Evaluation of Biomarkers to Quantify Liver Pathology in Patients With Presumed Non-Alcoholic Steatohepatitis at Baseline and Following Bariatric Surgery (BARI)
Phase: NA
Conditions: NASH - Nonalcoholic Steatohepatitis; NAFLD - Nonalcoholic Fatty Liver Disease
Sponsor: AdventHealth Translational Research Institute

 
 
 
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AdventHealth Orlando 
Translational Research Institute for Metabolism and Diabetes 
 
STUDY PROTOCOL TITLE 
Evaluation of Biomarkers to Quantify Liver Pathology in Patients with Presumed Non-Alcoholic 
Steatohepatitis at Baseline and Following Bariatric Surgery 
 
 
STUDY SPONSOR 
AdventHealth Orlando  
 
 
PRINCIPAL INVESTIGATOR 
Steven R. Smith, MD 
Chief Scientific Officer, AdventHealth Orlando Research 
Senior Investigator, Translational Research Institute 
301 E. Princeton St.  
Orlando, FL 32804 
407-303-7115 
 
 
 
 

 
 
 
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TABLE OF CONTENTS 
STUDY PROTOCOL TITLE ......................................................................................................... 1 
TABLE OF CONTENTS ................................................................................................................ 2 
LIST OF TABLES .......................................................................................................................... 5 
LIST OF FIGURES ........................................................................................................................ 5 
LIST OF ABBREVIATIONS ......................................................................................................... 6 
INTRODUCTION .......................................................................................................................... 8 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ....................................... 8 
STUDY OBJECTIVES ................................................................................................................... 9 
STUDY DESIGN.......................................................................................................................... 11 
Research Design ........................................................................................................................ 11 
Study Agent, Device, and/or Intervention Description ............................................................. 13 
Study Site(s)/Location(s) and Number of Subjects .................................................................. 13 
Multi-Site Research Logistics/Communication Plan ................................................................ 14 
Research Conducted in a Foreign Country ............................................................................... 14 
Community-Based Participatory Research ............................................................................... 14 
SUBJECT SELECTION ............................................................................................................... 14 
Vulnerable Populations ............................................................................................................. 14 
Inclusion Criteria ...................................................................................................................... 14 
Exclusion Criteria ..................................................................................................................... 15 
RESOURCES AVAILABLE ........................................................................................................ 18 
Site Initiation/Study Specific Training ..................................................................................... 18 
STUDY PROCEDURES .............................................................................................................. 18 
Subject Recruitment and Screening .......................................................................................... 18 
Consent Process ........................................................................................................................ 18 
Non-English Speaking Subjects ................................................................................................ 18 
Subjects who are Not Yet Adults (Infants, Children, Teenagers) ............................................. 18 
Cognitively Impaired Adults ..................................................................................................... 18 
Documentation of Informed Consent Process .......................................................................... 19 
Waiver of Written Documentation of Consent or Waiver of Consent ...................................... 19 
Prohibitions and Restrictions .................................................................................................... 19 
Randomization .......................................................................................................................... 19 
STUDY VISITS ........................................................................................................................ 19 
Screening Visit 1 (SV1) ........................................................................................................ 19 

 
 
 
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Screening Visit 2 (SV2) ........................................................................................................ 20 
Baseline Visit 1 (BLV1) ....................................................................................................... 20 
Baseline Visit 2 (BLV2) ....................................................................................................... 21 
Surgery (V3) – Day 1 ............................................................................................................ 21 
Post-Surgery Visits 
(V4) – Day 42 ±7 days (6 weeks) .................................................... 22 
(V5) – Day 84 ±7 days (12 weeks) ....................................................................................... 22 
(V6) – Day 182 ±14 days (26 weeks) ................................................................................... 22 
(V7) – Day 364 ±14 days (52 weeks) ................................................................................... 22 
DETAILED ANALYTICAL/CLINICAL PROCEDURES ..................................................... 22 
Physical Examination and Medical History .......................................................................... 22 
Drug, Alcohol, Tobacco Screen and AUDIT Questionnaire ................................................ 22 
Height, Body Weight, BMI Calculation ............................................................................... 22 
Vital Sign Measurements ...................................................................................................... 22 
Screening Laboratory Assessments ...................................................................................... 23 
Circulating Biomarkers ......................................................................................................... 23 
Magnetic Resonance Imaging and Spectroscopy (MRI) ...................................................... 24 
FibroScan® ............................................................................................................................ 24 
Quantitative Liver Function Test (HepQuant SHUNT Dual Cholate Liver Diagnostic Kit) 25 
Liver Biopsy.......................................................................................................................... 26 
STUDY DURATION ............................................................................................................... 26 
MATERIALS OF HUMAN ORIGIN:  COLLECTION, PREPARATION, HANDLING 
AND SHIPPING ....................................................................................................................... 27 
STUDY OUTCOME MEASURES (ENDPOINTS) .................................................................... 28 
DATA MANAGEMENT AND QUALITY PLAN ...................................................................... 28 
Data De-identification ............................................................................................................... 28 
Data Confidentiality, Storage, and Retention ........................................................................... 28 
Data Quality .............................................................................................................................. 29 
Data Sharing (Outside of AdventHealth) .................................................................................. 29 
SAMPLE SIZE DETERMINATION ........................................................................................... 30 
STATISTICAL ANALYSIS PLAN ............................................................................................. 30 
Summary Analyses ................................................................................................................... 30 
Analysis of PDFF and MRE ..................................................................................................... 31 
Multiplicity ............................................................................................................................... 31 
Missing Data ............................................................................................................................. 31 
Analysis Populations ................................................................................................................. 31 

 
 
 
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POTENTIAL RISKS AND BENEFITS ....................................................................................... 32 
POTENTIAL BENEFITS ......................................................................................................... 32 
POTENTIAL RISKS ................................................................................................................ 32 
Intravenous Lines/Blood Draws (e.g., Laboratory Samples) ................................................ 32 
Magnetic Resonance Imaging (MRI) and Spectroscopy ...................................................... 32 
FibroScan® ............................................................................................................................ 33 
Quantitative Liver Function Test (HepQuant SHUNT Liver Diagnostic Kit) ..................... 33 
Liver Biopsy.......................................................................................................................... 35 
Stool Collection .................................................................................................................... 36 
MITIGATION OF RISKS ........................................................................................................ 36 
Provisions to Protect the Privacy Interest of Subjects .............................................................. 36 
EARLY WITHDRAWAL OF SUBJECTS .................................................................................. 37 
Investigator Withdrawal of Subjects ......................................................................................... 37 
Subject Request for Withdrawal from Study ............................................................................ 37 
Data Collection and Follow-up for Withdrawn Subjects .......................................................... 37 
ADVERSE EVENT REPORTING............................................................................................... 37 
Adverse Events ......................................................................................................................... 37 
Recording and Notification of Adverse Events ........................................................................ 37 
Safety Monitoring Plan ................................................................................................................. 38 
Safety Monitoring ..................................................................................................................... 38 
Data and Safety Monitoring Board (DSMB) or Equivalent ..................................................... 38 
ETHICAL CONSIDERATIONS .................................................................................................. 38 
Sharing of Results with Subjects .............................................................................................. 38 
FUNDING SOURCE .................................................................................................................... 38 
PARTICIPANT STIPENDS OR PAYMENTS ............................................................................ 38 
PUBLICATION PLAN ................................................................................................................ 39 
REFERENCES ............................................................................................................................. 40 
APPENDIX A.  SCHEDULE OF EVENTS................................................................................. 43 
APPENDIX B.  ALCOHOL USE DISORDERS IDENTIFICATION TEST (AUDIT) 
QUESTIONNAIRE ...................................................................................................................... 45 
APPENDIX C. Liver Disease Score ............................................................................................. 46 

 
 
 
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LIST OF TABLES 
Table 1: Study Objectives and Endpoints ....................................................................................... 9 
 
 
LIST OF FIGURES 
Figure 1.  Decision Tree for Subject Eligibility ............................................................................ 17 
 

 
 
 
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LIST OF ABBREVIATIONS 
Abbreviation 
Definition 
ACE 
angiotensin converting enzyme 
AE 
adverse event 
ALT 
alanine aminotransferase 
AMRA 
Advanced MR Analytics AB 
APTT 
activated partial thromboplastin time 
AST 
aspartate aminotransferase 
AUDIT 
Alcohol Use Disorders Identification Test  
BMI 
body mass index 
CAP 
College of American Pathologists 
CAP™ 
Controlled Attenuation Parameter  
CBC 
complete blood count 
CJD 
Creutzfeldt-Jakob disease 
CMP 
comprehensive metabolic panel 
CSR 
clinical study report 
DNA 
deoxyribonucleic acid 
DSI 
Disease Severity Index derived from HepQuant SHUNT Dual Cholate Liver Diagnostic 
Kit 
DUA 
Data Use Agreement 
d4-cholate 
Cholic Acid-2,2,4,4-d4 
eCRF 
electronic case report form 
FAS 
full analysis set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HbA1C 
hemoglobin A1C 
HBsAg 
hepatitis B surface antigen 
HBV 
hepatitis B virus 
HCC 
hepatocellular carcinoma 
HCV 
hepatitis C virus 
HCVAb 
hepatitis C virus antibody 
HIV 
human immunodeficiency virus 
HSA 
human serum albumin 
ICH 
International Council on Harmonisation 
IHL 
intrahepatic lipid 
IND 
Investigational New Drug 
INR 
international normalized ratio 
IRB 
institutional review board 
IV 
intravenous 
LC-MS 
liquid chromatography-mass spectrometry 

 
 
 
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Abbreviation 
Definition 
LMS 
LiverMultiScan 
LSM 
Liver Stiffness Measurement 
MAR 
missing at random 
MELD 
model of end-stage liver disease  
MI 
multiple imputation 
MR 
magnetic resonance 
MRE 
magnetic resonance elastography 
MRI 
magnetic resonance imaging and spectroscopy 
MRI-PDFF 
magnetic resonance imaging proton density fat fraction 
MRS-IHL 
magnetic resonance spectroscopy intrahepatic lipid 
MTA 
Material Transfer Agreement 
NAFLD 
non-alcoholic fatty liver disease 
NASH 
non-alcoholic steatohepatitis 
PBS 
phosphate buffered saline 
PHI 
personal health information 
PID 
participant identification number 
PK 
pharmacokinetic 
PT 
prothrombin time 
PTH 
parathyroid hormone 
PTT 
partial thromboplastin time 
RNA 
ribonucleic acid 
RNA-Seq 
RNA sequencing 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SDV 
source data verification 
SOP 
standard operating procedure 
SSP 
study specific procedure 
TRI-MD 
Translational Research Institute for Metabolism and Diabetes 
TSH 
thyroid stimulating hormone 
T4 
thyroxine 
US 
United States 
vCJD 
variant Creutzfeldt-Jakob disease 
VCTE™ 
Vibration-Controlled Transient Elastography 
WI 
work instruction 
13C-cholate 
Cholic Acid-24-13C 
β-hCG 
beta subunit human chorionic gonadotropin 
 
 

 
 
 
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INTRODUCTION 
This document is a protocol for a human research study to evaluate imaging, circulating 
and microbiome biomarkers of liver pathology in patients with non-alcoholic fatty liver disease 
(NAFLD)/non-alcoholic steatohepatitis (NASH).  The described study will be conducted 
in compliance with the protocol, Good Clinical Practices (GCP) International Council 
on Harmonisation (ICH) Guidelines (E6) for GCP standards as adopted by the Food and Drug 
Administration (FDA) and associated Federal regulations, and all applicable institutional 
research requirements.   
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
The rise in prevalence of obesity across the United States (US) is accompanied by a host of 
cardiometabolic comorbidities, including NAFLD, which is now the most common cause of 
chronic liver disease in industrialized nations (Ahmed 2010, Loomba 2013).  Indeed, NAFLD 
affects approximately 20–30% of normal weight individuals and 57–98% of obese individuals 
depending on population and diagnostic modality (Vernon 2011).  Within the spectrum of 
NAFLD phenotypes, NASH is a condition that is distinguished from simple steatosis in that 
it includes necroinflammatory changes in addition to steatosis.  NASH is a burgeoning liver 
disease (estimated prevalence is 3–5% of the US population) for which no approved therapeutic 
treatment currently exists (Sanyal 2015).  
Approximately 25% of those diagnosed with NAFLD meet the histopathologic criteria for 
NASH (Cohen 2011), which unlike the clinically benign condition of simple steatosis, poses 
a higher risk of progression to cirrhosis and development of hepatocellular carcinoma (HCC).  
A major gap in the field is the inability to identify those who are at risk for progression from 
benign NAFLD to severe stages of NASH such as fibrosis, cirrhosis, and HCC.   
With treatment of NAFLD/NASH being a major public health objective, several promising 
therapies have recently entered advanced clinical trials.  However, one of the primary challenges 
hindering development of treatments for NAFLD/NASH is the lack of non-invasive tools 
to accurately diagnose and stage the disease.  Currently, most clinical trials examining chronic 
liver disease rely heavily on histologic evaluation of a liver biopsy sample. This diagnostic assay 
is associated with a number of drawbacks.  In addition to risk of complications associated with 
the invasive nature of the biopsy procedure, the limited volume of tissue collected by the biopsy 
introduces sampling error, while interpretation and scoring of the histology itself is a somewhat 
subjective process that can introduce variability to the assessment (Raitziu 2005).  Therefore, 
there is a recognized need within the field to develop a non-invasive alternative to the liver 
biopsy for diagnosing and staging NAFLD/NASH.  Such a development would enable the design 
of clinical trials to assess efficacy of prospective compounds to treat NASH without the need 
for liver biopsies, thereby improving safety 17and facilitating recruitment, which, in turn, would 
speed up the translation of new drugs to patients who are in need of treatment options. 
Several methods have been studied for non-invasive diagnosis of NAFLD and NASH, however 
none have been found to sufficiently correlate with stages of disease progression or NASH 
diagnosis to substitute for liver biopsy in either the clinical care of patients or selection of 
patients for clinical trials (Alkhouri 2012, Papagianni 2015, Renelus 2016).  Among these 
methodologies, medical imaging approaches have garnered significant interest within the 

 
 
 
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NAFLD research community given their potential to directly probe the extent of different 
pathophysiologic mechanisms within the liver itself (Dulai 2016, Hashemi 2016).   
Over the course of this study, longitudinal assessments of subjects with NASH will be performed 
using non-invasive imaging (FibroScan® and magnetic resonance imaging [MRI] methods) to 
evaluate the effects of bariatric surgery on the liver and on extra hepatic depots of adipose tissue.  
A battery of circulating, functional, and microbiome-based assays will also be employed to 
evaluate physiologic changes that occur in response to bariatric surgery (OWLiver Care analysis 
to discriminate between NAFLD and healthy liver;  OWLiver Care analysis to discriminate 
between NASH and simple steatosis;  Nordic Bioscience panel to interrogate fibrogenic activity; 
HepQuant SHUNT Dual Cholate Liver Diagnostic Kit test to monitor the effect of bariatric 
surgery on liver function; and stool sample assays of the gut microbiome  to assess the effect of 
bariatric surgery on the composition, functionality, and metabolites of the microbiota) (see the 
Research Design section for detailed information and background on these procedures and 
assessments).   
A population of patients undergoing standard of care bariatric surgery has been chosen because 
of the known effect of bariatric surgery on NASH.  Roux-en-Y gastric bypass is associated with 
decreased hepatic steatosis (with total resolution in 55.5% of patients) and resolution of hepatic 
inflammation and ballooning degeneration of hepatocytes (100.0 and 88.8% of patients, 
respectively), with histologic improvement including decreases in lobular inflammation 
and portal/lobular fibrosis (Barker 2006, Rabl 2012, Hafeez 2013, Schneck 2016), all in addition 
to functional liver recovery (Alizai 2015).   
STUDY OBJECTIVES 
The table below lists each objective along with its corresponding endpoint.  
 
Table 1: Study Objectives and Endpoints 
Co-Primary Objectives and Endpoints 
Objective 
Endpoint 
To evaluate the effect of bariatric surgery on liver fat 
in adults with non-alcoholic steatohepatitis (NASH) 
84 days (12 weeks) post-surgery  
Percent change from baseline in liver fat at 84 days 
(12 weeks) post-surgery, as assessed by magnetic 
resonance imaging proton density fat fraction 
(MRI-PDFF) 
To evaluate the effect of bariatric surgery on liver 
stiffness in adults with NASH 84 days (12 weeks) 
post-surgery  
Percent change from baseline in liver stiffness at 84 days 
(12 weeks) post-surgery, as assessed by magnetic 
resonance elastography (MRE) 
 
Secondary Objectives and Endpoints 
Objectives 
Endpoint 
To evaluate the effect of bariatric surgery on liver fat 
and liver stiffness in adults with NASH 84 days 
(12 weeks) post-surgery 
Percent change from baseline in liver fat and liver 
stiffness at 84 days (12 weeks) post-surgery, as assessed 
by FibroScan®-derived Controlled Attenuation 
Parameter (CAP™) and stiffness 

 
 
 
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Secondary Objectives and Endpoints 
Objectives 
Endpoint 
To evaluate the effect of bariatric surgery on liver fat 
and liver stiffness in adults with NASH over time 
post-surgery 
Percent change from baseline in liver fat and liver 
stiffness over time post-surgery (42 days [6 weeks], 
84 days [12 weeks], 182days [26 weeks], and 364 days 
[52 weeks]), as assessed by: 
• FibroScan®-derived CAP™ and stiffness   
• MRI-PDFF, magnetic resonance spectroscopy 
intrahepatic lipid (MRS-IHL), and MRE  
To evaluate the effect of pre-surgical dietary 
intervention on liver fat and liver stiffness in adults 
with non-alcoholic fatty liver disease (NAFLD) 
(entire study population) 
Percent change in liver fat and liver stiffness between 
screening and baseline, as assessed by: 
• FibroScan®-derived CAP™ and stiffness 
• MRI-PDFF and MRE 
 
Exploratory Objectives and Endpoints 
Objective 
Endpoint 
To evaluate the effect of bariatric surgery 
on extrahepatic adipose depots, skeletal muscle, 
and liver fibro-inflammatory state in adults with 
NASH over time 
Percent change from baseline, over time post-surgery 
(42 days [6 weeks], 84 days [12 weeks], 182 days 
[26 weeks], and 364 days [52 weeks]), in: 
• extrahepatic volumes of adipose tissue and skeletal 
muscle as assessed by MRI body composition profiling  
• corrected-T1 of the liver as assessed by 
LiverMultiScan MRI 
To evaluate the effect of bariatric surgery on kinetic 
measures of liver function in adults with NASH 
over time  
Percent change from baseline, over time post-surgery 
(42 days [6 weeks], 84 days [12 weeks], 182 days 
[26 weeks], and 364 days [52 weeks]), in Disease 
Severity Index (DSI) and %-shunt as assessed 
by HepQuant SHUNT 
To evaluate the effect of bariatric surgery on gut 
microbiome in adults with NASH over time 
Change from baseline, over time post-surgery (42 days 
[6 weeks], 84 days [12 weeks],182 days [26 weeks], 
and 364 days [52 weeks]), in stool sample: 
• composition (shotgun analysis of microbiome DNA)  
• functionality (shotgun analysis of microbiome RNA)  
• metabolites of microbiota  
To evaluate the effect of bariatric surgery 
on circulating metabolomics, protein biomarkers, 
and liver enzymes in adults with NASH over time 
Percent change from baseline, over time post-surgery 
(42 days [6 weeks], 84 days [12 weeks], 182 days 
[26 weeks], and 364 days [52 weeks]), in: 
• OWLiver, and OWLiver Care metabolomics panels 
from OWL 
• pro-collagen protein fragments panel from Nordic 
Bioscience 
• aspartate aminotransferase (AST) and alanine 
aminotransferase (ALT) enzymes 
To evaluate pre-surgery differences in biomarker 
signatures between adults with NASH and those 
with non-NASH NAFLD   
Difference measures between subject groups at baseline 
for: 
• FibroScan®-derived CAP™ and stiffness 
• magnetic resonance (MR)-derived measures 
• circulating biomarker measures  
• gut microbiome measures 
To assess predictive nature of measured biomarkers 
to non-invasively characterize disease state in adults 
with NAFLD (entire study population) 
Correlative relationship between disease pathobiology 
as assessed from histopathology measures of biopsy 
samples (liver) collected during bariatric surgery and any 
other secondary or exploratory endpoints  

 
 
 
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Exploratory Objectives and Endpoints 
Objective 
Endpoint 
To evaluate the effect of bariatric surgery on liver fat 
in adults with non-alcoholic fatty liver disease 
(NAFLD) (entire study population)  
Percent change in intrahepatic lipids by magnetic 
resonance spectroscopy (MRS-IHL) 
To evaluate the ability of a diffusion-based MRI 
method to non-invasively measure liver stiffness 
in adults with NAFLD (entire study population)  
Correlation between liver stiffness measures derived 
from diffusion-based MRI and those methods that impart 
a mechanical perturbation to tissue, namely: 
• FibroScan®-derived stiffness 
• MRE 
 
STUDY DESIGN 
Research Design 
This is a non-randomized, single-site, biomarker methodology study in subjects with 
presumed NAFLD and NASH undergoing bariatric surgery.  Participants will 
undergo bariatric surgery along with all pre- and post-surgery medical visits and standard 
of care laboratory assessments.   
Over the course of this study, longitudinal assessments of subjects with NASH 
or non-NASH NAFLD will be performed using a battery of non-invasive circulating, 
functional, imaging, and microbiome-based assays to evaluate physiologic changes that 
occur in response to bariatric surgery.   
The specific imaging to be employed in this study includes FibroScan® and MRI to 
noninvasively evaluate the effects of bariatric surgery on the liver and on extra-hepatic 
depots of adipose tissue.  The FibroScan® instrument applies a technique referred to as 
vibration-controlled transient elastography (VCTE™) to simultaneously derive measures 
of stiffness for the assessment of liver fibrosis, and of ultrasound wave attenuation for the 
assessment of liver steatosis (a measure referred to as the controlled attenuation 
parameter [CAP™]).  Magnetic resonance (MR) assessments will apply scanning 
methods to interrogate steatosis, inflammation, and fibrosis of the liver through measures 
derived from proton density fat fraction (MRI-PDFF), proton spectroscopy (MRS-IHL), 
T1 relaxometry using the LiverMultiScan (LMS) protocol, and MR elastography (MRE), 
respectively.  To assess effects of surgical intervention outside the liver, MRI evaluation 
of extra-hepatic adipose tissue volumes within visceral and subcutaneous regions, 
and skeletal muscle will be quantified. 
This study will also collect blood samples to investigate the effect of bariatric surgery 
on longitudinal profiles of several categories of circulating biomarkers and evaluate their 
utility in tracking NASH disease regression.    
Recent advances in metabolomic testing methodologies and associated analysis 
algorithms have led OWL Metabolomics to define lipidomic signatures for different 
NAFLD subtypes, and for differentiating NASH from simple steatosis (Barr 2012).  
Analysis of serum lipid metabolites by two separate logistic regression algorithms was 
conducted in a recent study of biopsy-proven NAFLD patients.  Findings from that study 
demonstrated that the OWLiver Care analysis is able to discriminate between NAFLD 

 
 
 
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and healthy liver, while the OWLiver analysis is able to discriminate between NASH 
and simple steatosis, both with AUROC >0.9 (Crespo 2016).  Similarly, assay 
development to quantify circulating proteins associated with tissue remodeling has led 
Nordic Bioscience to identify a panel to interrogate fibrogenic activity (Nielsen 2013).  
In a recent clinical study of patients with advanced hepatitis C, profiling markers related 
to both synthesis and degradation of the extracellular matrix (in the form of collagen 
pro-peptides and collagen degradation fragments) was found to serve a prognostic role by 
enabling identification of patients likely to respond to anti-fibrotic therapy 
(Nielsen 2015).  As similar fibrogenic processes are likely active in NAFLD patients that 
are at risk of advancing to NASH, there is interest to determine if the panel may provide 
similar prognostic value, and if assessed longitudinally, insight to therapeutic efficacy 
in advance of histological or clinical assessments.   
This study will also monitor the effect of bariatric surgery on liver function as 
characterized by the HepQuant SHUNT Dual Cholate Liver Diagnostic Kit test.  
This diagnostic test is being developed as a quantitative assay of liver function that 
involves the co-administration of stable isotopes of cholate:  13C-cholate 
[cholic acid-24-13C, IND #65,121] (administered intravenously [IV]); and 4d-cholate 
[cholic acid-2,2,4,4-d4, IND #65,123] (administered orally).  Following the dual-cholate 
administration, timed blood samples are collected over a 90-minute period.  Liquid 
chromatography-mass spectrometry (LC-MS) analysis of the samples will generate 
kinetic measures of cholate clearance.  Clearances of IV- and orally-administered cholate 
are liver-specific functions dependent upon systemic and portal blood flows, respectively, 
and hepatocyte uptake.  The primary output of the test is a summary parameter referred to 
as the Disease Severity Index (DSI), which is calculated from the clearance rates.  DSI 
values increase with impairment of cholate clearances and have been shown to correlate 
with increasing severity of liver disease and risk for adverse clinical outcomes 
(Helmke 2015).  A standard hepatic panel used clinically to monitor liver health will also 
be collected. 
The effect of bariatric surgery on gut microbiome will be assessed through stool sample 
assays for composition, functionality, and metabolites of microbiota (both stool and 
circulating).  Alterations to the intestinal microbiota have been found to contribute to the 
pathogenesis of many liver diseases, including NAFLD (Schnabl 2014).  Though the 
mechanism through which microbiota may negatively or positively influence the onset of 
NAFLD is not fully understood, it is plausible that investigation of the bacterial dysbiosis 
that arises in response to bariatric surgery may provide insight to: (1) novel approaches to 
inhibit pathways that promote disease progression in NAFLD, or (2) identification of 
non-invasive biomarkers to identify patients at risk for progression to NASH.   
The only non-standard of care procedures that will occur during bariatric surgery is a 
liver biopsy for histology and exploratory analyses.  In the post-surgery period, the only 
non-standard of care activities will be those related to the imaging and sample collection 
for monitoring of liver health with the specified biomarkers.  Research-related procedures 
occurring before and after surgery are detailed below. 

 
 
 
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Study Agent, Device, and/or Intervention Description  
To achieve one of our exploratory objectives, we will use the HepQuant SHUNT Liver 
Diagnostic Kit to quantitatively assess changes in liver function over time. The HepQuant 
SHUNT Liver Diagnostic Kit is an investigational device kit with an Investigational New 
Drug (IND) and Investigational Device Exemption (IDE) held by HepQuant, LLC. The 
US FDA originally in 2002 classified the HepQuant product as a drug. As such there are 
INDs for the drug components of the kit currently held by HepQuant, LLC: Cholic Acid-
24-13C (IND # 65,121) and Cholic Acid-2,2,4,4-d4 (IND # 65,123). In 2015, the FDA 
re-classified the HepQuant SHUNT Liver Diagnostic kit as a diagnostic device and drug 
combination product. Therefore, the FDA requires that new clinical trials apply for an 
IDE, not an IND.  Please refer to Investigator Brochure and email from manufacturer 
submitted with this package.  
 
As sponsor of the IND and IDE, HepQuant, LLC. is responsible for FDA filings. 
Investigator compliance with Federal Food, Drug, and Cosmetic Act and the 
implementation regulations (Title 21 of the Code of Federal regulations, 21 CFR Part 50, 
21 CFR Part 812, and 21 CFR Part 54) is defined in an Investigator Agreement with 
HepQuant, LLC. All investigational products will be stored in the secured pharmacy at 
the Translational Research Institute for Metabolism and Diabetes (TRI-MD). All 
products and procedures will be either handled and/or administered by trained clinical 
staff under the supervision of a study doctor.  
  
All other interventions are either standard of care for bariatric surgery or listed below for 
research purposes.  
 
Study Site(s)/Location(s) and Number of Subjects 
AdventHealth Orlando  site locations (campus, physician offices, etc.): 
• Celebration Health 
• Translational Research Institute for Metabolism and Diabetes 
Estimated number of participants:  35 
The study will enroll 25 participants with NASH for the full study and 10 participants 
with non-NASH [NAFLD or normal] to serve as controls.  The 10 non-NASH 
participants will have baseline measurements and perioperative biopsies only.  In the 
unlikely instance that biopsy results do not confirm NASH.  Those participants who do 
not have NASH diagnosis will not continue in the post-surgery study visits and may 
become part of the 10 participants non-NASH group (see Figure 1 below). Additional 
participants may be recruited if the analysis of the primary end point (12 weeks) reveals 
larger variability than anticipated.  

 
 
 
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Multi-Site Research Logistics/Communication Plan 
 
Not applicable. 
Research Conducted in a Foreign Country 
Not applicable. 
Community-Based Participatory Research 
 
Not applicable. 
SUBJECT SELECTION 
Vulnerable Populations 
Not applicable. 
Inclusion Criteria 
To be eligible for this study, participants must meet the following inclusion criteria: 
1. Age ≥18 years 
2. Undergoing bariatric surgery (Roux-en-Y or gastric sleeve only) as part of clinical 
care. 
3. Negative βhCG- level (female participants who become pregnant during the study 
will be withdrawn) 
4. MRI:  
• for initial assignment to NASH cohort – 
o MRI-PDFF ≥8% (data generated by TRI-MD), and 
o magnetic resonance elastography (MRE) ≥3.00 kPa (Chen 2011, Dulai 
2016, Loomba 2016, Costa-Silva 2018); or 
o MRE ≥ 2.70 kPa (Chen 2011) with at least one of the following risk 
factors for NASH: 
 type 2 diabetes 
 metabolic syndrome 
 ALT ≥ 1.5 times the upper limit of normal 
 Fibrosis-4 score suggestive of NASH (advanced fibrosis): Age ≤ 35: 
score not applicable; Age ≥ 36: > 2.67 (McPherson 2017)  
 AST to platelet ratio of ≥ 0.7, which is suggestive of NASH 
(significant fibrosis) (Lin 2011) 
 
• for initial assignment to NAFLD or normal participants cohort – 
o No requirement for minimum MRI-PDFF, and 
o MRE ≤ 2.50 kPa (Chen 2011, Dulai 2016, Loomba 2016, Costa-Silva 
2018) 
Final Cohort assignment to be confirmed based upon results of liver biopsy 
(See Figure 1) 
5. Histopathology assessment of liver biopsy that confirms NASH diagnosis 

 
 
 
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6. Hemoglobin A1c (HbA1c) ≤9.5% 
7. Fasting triglycerides ≤400 mg/dL (4.5 mmol/L) 
8. Body mass index (BMI) ≥35kg/m2 (– 0.5 kg/m2) when qualified for bariatric 
surgery 
9. Able to speak and understand written and spoken English 
10. Understands the procedures and agrees to participate by giving written informed 
consent  
11. Willing and able to comply with scheduled visits, laboratory tests, and other study 
procedures  
Exclusion Criteria 
Exclude participants from this study if any of the inclusion criteria are not met, or if any 
of the following are observed: 
1. Participation in other studies involving investigational drug(s) within 30 days 
prior to Screening Visit 1 
2. History of regular alcohol consumption exceeding 14 drinks/week for females 
or 21 drinks/week for males within the previous 6 months  
(1 drink = 5 ounces [150 mL] of wine, 12 ounces [360 mL] of beer, or 1.5 ounces 
[45 mL] of hard liquor) 
3. A total score of ≥8 on the Alcohol Use Disorders Identification Test (AUDIT) 
questionnaire (Appendix B), indicating harmful or hazardous ethanol 
consumption  
4. A positive urine drug test for illicit drugs 
5. Clinical evidence of hepatic decompensation, including, but not limited 
to esophageal varices, ascites, or hepatic encephalopathy 
6. Evidence of other forms of chronic liver disease (including laboratory tests 
as assessed by a sponsor-identified laboratory, and confirmed with a single repeat, 
if needed): 
 Hepatitis B virus (HBV): defined by presence of hepatitis B surface antigen 
(HBsAg) 
 Hepatitis C virus (HCV): As defined by a clinical history of previous 
diagnosis of Hepatitis C (treated or untreated) or a positive Hepatitis C 
antibody (HCVAb).  
 Human Immunodeficiency Virus (HIV) infection, defined as presence of HIV 
antibody 
 Known diagnosis of primary biliary cirrhosis, primary sclerosing cholangitis, 
autoimmune hepatitis, or overlap syndrome 
 Alcoholic liver disease 
 Known diagnosis of hemochromatosis 

 
 
 
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 Prior known drug-induced liver injury 
 Known or suspected hepatocellular carcinoma (HCC) or other liver cancer 
 History of liver transplant, current placement on a liver transplant list, 
or current model of end-stage liver disease (MELD) score >12 
 Histological presence of cirrhosis on a prior biopsy 
7. Diagnosis of type 1 diabetes mellitus  
8. Any malignancy not considered cured, except basal cell carcinoma and squamous 
cell carcinoma of the skin (a subject is considered cured if there has been no 
evidence of cancer recurrence in the previous 5 years) 
9. Use of drugs historically associated with non-alcoholic fatty liver disease 
(NAFLD) for ≥1 month in the previous year prior to Visit 3 (day of Surgery); 
examples include:  amiodarone, methotrexate, systemic glucocorticoids, 
tetracyclines, tamoxifen, estrogens at doses greater than those used for hormone 
replacement, anabolic steroids, valproic acid, other known hepatotoxins 
10. Subjects with history of severe claustrophobia impacting ability to perform MRI 
during the study without mild sedation/treatment with an anxiolytic 
11. Subjects who fulfill any of the contraindications for MRI; examples include metal 
implants, devices, paramagnetic objects contained within the body and excessive 
or metal-containing tattoos 
12. Unable to participate in MR assessments due to physical limitations of 
equipment tolerances (e.g., MRI bore size and 500-pound weight limit) based 
on Investigator’s judgment at screening 
13. Any person unable to lie still within the environment of the MRI scanner 
or maintain a breath hold for the required period to acquire images without mild 
sedation/treatment with an anxiolytic 
14. Subjects with any anatomical or pathological abnormality that would either 
preclude or tend to confound the analysis of study data, including any clinically 
significant abnormal findings on the MRI obtained at Screening Visit 2. 
15. Blood donation (excluding plasma donations) of approximately 1 pint (500 mL) 
or more within 56 days prior to Screening Visit 1 (participants may not donate 
blood any time during the study, through the final study visit)  
16. Known history or suspected hypersensitivity to human serum albumin, or its 
preparations.   
17. Investigator site staff members directly involved in the conduct of the study 
and their family members, site staff members otherwise supervised by the 
investigator, or subjects who are Pfizer employees, including their family 
members, directly involved in the conduct of the study 
18. Participants who are NPO status will not receive the HepQuant test 

 
 
 
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19. Presence of any condition that, in the opinion of the Investigator, compromises 
participant safety or data integrity or the participant’s ability to complete the 
study. 
 
The eligible population for enrollment in the full longitudinal study is subjects with 
biopsy-confirmed NASH.  Liver biopsies will be collected at the time of the bariatric surgery 
procedure; however, in advance of the surgery, screening criteria based on liver imaging 
assessments will be applied in a sequential manner in effort to help identify eligible subjects.   
The MR assessment performed at Screening Visit 2 will serve as the non-invasive qualification 
of subjects with NASH, which will be defined as those having a liver fat-fraction ≥8% and liver 
stiffness ≥3.00 kPa or MRE ≥ 2.70 kPa (Chen 2011) with at least one of the following risk 
factors for NASH: type 2 diabetes, metabolic syndrome, Screening labs or labs drawn within 30 
days prior to SV2 (ALT ≥ 1.5 times the upper limit of normal, AST to platelet ratio of ≥ 0.7, 
and/or Fibrosis-4 score suggestive of NASH: Age ≤ 35: score not applicable; Age ≥ 36: > 2.67 
(Lin 2011, McPherson 2017). See Appendix C for description of how the scores are calculated. 
The TRI-MD PDFF calculation will be used for determination of eligibility.  Only enrolled 
participants will have their MRI data sent to AMRA and Perspectum for PDFF analysis during 
the study timepoints indicated in Appendix A: Schedule of Events. During the bariatric surgery 
procedure, subject inclusion will be confirmed following histological analysis of the liver biopsy.  
Following bariatric surgery, qualifying subjects will undergo periodic assessments out to 52 
weeks post-surgery.  In addition, subjects who either (1) do not meet the screening criteria for 
presumed NASH prior to surgery, or (2) meet the initial screening criteria, but do not meet the 
biopsy criteria for NASH diagnosis, will be considered for inclusion in a non-NASH cohort 
whose baseline assessments and perioperative biopsies will serve as control data, but for which 
no post-surgical longitudinal assessments will be performed.   
A summary of the screening criteria for subject eligibility is provided in the decision tree 
diagram provided in Figure 1. 
 
 
Figure 1. Decision Tree for Subject Eligibility 
 

 
 
 
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RESOURCES AVAILABLE 
Site Initiation/Study Specific Training 
All TRI-MD faculty and staff assisting with the research will be trained on the protocol 
design, study procedures, and their duties and functions pertaining to this study.  
All training will be documented in accordance with AdventHealth Orlando ORA SOP 06 
(Research Personnel Selection, Qualification, Responsibilities, and Training) and 
WI.031.50.001 Approval, Distribution and Training Process for standard operating 
procedures (SOPs), work instructions (WIs), and study-specific procedures (SSP)-TRI-
MD.  These polices include plans for ongoing discussion of issues throughout the 
duration of the study such as reportable new information, implementing amendments, 
study progress, etc. 
STUDY PROCEDURES 
Subject Recruitment and Screening 
Recruitment methods utilized may include, but are not limited to, the following: 
electronic medical records searches; advertising in multiple media (such as print ads, 
flyers, brochures, posters); radio ads; television spots; and internet advertising (including 
social media).  All advertising materials will be submitted to the Institutional Review 
Board (IRB) for review prior to using or publishing them. 
Recruitment efforts will follow AdventHealth Orlando recruitment standard operating 
procedures (SOPs) for research.  AdventHealth Orlando employees will not be 
individually targeted nor excluded from study participation based on 
employment.  AdventHealth Orlando employees who engage the TRI-MD asking to 
participate in the study will be processed per standard consent procedures for 
participants.  In addition, during the consent process, the study staff will review standard 
consent language stating that an employee’s participation or lack of participation in the 
study will not affect their employment status or relationship with AdventHealth 
Orlando.    
Consent Process  
All study staff delegated the authority to obtain informed consent will follow HRP-802 
(Investigator Guidance: Informed Consent) and HRP-803 (Investigator Guidance: 
Documentation of Informed Consent). 
Non-English Speaking Subjects 
Not applicable as the study will only target English-speakers. 
Subjects who are Not Yet Adults (Infants, Children, Teenagers) 
Not applicable. 
Cognitively Impaired Adults 
Not applicable. 

 
 
 
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Adults Unable to Consent 
Not applicable. 
Documentation of Informed Consent Process 
Documentation of the informed consent process is required to establish that the 
participant was accurately and adequately informed and that no study-related procedures 
were initiated prior to obtaining informed consent.  A research team member will note 
in the source documentation the consent process, the date consent was obtained, and that 
consent was obtained prior to initiating any research procedures. 
Waiver of Written Documentation of Consent or Waiver of Consent  
Not applicable. 
Prohibitions and Restrictions  
During study participation through the final study visit, subjects may not: 
• donate blood 
• use any investigational agent 
• become pregnant (female subjects only) 
Randomization 
Not applicable.  
STUDY VISITS  
Procedures and assessments by visit are outlined below.  For additional details 
and specific time points, see the Detailed Analytical/Clinical Procedures section 
and Appendix A: Schedule of Events, respectively. 
Screening Visit 1 may occur during initial consultation at Celebration Health, or as a 
combination visit (SV1 and SV2) at TRI-MD.  The surgery (Visit 3) will occur at 
Celebration Health, with all remaining visits occurring at TRI-MD. 
Screening Visit 1 (SV1)  
The following procedures may be performed per standard of care during or after the 
initial consultation visit for determination of eligibility for bariatric surgery. The data will 
be collected as part of this research study: 
• physical examination 
• height and body weight measured 
• body mass index (BMI) calculated 
• vital signs measured 
• order provided for standard of care laboratory assessments  
o these tests may be repeated 2–3 weeks before Surgery 

 
 
 
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Signed informed consent will be obtained from interested participants who will then 
undergo the following procedures during their regularly scheduled initial consultation:  
• assessment of participant eligibility 
• demography collection 
• adverse event (AE)/concomitant medication review 
Screening Visit 2 (SV2) 
This visit will occur as soon as possible after SV1, or as a combination with SV1, upon 
final determination of eligibility for bariatric surgery and based on convenience for 
participant.  Any procedures required for eligibility that are not performed during SV1 
will be done at SV2. The following procedures will be performed after an overnight fast: 
• continued assessment of participant eligibility 
• physical examination 
• single supine vital signs 
• height and body weight measured 
o Height will be repeated to ensure all future BMI comparisons are based on 
TRI-MD’s calibrated scales and stadiometer.  
• body mass index (BMI) calculated 
• drug, alcohol, tobacco screen/AUDIT questionnaire 
• AE/concomitant medication review 
• blood collection for screening laboratories, including any the laboratory 
assessments at SV1 were not completed for any reason 
• urinalysis 
• urine pregnancy test 
• urine drug screen 
• FibroScan® 
• MRI 
 
Combination of Screening Visit 1 and Screening Visit 2 – if the initial 
surgery consultation at CH has been previously completed, SV1 and SV2 may be 
combined and completed at TRI-MD. All activities from SV1 and SV2 will be completed 
on the same day without duplicating activities.   
Baseline Visit 1 (BLV1) 
This visit will occur within 28 days after screening Visit 2 but prior the start of the 
standard of care pre-surgery liquid diet.  The following procedures will be performed 
after an overnight fast: 
• AE/concomitant medication review 
• body weight measured 
• BMI calculated 
• single supine vital signs measured 
• urine pregnancy test 

 
 
 
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• blood collection for hepatic panel 
• blood collection for OWLiver Care, OWLiver, and comprehensive metabolomic 
panel from OWL Metabolomics 
• blood collection for pro-collagen protein fragments panel from Nordic Bioscience 
• HepQuant SHUNT Dual Cholate Liver Diagnostic test 
• blood collection for biobanking 
• stool collection during visit or with an at-home kit after the visit 
• standard of care pre-surgery diet (will begin after Baseline Visit 1, but before 
Baseline Visit 2) 
Baseline Visit 2 (BLV2) 
This visit will occur within the final 5 days of the standard of care pre-surgery liquid diet. 
In the rare event that surgery is scheduled without sufficient time to collect BLV1 
endpoints, participants can proceed to BLV2 and continue in the study.  The following 
procedures will be performed after an overnight fast: 
• AE/concomitant medication review 
• body weight measured 
• BMI calculated 
• single supine vital signs measured 
• urine pregnancy test 
• blood collection for hemoglobin level 
• blood collection for hepatic panel 
• blood collection for OWLiver Care, OWLiver, and comprehensive metabolomic 
panel from OWL Metabolomics 
• blood collection for pro-collagen protein fragments panel from Nordic Bioscience 
• If BLV1 is not completed, stool collection will occur during this visit or with an at-
home kit after the visit 
• FibroScan® 
• MRI 
Surgery (V3) – Day 1 
The following procedures will be performed as part of standard of care for bariatric 
surgery: 
• body weight measured 
• BMI calculated 
• single supine vital signs measured 
• urine pregnancy test 
• Roux-en-Y or gastric sleeve will be performed using standard procedures by the 
bariatric surgeons at Celebration Health. 
The following additional research procedures will be performed: 
• Two liver biopsies for clinical histology and exploratory endpoints/biobanking 

 
 
 
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Post-Surgery Visits (V4) – Day 42 ±7 days (6 weeks) 
 
(V5) – Day 84 ±7 days (12 weeks) 
 
(V6) – Day 182 ±14 days (26 weeks) 
 
(V7) – Day 364 ±14 days (52 weeks) 
The following procedures will be performed after an overnight fast: 
• AE/concomitant medication review 
• body weight measured 
• BMI calculated 
• single supine vital signs measured 
• urine pregnancy test 
• blood collection for hepatic panel 
• blood collection for OWLiver Care, OWLiver, and comprehensive metabolomic 
panel from OWL Metabolomics 
• blood collection for pro-collagen protein fragments panel from Nordic Bioscience 
• HepQuant SHUNT Dual Cholate Liver Diagnostic test 
• blood collection for biobanking 
• home stool collection during visit or with an at home kit after the visit 
• FibroScan® 
• MRI 
DETAILED ANALYTICAL/CLINICAL PROCEDURES 
Physical Examination and Medical History 
A standard of care physical examination with collection of comprehensive medical 
history will be performed by a study physician, physician assistant, or nurse practitioner. 
Drug, Alcohol, Tobacco Screen and AUDIT Questionnaire 
Participants will be questioned regarding their consumption of illicit drugs, alcohol, 
and tobacco use.  Participants will also complete the Alcohol Use Disorders Identification 
Test (AUDIT) questionnaire to screen for harmful or hazardous ethanol consumption.  
Appendix B shows the questions the participants will be asked along with the scoring 
methods; the latter will not be provided to participants.  
Height, Body Weight, BMI Calculation 
Body weight (calibrated scale) and height will be obtained while in a gown, with 
undergarments, without shoes.  BMI will be calculated based on subject’s body weight 
and standing height [BMI = body weight in kg ÷ height in m2].  
Vital Sign Measurements 
Single supine measurements of vital signs will include pulse/heart rate, systolic/diastolic 
blood pressure, respiratory rate, and oral/tympanic temperature.  Pulse and blood pressure 
(using an appropriate size cuff for the subject’s arm circumference) will be measured 
with participants in a supine position after 5 minutes of rest.   

 
 
 
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Screening Laboratory Assessments 
The following laboratory assessments are utilized to assess eligibility during the 
screening process:   
• complete blood count (CBC) 
• platelet count 
• comprehensive metabolic panel (CMP) 
• lipid panel 
• prothrombin (may be prothrombin time [PT]/international normalized ratio 
[INR]/partial thromboplastin time [PTT]) 
• activated partial thromboplastin time (APTT) 
• HbA1c 
• thyroid stimulating hormone (TSH) with reflex to thyroxine (T4) 
• parathyroid hormone (PTH) 
• urine pregnancy test 
• urine drug test  
• HIV antibody 
• HBsAg (Hepatitis B antigen) screen 
• HCVAb (Hepatitis C antibody)  
These are used concomitantly to assess general patient health and determine study 
eligibility (Inclusion/Exclusion Criteria).   
 
Circulating Biomarkers 
Assessment of circulating biomarkers will be performed at scheduled visits as outlined 
in  Appendix A: Schedule of Events. 
Metabolomics:  A comprehensive panel of serum metabolites will be measured at OWL 
Metabolomics.  This panel of biomarkers allows for the identification and monitoring of 
disease progression / regression based on changes in the metabolomic signature of highly 
specific circulating lipid components.  The panel includes OWLiver®, OWLiver®Care, 
and approximately 600 additional metabolites for exploratory purposes.  This testing 
paradigm will allow for a comprehensive evaluation of changes in liver health 
and metabolic status after bariatric surgery/  
Hepatic Panel:  This panel includes total protein, albumin, globulin, albumin/globulin 
ratio, total bilirubin, direct bilirubin, alkaline phosphatase, alanine aminotransferase 
(ALT), and aspartate aminotransferase (AST).  It is a standard clinical test commonly 
used to determine the health of the liver.  
Pro-Collagen Protein Fragments:  At minimum, we will measure two pro-collagen 
protein fragments at Nordic Bioscience- ProC3 and ProC6.  Nordic Bioscience has 
developed a panel of biomarkers of extracellular matrix turnover that could provide 
important information about liver health including inflammation, disease prognosis, 
efficacy of interventions, and fibrogenesis.   

 
 
 
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We will bank additional samples for future assessment of novel biomarkers 
and mechanisms.   
Details regarding type of sample, matrix, tubes, volume and sample management 
conditions for collection will be described in the Laboratory Manual.   
Magnetic Resonance Imaging and Spectroscopy (MRI) 
Assessment by MRI will be performed at scheduled visits outlined in Appendix A: 
Schedule of Events and will include measures of liver fat fraction, liver stiffness, liver 
relaxometry, and body composition.  The goal of this test is to assess liver lipid content 
and stiffness, and perform volumetric fat quantitation using an Achieva 3T 
MRI/Multinuclear MRS (Philips, Andover, MA).  Scans will be performed under 
standardized conditions with participants in a supine position, using the quadrature body 
coil and the torsoXL coil.   
Standard MR images will be completed to obtain anatomical images for spectroscopy 
voxel and image slice localization and volumetric measurements of fat, muscle, organs, 
and bone in the participant’s abdomen.    Low resolution scans will be taken to determine 
appropriate positioning for high resolution images.  Imaging sequences will then be 
performed over the participant’s body, approximately spanning from the neck to the 
knees.  The MRE driver will be placed on the participant’s abdomen for the duration of 
the MR acquisition, and engaged during the MRE acquisition, for up to 3 total minutes.   
Images which are degraded due to motion may be repeated as time permits.  
The individual MRI scans and acquisition parameters are detailed in the Imaging Study-
Specific- Procedure (SSP) and Scan Parameters documents.  To minimize the potential 
for confounding factors to affect the measurements, MR scans will be conducted 
following a fast (except water) of at least 4 hours and as much as practically possible, at 
the same time of day relative to the screening assessment (±2 hours).  Attempts will be 
made to ensure longitudinal assessment of an individual subject are performed by the 
same study staff member. 
Total MRI scan time will be approximately 90 minutes. 
 
For eligible participants at SV2, and BLV2, V4, V5, V6 and V7 as indicated in Appendix 
A: Schedule of Events, images will be sent for external analysis. Images will be sent to 
Advanced MR analytics AB (AMRA) for adipose tissue distribution analysis and 
steatosis quantification from the PDFF scans and to Perspectum Diagnostics for 
quantitative, multiparametric MRI analysis using LiverMultiScan, to determine their 
proprietary corrected T1 values, iron content, and liver steatosis.  
 
FibroScan® 
Approximation of liver fat and stiffness by FibroScan® will occur at scheduled visits 
outlined in Appendix A: Schedule of Events.  Fibroscan® uses ultrasound based VCTE™ 
(vibration-controlled transient elastography) to obtain Liver Stiffness Measurement 
(LSM) and Controlled Attenuation Parameter (CAP™); LSM is correlated to liver 

 
 
 
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fibrosis stage, and CAP is correlated to hepatic steatosis.  By using an ultrasound 
transducer probe, a painless, mechanical impulse is delivered to the skin above the liver 
using a low frequency elastic wave (50 MHz).  The wave produces a mechanical 
deformation that moves towards and then through the liver.  The velocity of the shear 
wave in the liver is measured and is directly proportional to its stiffness (stiffer tissue, 
faster velocity).  Using a proprietary algorithm, the device determines whether the shear 
wave propagated into the liver and is valid.  The LSM, expressed in kilopascals (kPa), 
is only calculated on valid measures.  Liver stiffness measurement values range from 
1.5 to 75 kPa; lower values indicate a more elastic liver.  CAP™ measures the ultrasonic 
attenuation coefficient in the forward and return path through the liver at 3.5 MHz 
and is expressed in decibels/meter.  CAP™ is evaluated simultaneously with LSM using 
the same radio-frequency data and is only appraised if the LSM is “valid.”  The specific 
FibroScan® model being utilized is the 502 Touch (Echosens, Paris, France).   
Participants will lie on their back (supine) with their right arm raised in abduction 
to increase the intercostal space.  A water-based gel will be applied to the skin and the 
transducer probe will then be placed on the participant’s skin, in the intercostal space 
directly above the liver with slight pressure.  At least ten valid measurements will be 
taken and the results will be immediate.  To minimize the potential for confounding 
factors to affect the measurements, FibroScan® will be conducted following a fast 
(except water) of at least 4 hours and as much as practically possible, at the same time of 
day relative to the screening assessment (±2 hours).  Only study staff members who are 
trained in the use of FibroScan® are permitted to acquire measures of liver stiffness and 
CAP™.  Additionally, attempts will be made to ensure longitudinal assessments of 
an individual subject are performed by the same study site staff member.   
The test takes a total of about 10 minutes, with total scan time (including setup) of 
approximately 30 minutes.   
Details associated with the FibroScan® scanning protocol are documented in the Imaging 
SSP.   
Quantitative Liver Function Test (HepQuant SHUNT Dual Cholate 
Liver Diagnostic Kit) 
The HepQuant SHUNT Dual Cholate Liver Diagnostic Kit will be administered at 
scheduled visits outlined in Appendix A: Schedule of Events.   
An indwelling peripheral venous catheter is used to collect a baseline Pre-Dose blood 
sample.  Then, 20 milligrams of 13C-cholate is administered intravenously through 
the catheter, and 40 milligrams of d4-cholate is simultaneously administered orally, 
as detailed in the HepQuant Instructions for Use (IFU).  Five timed blood samples drawn 
at 5, 20, 45, 60, and 90 minutes after the simultaneous dosing are utilized to measure the 
cholate pharmacokinetic (PK) clearances.   
Total time for test setup and execution is expected to be 2 hours.  
Additional details on sample processing are documented in the Laboratory Manual. 

 
 
 
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Liver Biopsy 
The liver biopsy will be taken by the physician performing the bariatric surgery using 
a 14-gauge coring needle under direct visualization of the liver.  The biopsy is obtained 
through an incision that is made for the placement of a liver retractor.  The placement of 
the liver retractor is part of the standard procedure for bariatric surgery.  Two small 
biopsy samples of the liver will be taken and then the biopsy site will be cauterized 
to stop any bleeding that occurred due to the procedure.  One of the liver biopsies will be 
sent to a laboratory to be examined by a pathologist for diagnostic assessment of liver 
disease.  Liver histology samples may be sent to a second blinded pathologist for 
research purposes.  The second liver biopsy will be utilized for gene expression 
analysis via RNA sequencing (RNA-Seq).  Any remaining tissue will be archived for 
future analyses.   
 
A “Time Out” will be called during surgery where the surgeon and a research staff 
member will verify that the patient undergoing surgery is a research participant that is 
actively enrolled. The liver biopsy will not be collected for research purposes unless the 
time out is called. 
Collection of the liver biopsies will add approximately 10 minutes to the usual 
bariatric surgery procedure.   
   
Stool Collection 
A stool sample will be collected at the following time points: Baseline visit 1, Day 42 ±7, 
Day 84 ±7, Day 182 ±14, and Day 364 ±14 from participants in the NASH cohort.  
Sampling will occur only at the pre-surgery time point in the non-NASH cohort.  
Participants will be asked to provide the stool sample during their regularly scheduled 
study visits.  If the participant is not able to produce a sample, they will be 
provided with a stool specimen collection kit that includes all necessary supplies 
to collect the stool sample, a box with cold packs for transport and instructions 
for collection. Details of the stool collection kit and methods will be part of the an 
SSP and/or Laboratory Manual.   Once produced, participants will call the study 
coordinator to make arrangements for the sample to be dropped off.  If the 
participant is unable to drop off the stool sample, a courier will be set up to pick 
it up from their home. 
STUDY DURATION 
The overall study duration is anticipated to be up to 18 months.  This duration assumes 
a period of several months between the first baseline visit and the second baseline 
visit, and the longitudinal study period will then continue for 12 months. 
The study is projected to begin between May and July 2018, with study completion 
expected 1 year after recruitment of the final participant.  Recruitment of all participants 
is anticipated to take approximately nine months. 

 
 
 
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MATERIALS OF HUMAN ORIGIN:  COLLECTION, 
PREPARATION, HANDLING AND SHIPPING 
All biological materials will be obtained per the procedures described in this protocol.  
For all study procedures, SSPs will be prepared or established SOPs or Work Instructions 
will be used.  Samples will be collected and analyzed per the documented techniques 
established at the TRI-MD laboratory, Celebration Health and the AdventHealth Orlando 
Center for Diagnostic Pathology. 
Biospecimen samples will be stored in ultralow temperature freezers and liquid nitrogen 
dewars or other storage units located at the TRI-MD Laboratory Room 2404.  The 
TRI-MD facility is secured via key card and equipped with a back-up generator system.  
Laboratory personnel in the facility have 24/7 key-controlled access to the laboratory.  
Chain of custody of biospecimen samples is maintained through requisition forms 
and in the StarLIMS database.  Specimen tubes are coded, and specimen requests and 
distribution are documented.  Access to samples is limited to designated study team 
members as indicated on the delegation of authority log.  Biological materials will be 
maintained in a locked and secured area within the TRI-MD facility.  Physical access 
is limited by badge swipe assignments. 
The biopsy that will be used for histology will be stored at the AdventHealth Orlando 
Center for Diagnostic Pathology for 10 years per the College of American Pathologists 
(CAP) protocol.  Histology samples may be sent to a second pathologist for a blinded 
read for research purposes.   
A subset of de-identified samples will be provided to our collaborators at Pfizer for future 
use as defined below. 
Biospecimens collected for study-related endpoints, will be analyzed/tested at both 
AdventHealth Orlando and outside laboratories/institutions.  Laboratories that will 
analyze de-identified samples may include, but is not limited to:  HepQuant, OWL 
Metabolomics, Jackson Laboratories, and Nordic Bioscience.   
Additionally, remaining biospecimens will be archived for any additional 
hypothesis-related experimentation or testing for this study, which could not be predicted 
at the time the protocol was developed.  
Furthermore, if there are any left-over biospecimens after completing the above, they 
may be archived for the following:  other research (not for this study), but of any type 
(without limitation to disease, process, or research methods).  This other research can 
take place at AdventHealth Orlando or other institutions.   
Lastly, a predetermined amount of biospecimen samples will be collected specifically 
for archiving for future use, such as other research (not for this study), of any type 
(without limitation to disease, process, or research methods).  This other research can 
take place at AdventHealth Orlando or other institutions.   
The biospecimens collected for this study will be separated into biospecimen samples 
that will be used for the study and biospecimen samples that were collected to be 
archived for future use.  After study aims have been achieved and study related endpoints 
have been measured and analyzed, any remaining biospecimens will be stored at the 

 
 
 
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TRI-MD Laboratory Room 2404 and will also be considered as “archived biospecimens.”  
Archived biospecimens will be used for any additional hypothesis-related 
experimentation or testing for the purposes of this study, consistent with the original 
aims, which cannot be predicted at the time the protocol is developed due to the evolving 
nature of scientific exploration.   
Additionally, archived biospecimen samples may be stored indefinitely for future 
research.  Archived biospecimens could be used for separate research by both 
AdventHealth Orlando scientists and scientists outside of AdventHealth Orlando.  This 
would be allowed for research of any type (without limitation to disease, process, or 
research methods) if it has scientific merit as determined by the TRI-MD Scientific 
Review Committee.    For research outside of AdventHealth Orlando, transfer and chain 
of custody of the biospecimens are detailed in the Contract with the Funder and in 
associated Vendor Agreements.  In instances where these documents are not available, a 
Material Transfer Agreement (MTA) will be obtained, which will govern the transfer and 
chain of custody of the biospecimens outside of AdventHealth Orlando. 
STUDY OUTCOME MEASURES (ENDPOINTS) 
Please refer to Table 1.  
DATA MANAGEMENT AND QUALITY PLAN 
Data De-identification 
Participants will be enrolled using Cerner’s Patient Protocol Manager; the application 
assigns each participant a unique participant identifier, or “PID”.  This PID is a code 
consisting of a combination of numerals and letters, which serves as the identifier for this 
participant for this research study and links them back to their hospital medical record 
and their protected health information (PHI).  Access to the “link” between the PIDs, 
the PHI, and to the clinical data are only granted to the clinical research team as assigned 
on the Delegation of Authority Log.  All the clinical research data is recorded 
in a de-identified fashion onto our paper source documents, which is then transcribed 
into our electronic case report forms (eCRFs).  The eCRF is used for storage (a database) 
and facilitates analysis.  Clinical data generated by research devices also uses the PID, 
and once the data has been transformed into interpretable results, it is stored into the 
Clinical Research Database.Both storage locations are secured and only accessible to the 
assigned clinical research team.  The “link” will only be used to re-identify participants in 
the event of a serious adverse event (SAE) requiring identification of a PID and PHI to 
treat the participant.  The “link” will be stored in the Patient Protocol Manager and in 
Clinical Research Database, where only the TRI-MD research team has access.  These 
secure databases are stored/accessed on the AdventHealth Orlando password-protected 
computer network.  No one outside of AdventHealth Orlando investigators or researchers 
will have access to the databases.   
Data Confidentiality, Storage, and Retention 
The identity and personal health information will be kept confidential to the extent 
permitted by the applicable laws and/or regulations and will not be made publicly 

 
 
 
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available.  If results of this study are published or presented, identities will not be 
revealed.  Confidentiality will be maintained during and after the study.  This information 
is also included in the informed consent, which is discussed with each participant prior to 
enrollment. 
All paper sourced study documentation will be stored in the medical records room 
at TRI-MD, which is behind badge access doors, continuously locked, monitored 
and provides for limited access.  The data records may also be stored as electronic 
records.  This data is safeguarded so that only those on the research team have access 
to it via limited badge access to the medical records room (paper) or role relationship 
(electronic).  The electronic data is maintained under Adventist Information Technology 
security controls.   
The duration of study data retention will be determined by governing FDA regulations 
and/or sponsor contract mandate (if applicable).  TRI-MD retention policy is maintained 
in the Records Management Policy.  Electronic de-identified data will be kept 
indefinitely in our data warehouse.   
Data Quality 
Data quality and integrity will be governed by the system of ISO 9001 driven data 
management SOPs.  These SOPs delineate how quality is built into the identification, 
collection, handling, and processing of the data. 
The types of data collected may include data that are: (a) manually abstracted 
or electronically extracted from medical records, (b) observed in clinical exams, 
(c) obtained from laboratory and diagnostic tests, or from various biological monitoring 
or imaging devices, and (d) patient-reported items.  Each data type can be understood by 
its source and the associated collection mechanism.  Only the SOPs/SSPs and associated 
workflows applicable to the study will be used.   
Within the study, quality control steps may include: 
• Source Data Verification (SDV) 
• Query Management 
• Parallel Processing by a second party 
• Complete re-processing of a percentage of the data and comparing to the original  
• Change Control (if changes are made there will be documentation that covers change 
content, rationale, author, time and date) 
Data Sharing (Outside of AdventHealth Orlando)  
This is a AdventHealth-Orlando sponsored clinical study with Pfizer as the funder.  De-
identified results from this study will be shared with collaborators at Pfizer to conclude 
analysis and prepare data for publication.  However, only AdventHealth Orlando will 
have access to the clinical data and the code to identify the samples and make the link 
between lab and clinical data.  The source documentation will remain at TRI-MD.   
Some of the endpoint testing and post-acquisition data analysis will be conducted 
at outside laboratories/institutions including, but not limited to, OWL Metabolomics, 
HepQuant, Nordic Bioscience, Jackson Laboratories, Perspectum Diagnostics 

 
 
 
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and AMRA.  To perform these analyses/testing/etc. and to interpret results, certain data 
elements may need to be shared along with the biospecimen samples.  The governance of 
data sharing is detailed in the Contract with the Funder and in associated Vendor 
Agreements.  When these documents are not available, Data Use Agreements (DUAs) 
or Lab Services Agreements, as appropriate, will be obtained, which will identify the 
specific data elements to be shared (if any) and will govern the sharing of data related 
to this study.  Data will be de-identified, but a link/code is managed within an electronic 
research management system and maintained by a study coordinator.   
Should archived biospecimens be needed for research outside of AdventHealth Orlando 
that is beyond the details in the Contract with the Funder and in associated Vendor 
Agreements and certain data elements that are connected to the archived biospecimen 
samples are needed to conduct the research, then DUAs will be obtained.  The DUAs will 
identify the purpose for data sharing, the specific data elements to be shared, and will 
govern the sharing of data related to this study.  Data will be de-identified, however a 
link/code is managed within an electronic research management system and maintained 
by a study coordinator.   
SAMPLE SIZE DETERMINATION 
Sample size calculations are based on the two primary endpoints, percent change in PDFF 
and MRE.  Based on a literature review it is assumed that the mean percent change from baseline 
in PDFF at 12–16 weeks has a standard deviation of 30%, the mean change from baseline 
in MRE has a standard deviation of 1 kPa.  There is no formal hypothesis testing, however, 
the investigators would like to be able to detect a 30% change from baseline in PDFF 
and a 0.45 kPa change from baseline in MRE.  Using a 1-sided t-test with an alpha level of 0.1, 
there is greater than 99% power for the PDFF change and greater than 80% power for MRE.  
Investigators are also interested in detecting a positive correlation between the 2 measures.  
A test for a correlation greater than zero, with 25 subjects, has greater than 80% power to detect 
a correlation of 0.42 or greater.   
STATISTICAL ANALYSIS PLAN 
The following subsection outlines the statistical methods that will be used in the analysis of 
the study data.  Detailed descriptions will be included in the statistical analysis plan (SAP).  
Any deviations from the SAP will be described in the Clinical Study Report (CSR). 
Summary Analyses 
The number of subjects enrolled, completed, and discontinued from the study will be 
summarized.  For subjects who did not complete the study, the reasons for withdrawal 
from the study will be presented. 
Demographic and baseline characteristics will be summarized for all enrolled subjects.  
Continuous variables will be summarized using mean, median, minimum, maximum, 
quartiles, and standard deviation.  Categorical variables will be summarized using 
relative frequency.  Key demographic and baseline variables will be summarized 
and include, but are not limited to:  age, gender, race, ethnicity, height, weight, BMI, etc. 

 
 
 
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Analysis of PDFF and MRE 
Mixed model repeated measures analysis using all measured data will be used to explore 
changes over time, systematic differences, correlations with other endpoints, and time 
trends.  The models will include baseline values of the measurements and other baseline 
demographic information as deemed necessary.  Figures and tables will be presented.  
Details of the model and correlation structure will be detailed in the SAP. 
Linear mixed models will be used at each time point to calculate the intra-class 
correlation coefficient between MRE and PDFF.  The models will include baseline values 
of the measurements and other baseline demographic information as deemed necessary.  
The models will allow for different variance estimates for each test.  Correlations, least 
squares means, and differences in least squares means, along with their corresponding 
confidence intervals and standard errors, will be presented in tabular form. 
Imaging endpoints measured by other technologies as well as non-imaging endpoints will 
also be examined.  Results will be tabulated and graphed as appropriate.  Continuous 
variables will be summarized using mean, median, minimum, maximum, quartiles, 
and standard deviation.  Categorical variables will be summarized using relative 
frequency.  Mixed model repeated measures analyses using all measured data will be 
used to explore changes over time, systematic differences, correlations with other 
endpoints, and time trends.  The models will include baseline values of the measurements 
and other baseline demographic information as deemed necessary.  Intra-class 
correlations between endpoints may also be calculated.  Details of the model and 
correlation structure will be detailed in the SAP.   
Other exploratory and ad hoc analysis (if done) will be detailed in the CSR and/or SAP. 
Multiplicity 
Since this is an exploratory study intended to characterize PDFF and MRE measurements 
in a bariatric surgery population, no formal hypothesis testing is planned, therefore no 
multiplicity adjustments will be performed.  P-values will be treated as descriptive 
statistics. 
Missing Data 
Descriptive summary statistics will use all available data.  Repeated measures analyses 
will also use all available data and are expected to provide unbiased estimates under the 
assumption that data are missing at random (MAR) under the assumed model.  
Examination of informative missing data and sensitivity analyses may be performed if 
necessary and reported in the CSR.  Multiple imputation (MI) may be used for sensitivity 
or secondary analyses. 
Analysis Populations 
All enrolled patients will be included in the full analysis set (FAS).  The FAS will be 
used for all summaries and analyses unless otherwise specified in the SAP or CSR.   

 
 
 
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POTENTIAL RISKS AND BENEFITS 
POTENTIAL BENEFITS 
Participants will likely receive no direct benefit from taking part in this research study.  
However, the knowledge gained regarding liver disease in bariatric surgery patients may 
benefit public health as a whole.  In addition, patients will receive information about their 
liver health before and after surgery, which may provide some benefit.   
POTENTIAL RISKS 
Intravenous Lines/Blood Draws (e.g., Laboratory Samples) 
The placement of IV needles may cause transient pain, vasovagal syncope, and may also 
result in bruising, bleeding, and/or clotting at the site of needle insertion.  The application 
of direct pressure at the catheterization site will be used to help prevent these symptoms.  
There is a possibility that a catheter placement would be unsuccessful or need to be 
removed.  If this should occur, another catheter would be placed.  It is possible that this 
may occur more than once during participation in the protocol.  Staff who are trained 
and certified in the SOP will be used.  
The total blood collected from each participant will be approximately 545 mL over 
a 52 week follow up period.  This is inclusive of the approximately 90 mL collected 
at screening.  The total blood volume within the first 8 weeks is approximately 280 mL.   
Protection Against Risk 
• All venipuncture will be conducted by qualified staff using aseptic techniques to 
reduce these risks.   
Magnetic Resonance Imaging (MRI) and Spectroscopy 
There are no known biological risks associated with MRI and spectroscopy.  Some 
short -term discomfort may be experienced.  The short-term risks associated with MRI 
are minimal, but include heating, loud noises, and claustrophobia. Some of the images 
require the use a soft rubber-like clip attached to your finger to monitor the heart beat in 
order to compensate for cardiac motion.  There are some people who should not undergo 
MRI; the contraindication is largely based on the presence of metal objects within a 
person (i.e., pacemaker, aneurysm clip, metal fragments, etc.).   
Protection Against Risk 
• There will be strict safety screening per TRI-MD SOP 030.500.001 to ensure any 
people with contraindications are excluded from volunteering. 
• Optional MRI Acclimatization:   
If a participant voices any concerns with tolerability of MRI scans, an acclimatization 
session may be performed during a screening visit.  Participants will undergo an MRI 
acclimation session where they will be asked to lie in the MRI machine, in a manner 
similar to that required for MRI procedures in this study, to ensure participant 

 
 
 
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tolerability.  This screening will last up to 5 minutes, and no data will be collected 
during this acclimation session.   
Incidental Findings 
There will be no diagnostic analysis associated with any of the MRI sequences used 
in this protocol.  The images obtained at the screening visit will be sent for Radiology 
review to exclude any obvious medical abnormalities.  In the case where an 
unexpected anatomical or pathological abnormality is reported by the Radiologist, 
the participant will be excluded from the study and instructed to seek medical 
attention from their health care provider.  On subsequent study visits the images will 
not be sent for radiology review unless a medical abnormality is apparent on an 
image.  In this case, the images will first be reviewed by an investigator on this 
protocol.  If the abnormality is confirmed by radiology, then the participant will be 
instructed to seek medical attention from their health care provider.  The results of the 
radiology interpretation will become part of the participant’s medical record.   
FibroScan®  
There are no known risks associated with FibroScan®.   
Protection Against Risk 
Although there are no known risks, the FibroScan® test will be conducted only by 
individuals that have been trained and certified by the manufacturer.   
Quantitative Liver Function Test (HepQuant SHUNT Liver Diagnostic 
Kit) 
The following includes the known adverse events associated with the use of the 
HepQuant SHUNT Kit: 
1. Risks from the Test Compounds 
a. Allergic reaction to cholate compounds (theoretical – none yet reported) 
b. Allergic reaction to human serum albumin (HSA) 
c. Reactions could include: 
i. 
rash  
ii. having a hard time breathing 
iii. wheezing when you breathe 
iv. sudden drop in blood pressure 
v. swelling around the mouth, throat, or eyes 
vi. fast pulse 
vii. sweating 
viii. severe reactions are very rare but a severe reaction (called anaphylaxis) can 
lead to profoundly low blood pressure and even death 
2. Risks from the Indwelling Catheter 
a. Pain with placement of catheter 
b. Thrombosed vein 
c. Hematoma  

 
 
 
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Risks from the Test Compounds  
Cholates, labeled with stable (nonradioactive) isotopes, occur naturally and are not 
known to have any deleterious or adverse effects when given intravenously or orally in 
the doses used in HepQuant tests.  The serum cholate concentrations that are achieved by 
either the intravenous or oral doses are similar to the serum concentrations of bile acids 
that occur after the ingestion of a fatty meal.  Because cholates are naturally occurring 
with a pool size in humans of 1 to 5 g, the 20 and 40 mg doses of labeled cholates used 
in the HepQuant tests are unlikely to be harmful to a fetus.  However, the effects of these 
compounds on the fetus are not definitively known.  Possible adverse event associated 
with the cholate test compounds may include: allergic reaction.   
The two cholates used in the HepQuant test for this study are labeled with stable 
(non-radioactive) forms of carbon and hydrogen that are found in nature and can be 
measured in blood.  These forms of cholate have been used with FDA Investigational 
New Drug (IND) research studies since 2002, and their use in humans has been 
monitored since that time.  To date, the cholates used in this study have not been 
associated with any allergic reactions or side effects.  However, they are still considered 
experimental and there may be unknown risks. 
Human serum albumin is mixed with the 13C-cholate for IV injection.  Some 
individuals may have a known reaction to serum albumin, and will be excluded from 
this study.  Possible AEs from the HSA may include: allergic reactions.   
Rare Hypersensitivity Reactions have been reported to human serum albumin 
preparations and include anaphylaxis, severe anaphylaxis or anaphylactoid reactions, 
fever, chills, rash, urticaria, pruritus, angioneurotic edema, and erythema or flushing.  
Individuals who are hypersensitive to albumin preparations, any ingredient in the 
formulation, or components of the containers should be excluded from this substudy. 
Risk of Transmissible Diseases in Plasma-derived Preparations.  Because human albumin 
is prepared from pooled human plasma, there is a potential to pass human viruses 
(e.g., hepatitis viruses, HIV) to the recipient and may carry a risk of transmitting 
Creutzfeldt-Jakob disease (CJD) or its variant CJD (vCJD).  Through donor plasma 
screening and specific procedures like pasteurization to eliminate or inactivate any 
possible causes have reduced, but not entirely eliminated, the risk of transmission of 
disease causing agents.  Risk of transmission of viral disease with plasma-derived human 
albumin is considered extremely remote.  No causes of transmission of HBV, HCV, 
or HIV have been documented following use of commercially available human albumin.  
There are no documented cases of CJD or vCJD transmitted through plasma-derived 
preparations (including plasma-derived human albumin); theoretical risk for transmission 
of CJD with commercially available human albumin is considered extremely remote.   
However, no purification method has been shown to be totally effective in removing the 
risk of viral infectivity from plasma-derived preparations and because new blood-borne 
viruses or other disease agents may emerge which may not be removed or inactivated by 
current manufacturing processes, the risks of human albumin are not entirely known. 
Risks from Indwelling Intravenous Catheter 

 
 
 
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Placing the indwelling intravenous catheter will cause minor pain and discomfort.  
With any blood draw, there is a small risk of hematoma and a very small risk of a blood 
clot (1 in 100) or infection (1 in 1000).  HepQuant has performed the HepQuant SHUNT 
test or the prototypical dual cholate research test on over 500 individuals and most had 
the test multiple times.  There has never been an adverse event (AE) - the risk from the 
test is very small.  Possible adverse event from the placement of an indwelling catheter 
may include:  hematoma at injection site.   
Special Considerations in Bariatric Populations 
After bariatric surgery, stomach capacity is approximately 30 milliliters. Sugared 
beverages are not well tolerated after bariatric surgery. It is recommended that 4 meals be 
consumed per day. Prolonged fasting periods may not be tolerated. 
Other Considerations 
Beta blockers or angiotensin converting enzyme (ACE) inhibitors could affect the blood 
flow to the liver, and therefore could affect the flow of the HepQuant SHUNT cholate 
test compounds into the liver as well.  If the flow of blood to the liver was altered by 
these drugs, it could impact the SHUNT test results.  Additionally, because individual 
participants may take different amounts of these drugs, corrections for the different 
concentrations and their impact on blood flow to the liver cannot be made.  As such, 
participants who are currently taking either a beta blocker or an ACE inhibitor will be 
asked to delay taking their normal morning dose the day of their testing and until the 
90-minute test is completed.  Delaying these medications could cause a temporary 
elevation in blood pressure but the risk would be minimal, like that of participants that 
delay doses of medications in everyday life.  Participants will therefore be instructed to 
immediately take their morning dose after the final 90-minute blood draw has been 
completed.  Possible adverse events from delaying taking ACE inhibitor or beta blockers 
may include minimal change in blood pressure.   
Protection Against Risk 
• Individuals with a history or suspected hypersensitivity to human serum albumin, 
or its preparations will be excluded. 
• The HepQuant test has not been used on individuals who have undergone bariatric 
surgery.  Therefore, participants will be monitored closely.  If there are issues 
with tolerability, the test will be stopped and no additional tests will be attempted.  
In addition, the following procedures will be implemented: 
o The total volume of the oral cholate mixture will be 20–30 milliliters.   
o Only artificially sweetened beverages will be used to prepare the cholate 
solution. 
o Participants will be able to consume clear liquids during the procedure 
to mitigate the effects of fasting.  
Liver Biopsy 
In most instances, there are no complications in obtaining a liver biopsy.  
Performing the biopsy does introduce the risk of bleeding as well as a bile leak 
from the biopsy site.  It is also possible that inadvertent injury can occur to 

 
 
 
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surrounding organs from the biopsy needle.  There are no separate skin incisions 
or punctures that are required for this procedure and there should be minimal to 
no added pain from the procedure.   
 
Protection Against Risk 
• Bleeding and bile leak risks are minimized by having the biopsy performed under 
direct visualization of the liver, which allows for the physician to confirm 
adequate hemostasis and the absence of a bile leak. 
• Inadvertent injury to the surrounding organs from the biopsy need are minimized 
by having the biopsy conducted under direct visualization. 
• A pathologist will review the results of the liver biopsy histology and the 
subsequent results and information will be available to the participant.  
Significant findings will be reported the bariatric surgeon.  The results from 
this biopsy will become part of the participant’s medical record.   
 
Stool Collection 
There are no known risks with collecting stool.   
Protection Against Risk  
N/A 
MITIGATION OF RISKS  
Risk monitoring will be performed via a weekly or biweekly individual review of cases 
by the Principal Investigator, the study physician, and/or research coordinator, including 
progress or AEs occurring in the following: subject confidentiality, subject recruitment, 
informed consent process, data quality, and any external factors relevant for the safety of 
participants throughout the entire study.  In addition to these items, the study coordinator 
has been specifically instructed to make the Primary Investigator aware of all AEs, 
expected or unexpected that arise during the course of the study.  Wound healing after 
invasive procedures (i.e., IV line placement) will be monitored.  Laboratory tests are 
obtained during the screening visits of the study and any abnormal findings are sufficient 
to exclude individuals.  If an abnormal laboratory value is obtained, the research 
participant will be referred to their primary care physician for follow-up.  If illness 
or injury occurs during a study procedure, participants will be transported to 
the Emergency Room if needed.   
Provisions to Protect the Privacy Interest of Subjects 
Participants will be assigned unique identifiers for study-related records.  All precautions 
will be taken to make sure that only authorized individuals will access subject research 
records.  The collection of sensitive information about subjects will be limited to the 
minimum necessary to achieve the aims of the research, so that no unneeded sensitive 
information will be collected.   

 
 
 
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EARLY WITHDRAWAL OF SUBJECTS 
Investigator Withdrawal of Subjects 
Participation in the study may also be discontinued at any time at the discretion of the 
Principal Investigator, without participant consent if: 
• The Principal Investigator believes it necessary for a participant’s health or safety. 
• Participant has not followed study instructions. 
• Participant is not adherent to the intervention. 
• The TRI-MD has stopped the study. 
• Administrative reasons require a participant’s withdrawal. 
Participants will be withdrawn from the study if: 
• Participant uses any investigational agent (up to the final study visit). 
• Female participant has become pregnant. 
Subject Request for Withdrawal from Study 
Participation in this study is voluntary.  Participants may decide not to participate in this 
study or may withdraw from this study at any time without penalty or loss of benefits.  
If a participant leaves the study before the final regularly scheduled visit, he/she may be 
asked by the Investigator to make a final visit for ‘end-of-study’ procedures.  This is to 
ensure there are no safety concerns.   
Data Collection and Follow-up for Withdrawn Subjects 
If the study is prematurely terminated, or if a subject requests withdrawal from the study, 
or if the Principal Investigator withdraws a subject from the study, study data will be 
maintained in the research database up to the point of withdrawal.  This data will be 
included in subsequent analysis, as keeping these participants in the analysis is essential 
for study validity.   
ADVERSE EVENT REPORTING 
Adverse Events 
An AE is defined as both an expected side effect that is of a serious nature, or an 
unexpected side effect/event regardless of severity.  Each participant is evaluated for AEs 
at every study visit.  Any event that is reported to the study staff and meets the criteria 
of an AE will be documented as such and graded as to its attribution (unrelated to 
protocol, or possibly, probably, or definitely related to protocol) and severity (mild, 
moderate, or severe).  Any severe and/or unanticipated AE will be reported to the 
institutional review board (IRB) according to HRP 801 INVESTIGATOR GUIDANCE:  
Prompt Reporting Requirements.   
Recording and Notification of Adverse Events 
At each contact with the participant, the study team members will seek information on 
AEs by specific questioning and, as appropriate, by examination.  Information on all AEs 

 
 
 
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will be recorded immediately in the source document, and also in the appropriate AE 
module of the eCRF.  All clearly related signs, symptoms, and abnormal diagnostic 
procedures results will be recorded in the source document.   
Any event meeting the reporting guidelines per HRP 801 INVESTIGATOR 
GUIDANCE:  Prompt Reporting Requirements, will be reported to the AdventHealth 
Orlando IRB.   
Safety Monitoring Plan   
Safety Monitoring 
Adverse events will be documented and reported by the study coordinator, the Principal 
Investigator, and other TRI-MD staff.  Research and safety data will be reviewed by the 
Principal Investigator.  This review will take place at regular meetings with the research 
coordinator and study physician where the safety labs for each new participant will be 
reviewed.  Other items discussed will include progress or AEs occurring in the following:  
subject confidentiality, subject recruitment, and the informed consent process.  All AEs 
and unexpected and/or severe AEs will be reported to the AdventHealth Orlando IRB per 
HRP 801 INVESTIGATOR GUIDANCE:  Prompt Reporting Requirements.  The TRI-
MD has a standing committee that meets monthly to review all AEs in the clinical trials 
and will additionally be charged with review of the study.   
Data and Safety Monitoring Board (DSMB) or Equivalent 
Not applicable. 
ETHICAL CONSIDERATIONS 
Participation in this study is voluntary.  Participants may decide not to participate in this study 
or may withdraw from this study at any time without penalty or loss of benefits.  No vulnerable 
populations will be studied in this protocol.    
Sharing of Results with Subjects 
Participants will be offered the opportunity to meet with the Principal Investigator 
or designated medical staff to review the results of their laboratory assessments or other 
standard clinical data.  Copies of their testing results will be made available to the 
participants upon request.  In addition, the Principal Investigator or designated study staff 
will provide an overview of the study's outcome to the participant, when available, if he 
or she requests the information.   
FUNDING SOURCE 
This study will be externally funded by Pfizer Inc.   
PARTICIPANT STIPENDS OR PAYMENTS 
After the participant’s completion of all study visits, a total amount for the study will be $1,000-
1,375.00. A Mastercard® payment will be processed with the Dollar amount per visits completed 
as indicated in the table below. Mastercard® payments may take up to 3 business days to be 

 
 
 
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processed, once requested. In the event the participant is unable to complete all study visits the 
payment will be prorated.  
 
The payments are broken into two payments: Screening Visit 1 through Visit 5 (after completion 
of all visits through the 12-week time point), and Visits 6-7 (after completion of the remaining 
visits ending at the 52-week time point). 
Compensation for Baseline Visit 1 and Visits 4-7 will vary, depending whether a stool sample is 
provided and the dual cholate test is completed.  
Participants who agree to take part in this study will be paid for completed procedures according 
to the following schedule:   
 
Visit 
Total 
Screening Visit 1 
$50.00 
Screening Visit 2 
If both screening visits are combined, the payment will also be combined 
$125.00 
Baseline Visit 1   
Stool - $25.00; Dual cholate $50.00 
$50.00-125.00 
Baseline Visit 2 
$125.00 
Visit 3 (Surgery) 
$150.00 
Visit 4 
Stool - $25.00; Dual cholate $50.00 
$125.00-200.00 
Visit 5 
Stool - $25.00; Dual cholate $50.00 
$125.00-200.00 
Visit 6 
Stool - $25.00; Dual cholate $50.00 
$125.00-200.00 
Visit 7    
 Stool - $25.00; Dual cholate $50.00 
$125.00-200.00 
TOTAL 
$1,000-1,375.00 
 
PUBLICATION PLAN 
TRI-MD faculty and staff will adhere to POLICY-TRI-ADM-005 (Access to Clinical Trial Data 
for Publication Purposes).   

 
 
 
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REFERENCES 
1. Ahmed MH, Abu EO, Byrne CD. Non-alcoholic fatty liver disease (NAFLD): new challenge 
for general practitioners and important burden for health authorities? Prim Care Diabetes. 
2010; 4(3): 129–37. 
2. Loomba R, Sanyal AJ. The global NAFLD epidemic. Nat Rev Gastroenterol Hepatol. 
2013; 10(11): 686–90. 
3. Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural 
history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. 
Aliment Pharmacol Ther. 2011; 34(3): 274–285. 
4. Sanyal AJ, Friedman SL, McCullough AJ, Dimick-Santos L. Challenges and opportunities in 
drug and biomarker development for nonalcoholic steatohepatitis: findings and 
recommendations from an American Association for the Study of Liver Diseases–U.S. Food 
and Drug Administration Joint Workshop. Hepatology. 2015; 61(4):1392–405. 
5. Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions and new insights. 
Science. 2011; 332(6037): 1519–1523. 
6. Ratziu V, Charlotte F, Heurtier A, et al. Sampling variability of liver biopsy in nonalcoholic 
fatty liver disease. Gastroenterology. 2005; 128(7): 1898–1906. 
7. Alkhouri N, McCullough AJ. Noninvasive diagnosis of NASH and liver fibrosis within the 
spectrum of NAFLD. Gastroenterol Hepatol (NY). 2012; 8(10): 661–668. 
8. Papagianni M, Soforgianni A, Tziomalos K. Non-invasive methods for the diagnosis of 
nonalcoholic fatty liver disease. World J Hepatol. 2015: 7(4): 638–648. 
9. Renelus B, Foster T. Noninvasive evaluation of nonalcoholic fatty liver disease. Clin Liver 
Dis. 2016; 7(3): 45–47. 
10. Dulai PS, Sirlin CB, Loomba R. MRI and MRE for non-invasive quantitative assessment of 
hepatic steatosis and fibrosis in NAFLD and NASH: Clinical trials to clinical practice. 
J Hepatol. 2016; 65(5): 1006–1016. 
11. Hashemi SA, Alavian SM, Gholami-Fesharaki M. Assessment of transient elastography 
(FibroScan) for diagnosis of fibrosis in non-alcoholic fatty liver disease: A systematic review 
and meta-analysis. Caspian J Intern Med. 2016; 7(4): 242–252. 
12. Barker KB, Palekar NA, Bowers SP, Goldberg JE, Pulcini JP, Harrison SA. Non-alcoholic 
steatohepatitis: effect of Roux-en-Y gastric bypass surgery. Am J Gastroenterol. 2006; 
101(2): 368–373. 
13. Rabl C, Campos GM. The impact of bariatric surgery on nonalcoholic steatohepatitis. Semin 
Liver Dis. 2012; 32(1): 80–91. 
14. Hafeez S, Ahmed MH. Bariatric surgery as potential treatment for nonalcoholic fatty liver 
disease: a future treatment by choice or by chance? J Obes. 2013; 839275: 1-11. 
15. Schneck A-S, Anty R, Patouraux S, et al. Roux-en Y gastric bypass results in long-term 
remission of hepatocyte apoptosis and hepatic histological features of non-alcoholic 
steatohepatitis. Front Physiol. 2016; 7: 344. 

 
 
 
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16. Alizai PH, Wendl J, Roeth AA, et al. Functional liver recovery after bariatric surgery—
a prospective cohort study with the LiMAx test. Obes Surg. 2015; 25(11): 2047–2053. 
17. Barr J, Caballeria J, MArtinez-Arranz I, et al. Obesity-Dependent Metabolic Signatures 
Associated with Nonalcoholic Fatty Liver Disease Progression. J Proteome Res. 2012; 11: 
2521–2532. 
18. Crespo J, Martinez-Arranz I, Romero-Gómez M, et al. A non-invasive lipidomic test 
accurately discriminates non-alcoholic steatohepatitis from steatosis: A blind validation 
study. J Hepatology. 2016; 64(2): S478. 
19. Nielsen MJ, Nedergaard AF, Sun S, et al. The neo-epitope specific PRO-C3 ELISA measures 
true formation of type III collagen associated with liver and muscle parameters. Am J Transl 
Res. 2013; 5(3): 303–315.  
20. Nielsen MJ, Veidal S, Karsdal M, Patel K. Plasma Pro-C3 (N-terminal type III collagen 
propeptide) predicts fibrosis progression in patients with chronic hepatitis C. Liver Int. 2015; 
35: 429–437. 
21. Helmke S, Colmenero J, Everson GT. Noninvasive assessment of liver function. Curr Opin 
Gastroenterol. 2015; 31(3): 199–208. 
22. Schnabl B, Brenner DA. Interactkions between the intestinal microbiome and liver diseases. 
Gastroenterology. 2014; 146(6): 1513–1524. 
23. Bonder A, Afdhal N. Utilization of FibroScan in clinical practice. Curr Gastroenterol Rep. 
2014; 16(2): 372. 
24. Chen J, Talwalkar JA, Yin M, Glaser KJ, Sanderson SO, Ehman RL. Early detection of 
nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease by using MR 
elastography. Radiology. 2011; 259(3): 749–756. 
25. Dulai, P.S., C.B. Sirlin, and R. Loomba, MRI and MRE for non-invasive quantitative 
assessment of hepatic steatosis and fibrosis in NAFLD and NASH: Clinical trials to clinical 
practice. Journal of Hepatology, 2016. 65(5): p. 1006-1016. 
26. Loomba, R., et al., Novel 3D Magnetic Resonance Elastography for the Noninvasive 
Diagnosis of Advanced Fibrosis in NAFLD: A Prospective Study. Am J Gastroenterol, 2016. 
111(7): p. 986-94. 
27. Costa-Silva, L., et al., MR elastography is effective for the non-invasive evaluation of 
fibrosis and necroinflammatory activity in patients with nonalcoholic fatty liver disease. 
European Journal of Radiology. 2018; 98: 82-89. 
28. Lin, Z-H., et. al., Performance of the Aspartate Aminotransferase-to-Platelet Ratio Index for 
the Staging of Hepatitis C-Related Fibrosis: An Updated Meta-Analysis. Hepatology. 2011; 
53: 726-736. 
29. McPherson, S., et. al., Age as a Confounding Factor for the Accurate Non-Invasive 
Diagnosis of Advanced NAFLD Fibrosis. Am J Gastroenterol. 2017; 112(5): 740-751. 
30. Thomas MS, Newman D, Leinhard OD, Karlsson, A. Test-retest reliability of automated 
whole body and compartmental muscle volume measurements on a wide bore 3T MR 
system. Eur Radiol. 2014; 24(9): 2279–2291. 

 
 
 
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31. Karlsson A, Rosander J, Romy T, et al. Automatic and quantitative assessment of regional 
muscle volume by multi-atlas segmentation using whole-body water–fat MRI. J Magn Reson 
Imaging. 2015; 41: 1558–1569. 
32. West J, Leinhard OD, Romu T, et al. Feasibility of MR-based body composition analysis in 
large scale population studies. PLoS ONE. 2016; 11(9): e0163332. 
33. Banerjee R, Pavlides M, Tunnicliffe EM, et al. Multiparametric magnetic resonance for the 
non-invasive diagnosis of liver disease. J Hepatol. 2014; 60(1): 69–77. 
34. Pavlides M, Banerjee R, Sellwood J, et al. Multiparametric magnetic resonance imaging 
predicts clinical outcomes in patients with chronic liver disease. J Hepatol. 2016; 64(2):  
308–315. 
 

 
 
 
 
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APPENDIX A.  SCHEDULE OF EVENTS 
Description 
Screening 
Visit 1 
Screening 
Visit 2 
Baseline 
Visit 1 
Baseline 
Visit 2 
Surgery 
Post-Surgery 
Visit Number 
SV1 
SV2l 
BLV1m 
BLV2 
V3 
V4 
V5 
V6 
V7 
Day a 
Varies 
Varies 
Varies 
Varies 
1 
42 ±7 
84 ±7 
182 ±14 
364 ±14 
Physical Exam b  
X 
X 
 
 
 
 
 
 
 
Provider time to explain study 
X 
 
 
 
 
 
 
 
 
Assess participant eligibility  
X 
X 
 
 
X 
 
 
 
 
Informed consent c 
X 
 
 
 
 
 
 
 
 
Demography c 
X 
 
 
 
 
 
 
 
 
AE/ConMed review  
 
X 
X 
X 
 
X 
X 
X 
X 
Drug Alcohol Tobacco 
Screen/AUDIT questionnaire  
 
X 
 
 
 
 
 
 
 
Height and body weight d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Body Mass Index (BMI) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs b 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Screening Lab assessments 
(fasting) e 
 
X 
 
 
 
 
 
 
 
Urine pregnancy test 
 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis 
 
X 
 
 
 
 
 
 
 
Urine drug screen 
 
X 
 
 
 
 
 
 
 
HIV, HBV, HCV screen 
 
X 
 
 
 
 
 
 
 
Hepatic panel 
 
 
X 
X 
 
X 
X 
X 
X 
Hemoglobin 
 
 
 
X 
 
 
 
 
 
Pre-surgery diet f 
 
 
 
X 
 
 
 
 
 
 
SoC Bariatric surgery  
 
 
 
 
X 
 
 
 
 
Liver biopsy g 
 
 
 
 
X 
 
 
 
 
Blood collections for: 
OWL Metabolomics 
Nordic Bioscience 
 
 
X 
X 
 
X 
X 
X 
X 
Blood collections for: 
Biobanking h 
 
 
X 
 
 
X 
X 
X 
X 
Stool Collection  i 
 
 
X 
 
 
X 
X 
X 
X 
HepQuant SHUNT Dual Cholate 
Liver Diagnostic Kit j 
 
 
X 
 
 
X 
X 
X 
X 
IMAGING 
FibroScan® 
 
X 
 
X 
 
X 
X 
X 
X 
MRIk 
 
X 
 
X 
 
X 
X 
X 
X 
 
 

 
 
 
 
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SCHEDULE OF EVENTS, cont. 
Abbreviations:  AE = adverse event; AUDIT = Alcohol Use Disorders Identification Test; CH = Celebration Health; ConMed = concomitant medication; HBV = hepatitis B virus; 
HCV = hepatitis C virus; HIV = human immunodeficiency virus; MRE = magnetic resonance elastography; MRI = magnetic resonance imaging; SoC = standard of care; 
TRI-MD = Translational Research Institute for Metabolism and Diabetes.   
Note:  Screening Visit 1 (SV1) may occur either at CH or TRI-MD. Surgery (V3) occurs at CH. All other visits and procedures, including the combination screening visit 
(if applicable), occur at TRI-MD. 
a. 
Pre-surgery time points will vary based upon insurance requirements, length of diet, and other clinical needs.   
b. 
Performed by CH provider as a part of standard of care procedures at SV1 and at TRI-MD at indicated visits per our standard research procedures.   
c. 
Performed by CH study coordinator/CH provider.   
d. 
Height measured at SV1 and SV2 only; weight measured at designated visits for calculation of body mass index (BMI).   
e. 
Screening laboratory assessment are collected at SV2 or the combination SV1/SV2 visit and include:  complete blood count (CBC), platelet count, comprehensive metabolic 
panel (CMP), lipid panel, prothrombin (may include prothrombin time [PT]/international normalized ratio [INR]/partial thromboplastin time [PTT]), activated partial 
thromboplastin time (APTT), , hemoglobin A1c (HbA1c), thyroid stimulating hormone (TSH) with reflex to thyroxine (T4), parathyroid hormone (PTH).   
f. 
The pre-surgery diet varies and occurs as per standard of care.  BLV2 will occur during the last 5 days of the liquid diet.   
g. 
Liver biopsy for clinical histology and research.  Participants without non-alcoholic steatohepatitis (NASH) upon biopsy will not continue longitudinal assessment. The Liver 
Biopsy is the final criteria determining the cohort (NASH vs NON-NASH) 
h. 
Blood collections for biobanking:  plasma, serum, PAX DNA, P800 tube.  
i. 
Stool will be collected during clinic visit if possible. If not, a home stool collection kit will be sent home with participant.   
j. 
HepQuant SHUNT Dual Cholate Liver Diagnostic will be discontinued if there are any feasibility or tolerability issues.   
k. 
The TRI-MD liver fat percentage by Proton Density Fat Fraction (PDFF) will be used to ascertain eligibility. For participants that meet eligibility criteria at SV2, data will be 
sent to Perspectum and AMRA to generate PDFF and other liver endpoints from MRI data collected at SV2 and BLV2, V4, V5, V6 and V7.  
l. 
SV2 will occur as soon as possible after SV1. 
m. BLV1 will occur within a maximum of 28 days from SV2. 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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APPENDIX B.  ALCOHOL USE DISORDERS IDENTIFICATION TEST 
(AUDIT) QUESTIONNAIRE 
 

 
 
 
 
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APPENDIX C. Liver Disease Score Calculation 
 
To further evaluate potential risk of NASH, we will calculate two different scores based on 
bloodwork and clinical data we are already collecting.  
 
The AST to Platelet Ratio (APRI) score is calculated via the following formula:   
(AST in IU/L) / (AST Upper Limit of Normal in IU/L) / (Platelets in 109/L) 
 
The Fibrosis-4 (FIB-4) Index for Liver Fibrosis is calculated with the following formula: 
(Age x AST) / (Platelets x √(ALT))