Protein-enhanced Enteral Nutrition and Metabolomics in Critically Ill Trauma and Surgical Patients
Phase: PHASE3
Conditions: Nutrition Disorder, Trauma, Critical Illness
Sponsor: University of Washington

 
 
 
 
 
 
 
 
 
Study Protocol: 
Supplemental enteral protein in critically ill trauma and surgical patients: A randomized 
clinical trial 
NCT03170401 
December 15, 2023 
 
 
 

This document provides a description of the study protocol and analysis plan for this clinical 
trial.  
1. Overall design and conduct of this single center clinical trial. 
2. Data safety and monitoring plan. 
3. Modification to the study protocol specifically regarding pauses in enrollment and additional 
evaluation of safety and adverse events. 
4. Names and responsibilities of the study team members. 
5. Study sponsorship. 
6. Citations for study description and protocol. 
 
1 Overall design and conduct of the clinical trial 
This is a single center, randomized clinical trial in which critically ill trauma victims and surgical 
patients are identified, recruited, and randomized to either standard enteral nutritional support or 
to treatment with early supplemental enteral protein to at target amount of 2gm/kg/day. The 
treatment protocol/intervention was designed, tested and reported in a prospective, observational 
cohort study of 53 critically ill surgical patients.1 
1.1 Background and rationale 
Observational studies suggest that higher amounts of protein are associated with better outcomes 
in critically ill patients. However, clinical trials addressing the question of protein dosing in 
critically ill trauma patients have not been performed. Based upon expert opinion in addition to 
observational data, the 2016 ASPEN guidelines recommended that critically ill patients should 
receive 1.2 – 2.0 gm/kg/day of protein and potentially higher amounts in critically ill trauma and 
burn patients.2 Our trial is designed to test this hypothesis in trauma and surgical patients by 
randomizing to early supplemental enteral protein or to standard enteral nutritional support. 
1.2 Primary Research Question 
In critically ill trauma and surgical patients does receiving a higher amount (≥2.0 grams/kg/day) 
of protein compared to usual care (~1.0 gram/kg/day) improves biomarkers of nutritional status 
(serum transthyretin concentration) and short-term clinical outcomes (ventilator-free days and 
ventilator-associated pneumonia). 
1.3 Study hypotheses 
Compared to receiving standard enteral nutritional therapy, the administration supplemental 
enteral protein to target a total protein intake of 2 gm/kg/day leads to higher serum transthyretin 

concentrations during the 3rd week after admission to the intensive care unit and increase the 
number of ventilator-free days. 
1.4 Trial design 
A single center, randomized, open-label, clinical trial including subjects admitted to the 
trauma/surgical intensive care unit at Harborview Medical Center in Seattle, Washington, USA. 
A CONSORT style flow diagram will present the numbers of patients screened and all reasons 
excluded prior to randomization. This figure will also present the number randomized to each 
arm and the numbers included in the assessment of outcomes. 
1.5 Eligibility criteria: 
Published at https://clinicaltrials.gov/ct2 Study Details | Supplemental Enteral Protein in Critical 
Illness | ClinicalTrials.gov/show/NCT03160547. 
1.6 Study procedures 
Screening of all potential subjects will be done by the Registered Dietitian who is assigned to the 
Critical Care team and is a member of the research team. Potential study subjects will be 
identified upon admission to the critical care unit and considered for enrollment if they meet 
inclusion criteria. Once a subject has been identified, we will randomize that subject into one of 
the two treatment arms. We will randomly assign subjects to receive either additional enteral 
protein to achieve a total of 2g/kg/day of protein or to standard enteral nutritional support. Due to 
the limited published information and the variation across hospitals and within our own practice, 
both treatment groups can be considered standard care. At Harborview, there is no protocol 
utilizing one method of protein dosing vs the other.  Both are approaches used in our critically ill 
surgical and trauma patients. The University of Washington Institutional Review Board has 
approved this study with a waiver of documented consent as it is considered minimal risk (.  
We will use a randomized design to test whether supplementing enteral protein improves patient 
care and outcomes. Subjects will be followed until discharged from the hospital and clinical data 
will be obtained from the electronic medical record. The randomization will be done by 
previously sealed envelopes that contain an equal number of each treatment assignment.  
Subjects are randomized 1:1 to either no or additional enteral protein.   
Standard of care at our institution includes obtaining nutrition-related labs.  Vitamin C, Vitamin 
D, Zinc and other vitamins and micronutrients are typically measured within 24 hours of ICU 
admission and used to guide supplementation and treat any deficiencies.  Circulating 
transthyretin (TTHY), C-reactive protein (C-RP) are measured approximately weekly in patients 
requiring greater than 7 – 10 days of nutritional support. Similarly, we often obtain urine for 24-
hour total urinary nitrogen (TUN) measurements, and indirect calorimetry (this is a measure, not 
a lab procedure) after 7 – 10 days of support. Indirect calorimetry is used to adjust caloric intake 
and to better understand a patient’s general use of energy sources. In this trial the dietitian and 

clinical team may adjust caloric goals based upon indirect calorimetry. We will not adjust protein 
intake based upon TUN or TTHY measurements. 
In a subgroup of study subjects, we will also obtain serial blood and urine samples for analyses 
of metabolites.  Each blood sample collected will be 5ml and each urine sample will be l00ml. 
The samples will be obtained on Day 0 (defined as the day nutritional support is initiated), Day 1 
(24 hours later), Day 3 (72 hours later +/- 24 hours), Day 7-13, Day 14-20, Day 21-27, Day 28-
34.  If the subject is discharged prior to any of these timepoints, we would obtain the final 
sample on the day of discharge.  Only one sample will be collected during the above timepoints. 
Urine will be collected from the subject's indwelling catheter, if they do not have an indwelling 
catheter, no urine samples will be obtained. Blood samples will be obtained through an existing 
IV catheter if possible.   If the subject does not have an existing IV catheter, we will obtain the 
sample by venipuncture by either the bedside RN or the research RN.  We have found that 
without the ability to obtain the samples by venipuncture (with the subject’s permission), we are 
not able to collect the later samples, and this is affecting the data analysis.  We will stop all urine 
collection when the indwelling catheter has been removed. The blood and urine samples will be 
analyzed for approximately 200 metabolites as well as inflammatory biomarkers. 
Patients will be screened, evaluated, and randomized within 96 hours of admission to the ICU by 
the study dietitian with guidance from the principal investigator in the case of uncertainty 
whether the patient qualifies. Subjects will be randomized 1:1 to either supplemental enteral 
protein or standard enteral nutritional support. Randomization is performed using sealed 
envelopes with the treatment assignment. 
1.7 Sample Size Considerations 
Sample size calculation and statistical analysis of circulating transthyretin concentrations 
(Primary hypothesis): With 500 participants this study has 80% power to detect a difference of 
2.4 mg/dl in average circulating 
transthyretin levels during the third 
week between the protein 
supplementation group (assumed 
average circulating transthyretin level of 
14.4, and the standard of care group 
(assumed average circulating 
transthyretin level of 12.0, with a pooled 
standard deviation of 7.0). This 
conservatively assumes that there will be 
25% of the patients for whom the 
protein supplementation has no effect in 
that they are either well enough to have 
high circulating transthyretin levels or die prior to obtaining the primary outcome. Because the 

primary outcome is a surrogate marker, we do not plan for any formal interim analyses to 
evaluate efficacy or futility. Harborview Medical Center typically admits about 300 - 350 trauma 
victims per year who would meet eligibility criteria. With enrollment over 4 - 5 years, we 
anticipate that ~1500 patients will be eligible. Therefore, the rate of recruitment among eligible 
patients needs to be ~33%. The statistical analysis of the primary hypothesis will use a two-sided 
t-test with alpha of 0.05. We expect that less than 10% of patients will die prior to day 21. For 
these and for patients who are lost-to-follow-up prior to day 21, we will multiply impute the 
primary outcome separately for each group and incorporate previously observed circulating 
transthyretin levels (if available) and patient age information.  
Power calculation and statistical analyses of ventilator-free days (VFDs; secondary 
hypothesis): VFD is a relevant endpoint in critical care and critical care research. We will use 
this endpoint as our main clinical outcome as it generally reflects the development of 
complications and provides and objective estimate of the rate of recovery. VFD could be 
expected to be impacted by nutritional 
support in several important ways. The 
adjacent figure represents the difference 
in VFD between the groups that we have 
80% power to detect for a range of 
pooled VFD standard deviations and 
assuming no loss to follow-up (LTFU), 
15% LTFU and 25% LTFU, based upon 
the sample size selected for the primary 
hypothesis (n = 500). For patients who 
die while in the hospital, VFDs will be 
set to zero. For patients who are lost to 
follow-up (unlikely), VFDs will be 
multiply imputed separately by treatment group, using patient age and measures of injury 
severity (injury severity score, AIS scores).  
1.8 Interim analyses 
There are no planned interim analyses for this study and no early stopping rules were designed. 
1.9 Timing of final analysis 
All outcomes will be analyzed by arm once all data is collected, data cleaning is complete and 
after the statistical analysis plan (SAP) is finalized. See modifications to the study protocol for 
details regarding the final timing of data analysis that was based upon additional collection of 
data related to potential complications of the treatment intervention. 
1.10 Timing of outcome assessments and recording of nutritional intake 

All outcome assessments are based upon data from the index hospitalization. Daily nutrition 
intake was assessed only for up to a maximum of 28 days in the ICU. This included daily enteral 
formula type and volume, daily enteral caloric and protein intake, and daily intravenous fat (as 
propofol) and glucose calories administered.   
1.11 Confidence intervals and p-values 
95% confidence will be presented for selected key outcomes. P-values will be two-sided without 
adjustment for multiplicity. We will use the traditional two-sided p ≤ 0.05 to indicate statistical 
significance for the primary and other prespecified outcomes. For subgroup analyses we will 
report only confidence intervals. 
1.12 Description of general nutritional support for trial subjects 
Our approach to nutritional support and monitoring for subjects in this trial will follow the 
general approach that has been used in our intensive care unit and reported previously.3-5 Once 
deemed ready to receive enteral nutrition, the patient is prescribed an isotonic formula with 1 – 
1.5 kcal/ml. Continuous infusion starts at 20 ml/hour and the rate is advanced over 24 hours to an 
initial goal of 25 kcal/kg/day delivered by continuous infusion via an orogastric or nasogastric 
tube. Subsequent modifications of caloric and other nutritional prescriptions are performed by 
the ICU dietitian and include use of the Harris-Benedict equation and indirect calorimetry in 
appropriate patients after 5 – 7 days of mechanical ventilation.  Monitoring for intolerance is 
primarily by clinical examination. Diarrhea is managed first but modifying the enteral formula to 
include higher fiber and testing for C. difficile infection. 
1.13 Description of enteral protein supplementation treatment 
The dietitian will calculate energy and protein needs as indicated in 1.11 above. For subjects 
randomized to the supplemental protein, arm 2 gm/kg/day of Prosource (Medtrition, Lancaster, 
PA) will be administered via the nasal/oral feeding tube in 60 – 180 ml bolus infusions 2 – 4 
times per day, independently of the enteral formula received. That is, the daily target amount of 
protein will be administered as supplement boluses beginning on the day the patient is enrolled.  
Once the enteral formula infusion approaches the target rate then the amount of supplemental 
protein will be gradually decreased. Supplemental protein will be reduced by 50% once the 
patient received 75% of targeted caloric intake over the previous day (0700 – 0700). Once the 
patient reaches the target caloric intake for 48 continuous hours, the amount of supplemental 
protein will be decreased in order that the total protein prescribed equals 2 gm/kg/day. 
 
2 Data safety monitoring plan 
Safety of our intervention will be reviewed by an independent Data Safety Monitoring Board 
(DSMB) after at 2 timepoints during the trial. The committee will include surgical intensivists 
who also have experience with clinical and translational research.  Data for review will be 

prepared by the study analyst (Qian Qui) and study biostatistician (Susanne May). The review 
will be iterative. After review of the summary information, additional data from the EMR can be 
added at the request of the members. Members will also have access to individual patient/study 
subject records as needed for additional details. 
 
3 Modifications to the study from initial protocol 
3.1 Enrollment pauses 
There were 3 pauses in subject enrollment over the course of the clinical trial. The first pause 
was from August 8th – 27th, 2019. This pause was necessary while waiting for approval from the 
IRB for an updated blood sampling strategy that included more frequent blood sampling. The 
second pause was due to the COVID-19 pandemic, and we were required to stop non-critical 
activities at our hospital for 2 ½ months (3/10/2020 – 5/24/2020). Enrollment was also paused 
for 3 ½ month period while the hospital transitioned to a new electronic health record (EHR). 
This new EHR included a mechanism to record patient enrollment into clinical trials and we 
paused enrollment until that system was functional.  
3.2 Update to Institutional Review Board approval – August 2019 
On August 23, 2019, the protocol was updated following review by the full IRB committee. The 
IRB had initially provided this approval using the process of “expedited review”. When the study 
approval was migrated from the original “paper”-based format to the new Zipline, electronic 
format the IRB determined that review by the entire committee was required to permit waiver of 
documented consent for the frequency of blood sampling that we requested. This oversight on 
the part of the IRB led to a pause in enrollment from August 6th – 27th, 2019 (first pause 
described in 3.1). 
3.3 Expansion of data collection procedures 
In 2021, as we were approaching complete enrollment, we obtained approval from the University 
of Washington IRB to obtain data from the electronic medical record and from the institutional 
quality improvement databases to provide additional information regarding hospital acquired 
infections and other complications for study subjects. At this time, the IRB also approved our 
request to obtain a minimal dataset for patients who were potentially eligible but who were not 
enrolled in the trial. We included the following request in our modification: “We are requesting 
permission to retrospectively and prospectively enroll subjects who meet inclusion/exclusion 
criteria but are not planned for randomization.   We are requesting permission to obtain this data 
from the retrospective group to the start of this study. We will identify patients who met 
enrollment criteria, but were not randomized, beginning on 11/17/2016 (date first randomized 
subject was enrolled) through the present date. For these retrospectively identified subjects, we 
will obtain data from the EMR. Prospective: Beginning with approval of this MOD, we will 

collect data from the EMR from patients who met enrollment criteria, but not randomized. This 
group will be identified prospectively, and data collected upon discharge from the hospital. The 
purpose of obtaining information for these eligible, but not randomized subjects is to be able to 
create an accurate CONSORT diagram, which will be required for reporting/publication of any 
data from this trial. This information will also be helpful for future grant submissions.” 
3.4 Additional assessments of adverse events and complications 
Following the completion of enrollment, initial data collection and analyses, we observed more 
respiratory adverse events in the supplemental protein treatment arm than the control arm (see 
manuscript Table 3: Safety, complications, and adverse events). Both reintubation rates and 
possible aspiration rates were higher. To better understand whether subjects had aspirated gastric 
contents, the timing of this aspiration, whether it caused deterioration in gas exchange or lead to 
pneumonitis or pneumonia, we reviewed the medical records directly. Briefly, 2 co-investigators 
independently reviewed medical records and were asked to “adjudicate” whether a subject had 
clear evidence of tracheobronchial aspiration of gastric contents. All instances of reintubation or 
possible aspiration (see eMethods for definitions) were reviewed. Adjudicated aspiration was 
assigned if there was consensus assignment by the 2 reviewers. If there was disagreement, a 
third, independent reviewer made the assignment. All reviewers were blinded to the treatment 
assignment. These reviews were conducted from 1/4/2023 – 4/1/2023. The data were then 
merged with the research database and submitted to a study statistician (Siobhan Brown) for 
analysis. 
4. Names and responsibilities of the study team members 
Grant E. O’Keefe, MD. Professor of Surgery, University of Washington. Lead investigator. 
Obtained funding from NIGMS, Designed the trial and co-designed the treatment intervention 
with Marilyn Shelton. Oversaw study subject enrollment and assisted with determination of 
potential study subject eligibility. Responsible for reviewing all data analyses and drafting the 
manuscript. 
Siobhan P. Brown, PhD. Senior Research Scientist. Department of Biostatistics. University of 
Washington. Created the statistical analysis plan in consultation with Grant O’Keefe and Susanne 
May. Conducted the statistical analyses for the manuscript and prepared the summary tables 
based upon these analyses. Also performed critical review of the manuscript. 
Qian Qui, MBA. Harborview Injury Prevention and Research Center, University of Washington. 
Responsible for overall data management over the course of the clinical trial. This included 
extraction of data from the EMR databases, cleaning the data and generating scores (APACHE, 
SOFA, etc.) from the raw data in the EMR. Also linked data from the various data sources (EMR 
derived data, trauma registry and other hospital registries and the nutritional data collected 
during the trial) to create the dataset used for the final analyses. Prepared data for the DSMB. 
Critical review and revision of the manuscript. 

Marilyn Shelton, RD. Clinical Dietitian, Harborview Medical Center. Co-designed the treatment 
intervention with Grant O’Keefe. Identified potential study subjects for inclusion. Determined 
nutritional intake goals (calories and protein) for enrolled subjects. Recorded daily nutritional 
intake while subjects were receiving care in the intensive care unit up to a maximum of 28 days. 
Critical review and revision of the manuscript. 
Erika K Bisgaard, MD, Assistant Professor. Department of Surgery. University of Washington. 
Responsible for health record review and adjudication of aspiration. Critical review of the 
manuscript. 
Alex Malloy, MD. Clinical Instructor. Department of Surgery. Responsible for health record 
review and adjudication of aspiration. Critical review of the manuscript. 
Jamie Robinson, MD. Clinical Instructor. Department of Surgery. University of Washington. 
Responsible for health record review and adjudication of aspiration. Critical review of the 
manuscript. 
Dan Robiuk, MD. Clinical Instructor. Department of Surgery. University of Washington. 
Responsible for health record review and adjudication of aspiration. Critical review of the 
manuscript. 
Ida Wilson, MD. Clinical Instructor. Department of Surgery. University of Washington. 
Responsible for health record review and adjudication of aspiration. Critical review of the 
manuscript. 
Susanne May, PhD. Professor, Biostatistics. University of Washington. Initial design of the 
clinical trial, including sample size estimates and power analyses for secondary outcomes. 
Assisted with the completion of progress reports and with the preparation of data and 
consultation as part of the data safety reviews. Critical review of the manuscript. 
 
5. Study sponsorship 
This study was supported, in part, but National Institutes of Health Grant 1RO1GM127790 to the 
University of Washington (PI: Grant O’Keefe). The funding agency had no role in any aspects of 
the design of the trial or the analyses of data.  
 
6. Citations for study description and protocol 
1. 
O'Keefe GE, Shelton M, Qiu Q, Araujo-Lino JC. Increasing Enteral Protein Intake in 
Critically Ill Trauma and Surgical Patients. Nutr Clin Pract. Oct 2019;34(5):751-759. 
doi:10.1002/ncp.10256 
2. 
McClave SA, Taylor BE, Martindale RG, et al. Guidelines for the Provision and 
Assessment of Nutrition Support Therapy in the Adult Critically Ill Patient: Society of Critical 
Care Medicine (SCCM) and American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.). 
JPEN J Parenter Enteral Nutr. Feb 2016;40(2):159-211. doi:10.1177/0148607115621863 

3. 
O'Keefe GE, Shelton M, Cuschieri J, et al. Inflammation and the host response to injury, 
a large-scale collaborative project: patient-oriented research core--standard operating procedures 
for clinical care VIII--Nutritional support of the trauma patient. J Trauma. Dec 2008;65(6):1520-
8. doi:10.1097/TA.0b013e3181904b0c 
4. 
Parent B, Seaton M, O'Keefe GE. Biochemical Markers of Nutrition Support in Critically 
Ill Trauma Victims. JPEN J Parenter Enteral Nutr. 02 2018;42(2):335-342. 
doi:10.1177/0148607116671768 
5. 
Chung CK, Whitney R, Thompson CM, Pham TN, Maier RV, O'Keefe GE. Experience 
with an enteral-based nutritional support regimen in critically ill trauma patients. J Am Coll Surg. 
Dec 2013;217(6):1108-17. doi:10.1016/j.jamcollsurg.2013.08.006