patients discharged to home 
(pre-hospitalization level of care) from the study hospital. This endpoint will be 
analyzed only in survivors using SACE methods because healthcare facility length of 
stay in those who die during the follow-up period is non-informative for this endpoint. 
  
3. Alive and home at day 90: This endpoint is the proportion of participants who have 
survived and are present at home, defined as pre-hospitalization level of care, at day 90. 


 
4. Ventilator free days (VFD) to day 28: VFD depends on both duration of ventilation and 
mortality through study day 28. In participants who survive 28 days, VFD is defined as 28 
minus duration of ventilation. Duration of ventilation is counted from the first study day of 
assisted breathing through the last day of assisted breathing provided the last day is prior to 
day 28. Otherwise, it is counted from the first study day of assisted breathing through day 
28. For participants discharged with assisted ventilation (e.g., to LTAC facility) prior to day 
28, a phone call will be required to assess ventilator status at day 28. Participants 
discharged prior to day 28 (but not to home) on unassisted breathing will be assumed to 
remain on unassisted breathing through day 28. Isolated periods of ventilation briefer than 
24 hours for surgical procedures and ventilation solely for sleep disordered breathing do not 
count towards duration of ventilation. In participants who never require assisted breathing, 
duration of ventilation is zero. Participants who do not survive 28 days will be assigned zero 
VFD. VFD is undefined in participants with chronic/home mechanical ventilation (except 
solely for sleep disordered breathing) and they will be excluded from this analysis.  
 
5. Time to mortality to day 90: Time to mortality will use best available data on vital status 
and date of death and censor based on last known alive date for participants with missing 
vital status. 
 
Physiological Endpoints 
 
6. Development and severity of ARDS to day 7: We will determine the presence and 
severity of ARDS for each day of mechanical ventilation to day 7 using the following 
approach: 
a. For each ventilator day: if ABG available between 2:00 AM and 8:00 AM, measure P/F 
(PaO2, FiO2 and PEEP) for all ABGs during this time window daily to day 7. Or, for 
ventilator days that no ABG available between 2:00 AM and 8:00 AM, determine lowest 
imputed P/F from measured S/F (SpO2, FiO2, and PEEP).  See Appendix B for P/F 
imputation table. 
b. For participants with P/F <300 or imputed P/F <300, FiO2 ≥40%, and PEEP ≥5 cm H2O, 
determine if hypoxemia is valid, acute, and not fully explained by CHF or fluid overload 
c. If yes to item (b), local investigators will: 
i. 
Review the first CXR (or CT) performed on each ventilated day with valid P/F or 
imputed P/F <300 (to day 7). If no chest imaging studies are present that day, site 
investigators may review imaging one day before or after to determine if ARDS 
imaging criteria met. 
ii. 
Assess if the images are consistent with ARDS (bilateral opacities not fully explained 
by effusions, lobar/lung collapse, or nodules). If equivocal, the reviewing investigator 
will adjudicate with additional investigators. 
 
7. Change in organ failure severity to day 7: We will measure acute organ failure severity 
daily to day 7 for three key organ systems: acute hypoxemic respiratory failure, acute kidney 
injury, and cardiovascular failure (i.e., shock). We will measure acute hypoxemic respiratory 
failure using data from the ARDS assessment, except this outcome does not require chest 
imaging consistent with ARDS. We will measure acute kidney injury by highest daily 
creatinine values or new use of dialysis/ renal replacement therapy (chronic dialysis 


participants are excluded). We will measure cardiovascular failure by highest daily dose of 
vasopressors (epinephrine, norepinephrine, dopamine, phenylephrine, or vasopressin). 
 
8. Plasma 25OHD levels: In addition to the screening vitamin D test (for trial entry), we will 
measure gold-standard plasma 25OHD levels at baseline for all randomized participants 
and at day 3 in the first 300 randomized participants to confirm biomarker response to the 
intervention. The gold-standard test will be done in batch using LC/MS/MS methods. 
Individual results will not be available to investigators and coordinators to avoid unblinding. 
Based on pre-specified rules and in consultation with the DSMB (who will have access to 
additional adverse event data), we may require changes to the vitamin D dose or extend the 
duration of 25OHD/calcium monitoring. 
 
9. Plasma IL-6 levels: We will measure plasma IL-6 levels at baseline and day 3 in the first 
300 randomized participants in batch concurrent with 25OHD testing. IL-6 is a pro-
inflammatory cytokine associated with increased incidence and severity of ARDS severity 
and worse clinical outcomes. Vitamin D may improve outcomes in critically ill patients by 
reducing levels of IL-6 (either causal association or as a result of improved recovery) 
 
Safety Endpoints 
 
10. Calcium levels to day 14: Because the half-life of 25OHD is approximately 2 weeks, we will 
record clinically available serum or ionized calcium levels through day 14 for all randomized 
participants. In addition, we will measure baseline and day 3 in the first 300 randomized 
participants in real time, either as part of routine clinical care or as a research procedure if 
not available. Day 3 is the when 25OHD levels peak in ICU patients given 540,000 IU of 
vitamin D.1,49 While no prior study of acute vitamin D repletion, using similar high doses as 
our trial, has observed clinically important hypercalcemia, we will confirm these findings in 
the VIOLET trial. Because little change in calcium levels is anticipated, access to individual-
level calcium results will not unblind study staff. During the trial, the DSMB may seek 
additional subject measurements for safety assessment, though this is not anticipated. 
 
11. Kidney stones to day 90: While an association between hypervitaminosis D and kidney 
stones has been suggested, the relationship with high dose vitamin D supplementation and 
kidney stones remains controversial. We will assess for incident kidney stones by chart 
review at the end of the hospitalization and by self-report at the 90 day phone call for those 
discharged from the hospital prior to day 90. 
 
12. Fall-related fractures to day 90: The association between acute vitamin D 
supplementation and falls/fractures remains unclear. Most data suggest that high dose 
vitamin D in healthy outpatients may improve muscle function, balance, and bone mineral 
density, and thus decrease fall-related fractures, but other data suggest that high dose 
vitamin D supplementation may actually increase the incidence of falls/fractures. Because of 
this uncertainty and limited data in hospitalized patients, we will assess for incident fall-
related fractures by chart review at the end of the hospitalization and by self-report at the 90 
day phone call for those discharged from the hospital prior to day 90.  


 
 
2.11 Subgroups 
 
The primary analysis will be based on randomized participants with baseline 25OHD levels <20 
ng/mL by LC/MS/MS (confirmed vitamin D deficient cohort). Secondary analysis will be based 
on the screened vitamin D deficient cohort (all randomized patients). A priori subgroups for 
analysis will include age, sex, race/ethnicity, residence (independent vs. long-term care facility), 
BMI, pre-hospitalization vitamin D supplementation, baseline 25OHD level (by the LC/MS/MS 
method), baseline renal function, ARDS risk factor (e.g., infectious vs non-infectious), LIPS 
score, pre-randomization mechanical ventilation status, pre-randomization presence of ARDS, 
and source of ICU admission.  
 
3. Main Manuscript and Statistical Methods 
The primary analysis will be based on randomized participants with baseline 25OHD levels <20 
ng/mL, as measured by LC/MS/MS (confirmed vitamin D deficient cohort). Additional 
analyses will be based on all randomized participants (screened vitamin D deficient cohort).  
 
The main manuscript will include the following tables and figures. 
 
 
Figure. CONSORT Diagram 
 
Table. Baseline Characteristics (confirmed deficient cohort) 
• 
Demographics 
• 
Co-Morbidities—Charlson, BMI, eGFR, pre-hospital level of care 
• 
VIOLET Risk Factors 
• 
Location—intent to admit, ICU destination 
• 
Illness Severity—SOFA, LIPS, mechanical ventilation, ARDS, vasopressor use, vital 
signs, WBC, lactate 
• 
Baseline vitamin D (home meds, vitD level by LCMS, calcium level) 
 
Figure. Change in LCMS from Day 0 to Day 3 for (screened deficient cohort) 
Summary data in text of results or Supplementary Material 
 
Table. Primary and Main Secondary Endpoints (confirmed deficient cohort) 
 
Primary 
• 
90 day all-cause, all-location mortality 
Secondary Clinical 
• 
Hospital mortality to day 90 
• 
Alive and home (prior level of care) at day 90 
• 
All-cause, all-location 28 day mortality 
• 
Hospital LOS 
• 
Healthcare facility LOS 
o Include proportion that went to healthcare facility (vs home) separately 
• 
VFD to day 28 


o Include proportion ventilated to help with interpretation 
Secondary Physiological 
• 
Respiratory failure 
o New mechanical ventilation ever to day 7 (Y/N) 
o Lowest P/F in the first 7 days if on mechanical ventilation 
• 
ARDS 
o New ARDS ever to day 7 (Y/N) 
o ARDS severity by Berlin definition at time of new ARDS diagnosis1 
o Lowest P/F in the first 7 days if ARDS 
• 
AKI to day 7 
o New RRT ever to day 7 (Y/N) 
o Worst AKI severity in first 7 days (no, mild, moderate, or severe AKI by KDIGO 
criteria2 
o Highest creatinine in first 7 days 
• 
Cardiovascular failure to day 7 
o New vasopressor use ever to day 7 (Y/N) 
o Highest CV SOFA score in first 7 days 
 
• 
IL-6 to day 3 
Safety 
• 
Calcium to day 14 
o Daily comparison to day 14 
o Hypercalcemia ever during the first 14 days (Y/N) 
• 
Kidney stones to day 90 
• 
Falls to day 90 
• 
Fall-related fractures to day 90 
• 
EQ-5D-5L at day 90 (adjusted for baseline) 
 
Figure. Time to Mortality to Day 90 (confirmed deficient cohort) 
 
Figure. 90-day Mortality by Baseline Vitamin D Level for (screened deficient cohort) 
 
3.1 Statistical Methods for Main Manuscript 
All analyses are intention to treat, that is, all randomized participants within each cohort 
(screened deficient, confirmed deficient) will be included in the analyses specified for each 
cohort. The confirmed deficient cohort is the primary efficacy cohort per the protocol. 
 
All significance testing will be two-tailed at alpha = 0.05 with no adjustment made for multiple 
comparisons3 
 
With the exception of the primary outcome, analysis is based on all available data with no 
imputation of missing data.  
 
The causal clinical effect of treatment on outcomes defined only in patients who are on study is 
complicated by potential case mix imbalance between the treatment groups due to differential 
recovery and mortality. However, the proposed statistical methods for these outcomes maintain 
the correct comparison-wise Type I error rate under the global null of no treatment effect 
whatsoever on any patient. 


 
3.2 Primary Endpoint 
The primary outcome of all-cause, all-location mortality to day 90 will be compared between 
treatment groups on the risk difference scale using a generalized linear model with a binomial 
distribution function and identity link function. This is equivalent to a Z-test comparing the 
difference between two proportions but extends naturally to assessment of sub-group by 
treatment interactions on the risk difference scale. 
 
For participants that have missing 90-day mortality, we will use the Centers for Disease Control 
and Prevention’s National Death Index (NDI) to determine vital status, using each patient’s 
social security number (SSN) for an exact NDI match or other identifying information if SSN 
unavailable. If none of the patients with missing 90-day mortality are found in the NDI to have 
died within 90 days we will assume they are all alive at 90 days. If any are known to be 
deceased, we will impute 90-day mortality for the remaining patients with unknown status using 
a multi-state model to estimate the transition probabilities across recorded states (in hospital, 
post hospital facility, home, death). In addition, we will include a sensitivity analysis based on 
the assumption that the probability of death increases with elapsed time since last follow up. 
 
 
 
3.3 Secondary Endpoints 
3.3.1Clinical Endpoints 
Hospital mortality to day 90, and alive and home (prior level of care) at day 90, and 28 day 
mortality will be compared between treatment groups using Pearson’s chi-square test. 
 
Hospital length of stay and healthcare facility length of stay will be analyzed only in survivors to 
day 90. We will use survivor average causal effect (SACE) methods to estimate each outcome 
among participants that would have survived in both treatment and control groups4. The SACE 
methodology includes a model for predicting survival using baseline covariates. The covariates 
used will be sex, age, Charlson, SOFA, baseline 25OHD as measured by LCMS, and 
race/ethnicity (non-Hispanic white versus other). 
 
Ventilator-free days to day 28 will be compared between treatment groups using a t-test. 
 
3.3.2 Physiological Endpoints 
Binary endpoints will be compared between treatment groups using Pearson’s chi-square test. 
 
Ordinal endpoints will be will be scored as 1,2,3, … and compared between treatment groups 
using the CMH mean score test.  
 
Continuous endpoints will be compared between treatment groups using a t-test with two 
exceptions. The highest creatinine and CV SOFA score will be analyzed controlling for baseline 
value using repeated measures ANOVA with a treatment by time interaction and shared 
intercept at time 0 (baseline).  This model is equivalent to ANCOVA controlling for baseline but 
is able to make use of all available data. 
 
 


3.3.3 Safety Endpoints 
Calcium levels to day 14 will be compared daily between treatment groups using a t-test. Binary 
occurrence of hypercalcemia (as defined by the protocol) to day 14 will be compared between 
treatment groups using Pearson’s chi-square test. 
 
Kidney stones to day 90 will be compared between treatment groups using Pearson’s chi-
square test. 
 
Falls and fall-related fractures to day 90 will be compared between treatment groups using 
Pearson’s chi-square test. 
 
EQ-5D-5L at day 90 (adjusted for baseline) will be analyzed only in survivors to day 90. We will 
use survivor average causal effect (SACE) methods to estimate the outcome among 
participants that would have survived in both treatment and control groups as described above. 
 
3.3.4 Baseline Characteristics 
Summary statistics will be tabulated to show the distribution in each treatment group and in 
aggregate. Categorical variables will be summarized as frequency and percent and continuous 
outcomes as mean and standard deviation or median and interquartile range as appropriate. No 
p-values testing for a baseline difference between treatment groups will be presented 
 
3.3.5 Figures 
A log-rank test will be used to compare the time to 90 day mortality in the two treatment groups. 
 
A quadratic smoothing spline in each treatment group will be used to display the relationship 
between the primary mortality outcome and baseline 25OHD level as measured by LCMS. 
Pointwise bootstrap confidence intervals for the difference between the two curves will be 
calculated at LCMS values of 5, 10, 15, 20, 25, and 30. 
 
4. Supplementary Materials Outline and Statistical Methods 
The supplementary materials will include the following tables and figures. 
 
Table. Baseline Characteristics for confirmed not deficient (LCMS≥20), confirmed 
deficient, and screened deficient (all randomized) 
• 
Demographics 
• 
Co-Morbidities—Charlson, BMI, eGFR, pre-hospital level of care 
• 
VIOLET Risk Factors 
• 
Location—intent to admit, ICU destination 
• 
Illness Severity—SOFA, LIPS, mechanical ventilation, ARDS, vasopressor use, vital 
signs, WBC, lactate 
• 
Baseline vitamin D (home meds, vitD level, calcium level) 
 
Table. Study Initiation/Dosing for confirmed deficient and screened deficient (all 
randomized) 
• 
Inclusion to Randomization (time) 
• 
Randomization to Drug Administration (time) 
• 
Study Drug Dosing 
• 
Study Drug Tolerance 


 
Figure. Subgroup Analysis of Primary Endpoint (confirmed deficient) 
• 
Age: <60, ≥60 
• 
Sex: M, F  
• 
Race/ethnicity: NH white, NH black, Hispanic, other  
• 
Residence: independent, long-term care facility  
• 
BMI: <20, 20-24.9, 25-29.9, 30-34.9, ≥35 
• 
Pre-hospitalization vitamin D supplementation: none, 1-800 IU/day, >800 IU/day 
• 
Baseline 25OHD level (by the LCMS): <12, 12-19, 20-29, ≥30 AND <10, 10-19, 20-29, 
≥30 AND <20, ≥20 
• 
Baseline renal function: eGFR 15 (or on dialysis), 15-29, 30-59, 60-89, ≥90 
• 
ARDS risk factor: sepsis, pneumonia, shock, mechanical ventilation, other AND 
infectious, non-infectious 
• 
LIPS score: <4, ≥4 
• 
Pre-randomization mechanical ventilation status: Y, N  
• 
Pre-randomization presence of ARDS: Y, N  
• 
Source of ICU admission: ED, other 
• 
ICU destination: MICU, other 
• 
Modified intention to treat (received full study drug dose without vomiting within 1 hour): 
yes, no 
 
Figure. Subgroup Analysis of Primary Endpoint for screened deficient (all randomized) 
• 
Age: <60, ≥60 
• 
Sex: M, F  
• 
Race/ethnicity: NH white, NH black, Hispanic, other  
• 
Residence: independent, long-term care facility  
• 
BMI: <20, 20-24.9, 25-29.9, 30-34.9, ≥35 
• 
Pre-hospitalization vitamin D supplementation: none, 1-800 IU/day, >800 IU/day 
• 
Baseline 25OHD level (by the LCMS): <12, 12-19, 20-29, ≥30 AND <10, 10-19, 20-29, 
≥30 AND <20, ≥20 
• 
Baseline renal function: eGFR 15 (or on dialysis), 15-29, 30-59, 60-89, ≥90 
• 
ARDS risk factor: sepsis, pneumonia, shock, mechanical ventilation, other AND 
infectious, non-infectious 
• 
LIPS score: <4, ≥4 
• 
Pre-randomization mechanical ventilation status: Y, N  
• 
Pre-randomization presence of ARDS: Y, N  
• 
Source of ICU admission: ED, other 
• 
ICU destination: MICU, other 
• 
Modified intention to treat (received full study drug dose without vomiting within 1 hour): 
yes, no 
 
Figure. Time to Mortality to Day 90 for screened deficient (all randomized) 
• 
Baseline LCMS<12 
• 
Baseline LCMS 12 to <20 
• 
Baseline LCMS ≥20 
 
Table. Primary and Main Secondary Endpoints for screened deficient (all randomized) 
 


Table. Adverse Events and Safety Outcomes for confirmed deficient, confirmed not 
deficient (LCMS≥20), and screened deficient (all randomized) 
 
4.1Statistical Methods for Supplementary Materials  
Statistical methods for the supplement will be the same as those for similar outcomes in the 
main manuscript 
 
4.2 Subgroup Analyses of Primary Endpoint 
For the primary outcome, interactions between treatment groups and will be analyzed on the 
risk difference scale using a generalized linear model with a binomial distribution function and 
identity link function including a treatment effect, a sub-group effect, and a treatment by sub-
group interaction. 
 
4.3 Adverse Events 
Adverse events will be analyzed using weighted Poisson regression with non-serious events 
weighted by one and serious events weighted by two. Events rather than patients will be the unit 
of analysis. Adverse events will be grouped and analyzed separately by MeDRA system organ 
classes 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


References 
1. Acute Respiratory Distress Syndrome The Berlin Definition The ARDS Definition 
Task Force JAMA. 2012;307(23):2526-2533 
2. Official journal of the American society of nephrology VOLUME 2 | ISSUE 1 | 
MARCH 2012 http://www.kidney-international.org 
3. Multiplicity Considerations in the Design and Analysis of Clinical Richard J. Cook and 
Vern T. Farewell. Journal of the Royal Statistical Society. Series A (Statistics in 
Society), Vol. 159,No. 1 (1996), pp. 93-110) 
4. Hayden D, Pauler DK, Schoenfeld D. An estimator for treatment comparisons among 
survivors in randomized trials. Biometrics. 2005;61(1):305-310