The Role of Very Low Calorie Ketogenic Diet (VLCKD) in Patients Affected by Non-Alcoholic Steatohepatitis (NASH) With Significant Fibrosis (KETONASH)
Phase: NA
Conditions: NASH, NAFLD, Obesity, Liver Fibrosis
Sponsor: University of Bologna

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
"MULTICENTRIC,
 
PROSPECTIVE,
 
OPEN,
 
RANDOMISED,
CONTROLLED,  AND  INTERVENTIONAL  STUDY  PROTOCOL  THAT
DOES NOT INVOLVE THE USE OF MEDICINAL PRODUCTS"
Study Code: KETONASH
ClinicalTrial.gov (NCT) number: TBD
Study title:
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD)
in  Patients  Affected  by Non-Alcoholic  Steatohepatitis
(NASH) with Fibrosis"
Author(s):
    Federico Ravaioli
Study Code:
    KETONASH
NCT number:
    TBD
Version Date:
    23.11.2023
Version:
    4.0
This document is the property of the study coordinator. Its content is CONFIDENTIAL and
must not be disclosed, disseminated, copied, or used without written authorisation.
1
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
SIGNATURE PAGE
Study Title:
   "The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected
by Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
Author(s):
   Federico Ravaioli
Study Code:
   KETONASH
NCT number:
   TBD
Version Date:
   23.11.2023
Version:
   4.0
Author(s) signature(s):
This protocol has been written and will be conducted in accordance with good clinical practice and
applicable national regulations.
Dr. Federico Ravaioli
(Author)
_________________________________
Date: 24.11.2023
Principal Investigator's and Collaborators' signatures:
I declare that I have read the protocol and consent to conduct this clinical study in accordance with
all protocol requirements and Good Clinical Practice Guidelines.
Prof. Fabio Piscaglia
(Principal Investigator)
_________________________________
Date: 24.11.2023
Dr.
 
Federico
 
Ravaioli
(Scientific Supervisor)
_________________________________
Date: 24.11.2023
Prof.  Maria  Letizia  Petroni
(Scientific Supervisor)
_________________________________
Date: 24.11.2023
2
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
TABLE OF CONTENTS
PROTOCOL SIGNATURE PAGE…………………………………………………………………………. 2
TABLE OF CONTENTS……………………………………………………………………………………. 3
PROTOCOL SYNOPSIS…………………………………………………………………………………… 5
LIST OF ABBREVIATIONS AND ACRONYMS………………………………………………………….. 8
INTRODUCTION AND RATIONALE……………………………………………………………………….9
STUDY OBJECTIVES……………………………………………………………………………………. 13
STUDY POPULATION……………………………………………………………………………………. 14
Inclusion and exclusion Criteria……………………………………………………………………………………... 14
Study Withdrawal Criteria……………………………………………………………………………………………. 15
STUDY DESIGN………………………………………………………………………...………………… 15
Identification, Screening, and Randomization (Visit 1)…………………………………………………………… 16
Dietary Intervention (Visits 2-8)……………………………………………………………………………………... 18
Maintenance Phase (Visits 9-13)……………………………………………………………………………………. 20
Study End (Visit 15)…………………………………………………………………………………………………... 20
Physical Activity……………………………………………………………………………………………………….. 20
Psychological-Motivational Support………………………………………………………………………………… 20
STUDY PROCEDURES………………………………………………………………………………….. 21
Medical Visits and Dietary Consultations…………………………………………………………………………... 21
Body Composition Assessment via Bioelectrical Impedance Analysis (BIA)…………………………………... 22
Liver Biopsy…………………………………………………………………………………………………………… 23
Abdominal  Ultrasound  with  Transient  Elastography  (FibroScan®)  and  CAP™ (Controlled  Attenuation
Parameter)
…………………………………………………………………………………………………………………….. 24
Laboratory Tests and Serum Biomarkers………………………………………………………………………….. 26
Summary of Study Procedures……………………………………………………………………………………… 27
MEASUREMENT OF STUDY OBJECTIVES…………………………………………………………... 27
Expected Results……………………………………………………………………………………………………... 28
Study Duration………………………………………………………………………………………………………… 28
STATISTICAL ANALYSIS PLAN…………………………………………………………………………. 28
Sample Size Determination………………………………………………………………………………………….. 28
Statistical Analysis……………………………………………………………………………………………………. 28
Interim Analysis……………………………………………………………………………………………………….. 29
DATA COLLECTION………………………………………………………………………………………. 29
3
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
Electronic Data Capture Form (eCRF)……………………………………………………………………………... 29
Data Management and Data Quality……………………………………………………………..………………… 29
STUDY COSTS……………………………………………………………………………………………. 31
REGULATORY AND ETHICAL ASPECTS……………………………………………………………... 31
Statements of Compliance…………………………………………………………………………………………... 31
Ethics Committee……………………………………………………………………………………………………... 31
Informed Consent Management…………………………………………………………………………………….. 31
Confidentiality and Data Protection…………………………………………………………………………………. 32
Insurance………………………………………………………………………………………………………………. 32
Data Use and Results Publication…………………………………………………………………………….……. 32
REFERENCES…………………………………………………………………………………………….. 33
4
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
PROTOCOL SYNOPSIS
Study Code
KETONASH
NCT Number
TBD
Principal Investigator
Prof.  Fabio  Piscaglia:  Internal  Medicine  Unit  for  Hepatobiliary  and
Immunoallergological Diseases, IRCCS Hospital-University of Bologna,
Sant’Orsola Polyclinic
Study Purpose
The purpose of the KETONASH study is to evaluate, in patients with
metabolic-associated fatty liver disease (MAFLD) with non-alcoholic
steatohepatitis  (NASH) and significant liver fibrosis (F1-F2-F3), the
effect of a very-low-calorie ketogenic diet (VLCKD) compared to that of
a  standard  low-calorie  diet  (standard  Mediterranean  LCD  -  in
accordance  with  the  European  Association  for  the  Study  of  the
Liver/European Society for Clinical Nutrition and Metabolism guidelines
on MAFLD/NAFLD).
Primary Objective
• Histological improvement in terms of liver fibrosis without worsening
of NASH
• Histological improvement of NASH without worsening of liver fibrosis
Study Type
Multicentric,  Prospective,  Open,  Randomised,  Controlled,  and
Interventional, with no medicinal use
Study Phase
N/A
Study Design
5
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
Study Coordinator
Internal  Medicine  Unit  for  Hepatobiliary  and  Immunoallergological
Diseases,  IRCCS  Hospital-University  of  Bologna,  Sant’Orsola
Polyclinic
Study  Duration  per
Patient
The study has a duration of 12 months from patient enrollment and
randomisation.
Planned Follow-up
N/A
Population
Patients with a current or past diagnosis of metabolic-associated fatty
liver disease (MAFLD/NAFLD/MAFLD) formulated in accordance with
the European Association for the study of the Liver guidelines, with
significant  liver  fibrosis  (F2-F3)  and  non-alcoholic  steatohepatitis
(NASH)
Sample Size Estimate
42 patients (2:1 randomisation)
Number
 
of
 
Study
Centers
3 centers (including Coordinator)
Randomisation
Two arms, 2:1 randomisation stratified by diabetes mellitus and gender
Participants
Inclusion Criteria:
• Patients aged ≥18 years with histological diagnosis of NASH with
evidence  of  stage  1,  2,  or  stage  3  fibrosis  (F1/F2/F3)  (defined
according to NASH CRN[1]) obtained no more than 6 months before
enrollment;
• Stable weight for more than  6 months with BMI between 30-40
kg/m2;
• Patients in whom it is safe and feasible to proceed with liver biopsy
and who consent to undergo liver biopsy after 12 months of enrollment
to assess the effect of dietary treatment;
• Obtained informed consent.
Exclusion Criteria:
6
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
• BMI <30 or BMI >40
• Presence of evolved chronic liver disease into cirrhosis (histological
F4 or elastometric LSM >14 kPa)
• Type 1 diabetes mellitus
• Model for End-stage Liver Disease (MELD) score >12, AST or ALT
≥5× ULN, HbA1c >9.5%, INR ≥1.4, creatinine >1.5 mg/dl, platelets
<100,000/mm3, and total bilirubin >1.5 mg/dl.
•  Concurrent  presence  of  any  other  known  chronic  liver  disease
beyond  MAFLD/NAFLD,  such  as  alcoholic  liver  disease,  viral
(HCV/HBV),  cholestatic-autoimmune  (PBC/PSC/AIH),  Wilson's
disease,  hemochromatosis,  drug-induced  liver  injury  (DILI),  or  the
presence or suspicion of hepatocellular carcinoma (HCC);
•  Average  alcohol  consumption  exceeding  4/2  units/day
(males/females) in the preceding 6 months and a history of excessive
alcohol consumption in the last 5 years;
• Previous or planned liver transplant, bariatric surgery, ileal resection,
or biliary diversion;
• History of acute cholecystitis and biliary obstructions (cholangitis);
• Recent (in the last 12 months) or concurrent use of agents known to
cause hepatic steatosis (long-term systemic corticosteroids [>10 days],
amiodarone,  methotrexate,  tamoxifen,  tetracyclines,  high-dose
estrogens, valproic acid);
•  Recent  (in  the  last  3  months)  change  in  the  dose/regimen  or
introduction of Vitamin E (at doses ≥400 IU/day), ursodeoxycholic acid
(UDCA), betaine, S-adenosyl methionine, silymarin, or pentoxifylline;
• Presence of psychiatric disorders and/or diagnosis of any eating
disorder;
• Life expectancy <6 months.
Interim Analysis
No interim analyses are planned.
7
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
LIST OF ABBREVIATIONS AND ACRONYMS
AASL
American Association for The Study Of Liver Diseases
ICW
Intra Cell Water
AGA
American Gastroenterology Association
IgA
Immunoglobulin A
AIH
Autoimmune Hepatitis
IgG
Immunoglobulin G
ALT
Alanine Amino Transferase
IgM
Immunoglobulin M
AST
Aspartate Amino Transferase
BMI
Body Mass Index
BCM
Body Cellular Mass
INR
International Normalized Ratio
BDI
Beck Depression Inventory
IPAQ
International
 
Physical
 
Activity
Questionnaire
BES
Binge Eating Scale
LCD
Low Calorie Diet
BIA
Body Impedance Analysis
LDL
Low-Density Lipoprotein
BIVA
Body Impedance Vectorial Analysis
LSM
LiverStiffnessMeasure
BMI
Body Mass Index
MAFLD
Metabolic Associated Fatty Liver Disease
BMR
Basal Metabolic Rate
MELD
Model for end-stage Liver diseases
CAP
Controlled Attenuation Parameters
MM
Muscle Mass
CONSORT
Consolidating Standards Of Reporting Trials
NAFLD
Non-Alcoholic Fatty Liver Disease
DILI
Drug-InducedLiverInjury
NASH
Non-AlcoholicSteatohepatitis
DNA
Deoxyribonucleic acid
NFS
NAFLD Fibrosis Score
EASL
European Association for The Study of The Liver
NHANE
S
National
 
Health
 
and
 
Nutrition
Examination Survey
ECW
Extra Cell Water
PBC
PrimaryBiliaryCholangitis
EDTA
Ethylenediaminetetraacetic acid
PSC
PrimarySclerosingCholangitis
EFSUMB
European Federation of Societies for Ultrasound in Medicine
and Biology
RCT
RandomizedControled Trial
ESPEN
European Society for Clinical Nutrition and Metabolism
RNA
 Ribonucleic acid
FFT
Free Fat Mass
STAI
State-Trait Anxiety Inventory
FLI
Fatty Liver Index
T2D
Type 2 Diabetes
FM
Fat Mass
TBW
Total Body Water
Hb1Ac
glycated haemoglobin
TSD-
OC
SIO Obesity-Related Disability Test
HBV
Hepatitis B Virus
TSH
ThyroidStimulatingHormone
HCC
Hepato Cellular Carcinoma
UDCA
Ursodesossicolic Acid
HCV
Hepatitis C Virus
VLCD
Very Low-Calorie Diet
HDL
High-Density Lipoprotein
VLCKD
Very Low-Calorie Ketogenic Diet
HRQoL
Health-Related Quality Of Life
8
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
INTRODUCTION AND RATIONALE
Non-Alcoholic Fatty Liver Disease (NAFLD/MAFLD)
Non-alcoholic fatty Liver Disease (NAFLD) is a spectrum of diseases characterised by the
accumulation  of  lipids  in  the  liver  parenchyma.  It  includes  simple  steatosis  but  can
progress to non-alcoholic steatohepatitis (NASH) and advanced fibrosis, cirrhosis, and
hepatocellular carcinoma [2]. Since it is widely associated with insulin resistance and
related metabolic disorders, NAFLD has recently been renamed Metabolic Associated
Fatty Liver Disease (MAFLD) [3]. MAFLD/NAFLD is also an independent risk factor for
type 2 diabetes, cardiovascular diseases, and mortality [4].
MAFLD/NAFLD  is  one  of  the  most  common  chronic  liver  disorders  [5].  The  global
prevalence is estimated to be increasing, currently around 25%, highest in the Middle East
(32%) and South America (31%), followed by Asia (27%), the United States (24%), Europe
(23%), and Africa (14%) [3]. The prevalence in the United States is expected to increase
from 83.1 million in 2015 to 100.9 million in 2030, with increasingly early diagnoses in the
adolescent population (estimated prevalence from 3% to 18%) [6].
The aetiology of MAFLD/NAFLD is predominantly related to dietary habits but also to
endocrine or pharmacological causes that lead to the accumulation of lipids in the liver,
promoting  metabolic  alterations  that  contribute  to  hyperglycemia,  dyslipidemia  and
hyperinsulinemia[7]. These alterations further increase liver damage by regulating adipose
tissue lipolysis, hepatic lipid absorption, lipogenesis, and beta-oxidation [8].
A cohort study reported that the major independent predictors of the severity of fatty liver
are poor adherence to the Mediterranean diet, high body mass index (BMI), and the
homeostasis model assessment of insulin resistance (HOMA-IR) [9].
Therefore, MAFLD/NAFLD considered the hepatic component of the metabolic syndrome,
is  closely  associated  with  insulin  resistance,  obesity,  and  type  2  diabetes  (T2D).
Approximately  70%  -  80%  of  obese  and  diabetic  patients  with  MAFLD/NAFLD  are
positively  correlated  with  a  sedentary  lifestyle,  promoting  hyperglycemia  and
hypertriglyceridemia, which in turn promote T2D and cardiovascular diseases[10, 11].
The  pathogenesis  to  progress  assumes  the  presence  of  lipid  accumulation,  insulin
resistance, and inflammation, but the underlying mechanisms, including those driving
disease progression, still need to be fully understood to date.
9
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
Fatty acids derived from lipogenesis and de novo synthesis circulate in peripheral tissues,
including the liver and adipose tissue, where they accumulate, causing insulin resistance.
Adipose tissue acts as a mediator of systemic lipid storage and secretes hormones and
adipokines. Adiponectin is a specific adipokine that regulates fatty acid oxidation and
inhibits  lipid  accumulation  in  both  adipose  tissue  and  the  liver,  maintaining  glucose
homeostasis, including liver insulin sensitivity[12]. Recent studies have shown that serum
adiponectin levels are lower in patients with MAFLD/NAFLD than in healthy individuals.
Hypoadiponectinemia  compromises  fatty  acid  metabolism,  promoting  a  chronic
inflammatory state in the liver. Therefore, maintaining adiponectin levels may prevent
patients with MAFLD/NAFLD from developing inflammation and fibrosis[13].
The progression of MAFLD/NAFLD to NASH and, therefore, the pathogenesis of the latter
is based on the two-stage hypothesis: The first stage is insulin resistance associated with
high blood lipid levels, leading to fatty liver. The second stage involves oxidative stress,
lipid peroxidation, and mitochondrial dysfunction essential for the progression to non-
alcoholic steatohepatitis (NASH). With the identification of more advanced mechanisms, it
has been demonstrated that NASH develops through a multifactorial process that includes
not only insulin resistance, oxidative stress, and genetic factors but also nutritional factors,
lifestyle, and changes in the intestinal microbiota. Therefore, it becomes important to treat
MAFLD/NAFLD to prevent its progression and the associated metabolic diseases[14–17].
MAFLD/NAFLD is the negative consequence of ectopic hepatic lipid accumulation in the
presence  of  insulin  resistance,  characterised  by  the  deposition  of  triglycerides  in
hepatocytes. A close connection exists between visceral adiposity and a Western diet high
in carbohydrates and fats[18–20]. Visceral fat stimulates the release of pro-inflammatory
cytokines  and  maintains  insulin  resistance.  Diets  rich  in  saturated  fats  and  simple
carbohydrates release fatty acids into the liver, stimulating lipogenesis, oxidative stress,
and lipotoxicity[21].
Ketogenic Diets with Very Low Caloric Intake (VLCKD)
Since the 1960s, low-carbohydrate diets (LCKD) have been utilised for obesity treatment.
While the origins of the ketogenic diet as a medical therapy for managing drug-resistant
epilepsy date back to the early 1900s, the very-low-calorie ketogenic diet (VLCKD) has
emerged as a promising option for achieving significant weight loss in a short period[22]. It
has  been  observed  that  diet-induced  ketosis  positively  influences  metabolic  and
10
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
inflammatory  markers  (HbA1c,  fasting  glucose,  insulin  levels,  blood  lipid  levels,  liver
enzymes, and  advanced  fibrosis) in  type  2  diabetic (T2D) patients, aiding  in  weight
management as well[22–25].
Ketogenesis, a physiological process occurring through hepatic beta-oxidation of fatty
acids, leads to the production of energy in the form of ketone bodies. These water-soluble
molecules are produced when hepatic glucose and glycogen reserves are minimised
(during fasting, pathological states, excessive physical exercise, etc.). The onset of ketosis
stabilises insulin levels, and in recent decades, the ketogenic diet has shown promising
results for related pathological conditions[23].
Stored energy as fat in adipose tissue is released as acetyl-CoA and converted into
intermediate  substrates.  When  insulin  levels  are  low,  stored  fat  in  adipose  tissue
undergoes lipolysis through hormone-sensitive lipase. Once released, free fatty acids
undergo beta-oxidation in hepatic mitochondria to produce acetyl-CoA for ketone body
generation:  acetoacetate,  beta-hydroxybutyrate,  and  -  in  lesser  amounts  -  acetone,
inducing a state of nutritional ketosis. This can lead to a metabolic shift. Reduced insulin
release promotes a metabolic shift towards lipid oxidation, utilising excess fatty acids and
ketones to produce energy, sparing muscle mass, and improving insulin sensitivity[22, 23].
A systematic review and meta-analysis were conducted to assess the efficacy and safety
of VLCKD in overweight and obese patients[26]. The reported studies associated VLCKD
with weight loss when the ketogenic phase lasted at least four weeks, and the lost weight
was maintained  in  the  subsequent follow-up  for up  to  two  years.  No  changes were
observed in LDL cholesterol, HDL cholesterol, serum creatinine, serum uric acid, and
serum potassium, while serum sodium increased. Current nutritional strategies are only
sometimes effective in long-term weight loss and maintenance. Very low-calorie ketogenic
diets  (VLCKD),  although  requiring  support  from  further  studies,  are  emerging  as  a
successful nutritional model for obesity management. The rapid weight loss enhances
patient motivation, and increased satiety promotes dietary compliance[26].
Additionally, VLCKDs preserve lean mass, which is crucial for glucose metabolism and
resting energy expenditure. Bezerra, Bueno et al. examined 13 randomised controlled
trials  (RCTs)  involving  a  total  of  1569  subjects,  comparing  VLCKD  (<50  g
carbohydrates/day) with a low-fat diet (<30% kcal) in overweight and obese adults. The
included RCTs had a minimum follow-up of 12 months. The primary outcome was body
weight, and secondary goals included lipid profile (triglycerides, HDL, LDL), systolic and
11
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
diastolic blood pressure, fasting glucose, insulin, HbA1c, and CRP. Analyses showed that
VLCKD achieved more significant long-term reductions in body weight, triglycerides, and
diastolic pressure, along with more significant increases in HDL and LDL cholesterol
compared to a low-fat diet[27, 28].
It is, therefore, crucial to identify recommendations for the correct use of this therapeutic
approach[29, 30]. Recently, the consensus of the Italian Society of Endocrinology (SIE)
has indicated the use of VLCKD as a dietary treatment for overweight/obese patients with
MAFLD/NAFLD[31].
VLCKD in NAFLD
Currently, all the latest guidelines from the European Society for Clinical Nutrition and
Metabolism (ESPEN)[32, 33], as well as those from the European Association for the
Study of the Liver (EASL), American Association for the Study of Liver Diseases (AASLD),
and  American  Gastroenterology  Association  (AGA)[2,  6,  30,  34],  conclude  that  the
therapeutic  goal  in  patients  with  MAFLD/NASH  is  significant  weight  loss  through  a
hypocaloric diet of 1200-1500 kcal/day. Among the diets with more substantial evidence,
patients with MAFLD/NAFLD should follow the Mediterranean Diet, minimising the intake
of  saturated  fatty  acids  and  red  and  processed  meats  and  eliminating  consuming
commercial products containing fructose[34].
However, how much weight loss should be targeted in NAFLD therapy? Vilar-Gomez E. et
al., conducting a prospective study on nearly 300 patients with histological diagnosis of
NASH, demonstrated that a weight loss of ≥5%, ≥7%, and ≥10% maintained over time (52
weeks) leads to the improvement of hepatic steatosis, resolution of NASH, and regression
of hepatic fibrosis, respectively[24, 35]. However, despite the use of best practices in our
centre, only 28.1% of patients manage to lose at least 5% of their weight, and just over
13% achieve a weight loss of at least 10% of the initial weight at 12 months[36].
Given the significant weight loss in obese patients, VLCKD could also positively impact
MAFLD/NAFLD due to its shallow carbohydrate content. Although it is not known whether
ketosis itself plays an additional role, various mechanisms could be directly connected to
ketosis, leading to a direct improvement in NAFLD[23]. Currently, few studies considering
the  role  of  VLCKD  in  the  context  of  MAFLD/NAFLD  have  ever  evaluated  hepatic
histological  changes  after  this  dietary  approach.  The  available  data  show  improved
surrogate parameters in uncontrolled studies[37].
12
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
In light of such limited evidence regarding the role of VLCKD diet therapy in improving
histological parameters in patients with MAFLD/NAFLD, we aimed to assess the role of the
VLCKD diet in patients with MAFLD/NAFLD complicated by significant hepatic fibrosis (F2-
F3) and NASH compared to a standard hypocaloric dietary approach.
STUDY OBJECTIVES
The purpose of the KETONASH study is to evaluate, in patients with metabolic liver
disease (MAFLD) presenting with non-alcoholic steatohepatitis (NASH) and significant
hepatic  fibrosis  (F1-F2-F3),  the  effect  of  a  very-low-calorie  ketogenic  diet  (VLCKD)
compared  to  that  of  a  standard  low-calorie  diet  (standard  Mediterranean  LCD)  -  in
accordance with the most recent guidelines on MAFLD/NAFLD[2, 6, 30, 34].
The primary objectives of the study are to assess patients with MAFLD/NAFLD/MAFLD
treated with VLCKD compared to standard LCD:
1. Improvement in histological terms of hepatic fibrosis without worsening of NASH.
2. Improvement in NASH without worsening of hepatic fibrosis.
The secondary objectives include the evaluation of changes in histological, biochemical,
and imaging parameters in patients treated with VLCKD compared to standard LCD,
specifically assessing:
1. Individual histological components that comprise the NIH NASH CRN Score and/or FLIP
SAF score (steatosis, ballooning, lobular inflammation, fibrosis, etc.).
2. Liver biochemical tests (transaminases, GGT, alkaline phosphatase, total proteins,
albumin, etc.).
3. Composite liver biochemical biomarkers such as Fibrosis-4 (FIB-4), MAFLD/NAFLD
fibrosis score (NFS), Fatty Liver Index (FLI), and FAST Score.
4. Physical liver fibrosis biomarkers through Transient Elastography (Fibroscan, Echosens,
France).
5. Physical biomarkers of hepatic steatosis evaluated by
    a) qualitative method (mild, moderate, severe) hepatic ultrasound.
    b) quantitative  method  using  Controlled  Attenuation  Parameter (CAP, Echosens,
France).
13
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
6.  Anthropometric  parameters  (BMI,  circumferences),  strength  (handgrip),  and  body
composition (bioimpedance and skinfold thickness).
7. Questionnaires on quality of life (Health-related quality of life, HRQoL), lifestyle (physical
exercise/sedentary habits), and liver disease-related events (NASH-CHECK, CLDQ).
8. Tolerance, occurrence of side effects, and compliance of patients undergoing diet
therapy with VLCKD.
STUDY POPULATION
The study population consists of patients referred to the Coordinating Center (including
satellite  centers)  with  a  current  or  past  diagnosis  of  metabolic  liver  disease
(MAFLD/NAFLD)  formulated  in  accordance  with  the  most  recent  guidelines  on
MAFLD/NAFLD [2, 6, 30, 34]. The inclusion and exclusion criteria align with the most
recent  trials  [38–40]  in  patients  with  MAFLD/NASH  and  international  expert
recommendations [41, 42].
Inclusion criteria:
-  Patients  aged  ≥18  years  with  histological  diagnosis  of  MAFLD/NAFLD/NASH  with
evidence of stage 1, 2, or stage 3 fibrosis (F1/F2/F3) (defined according to the NASH
Clinical Research Network - NASH CRN criteria [1]) obtained no more than 6 months
before enrollment.
- Stable weight for over six months with a BMI between 30-40 kg/m2.
- Patients in whom it is safe and functional to proceed with liver biopsy and who consent to
undergo liver biopsy after 12 months of enrollment to assess the effect of diet therapy.
- Informed consent obtained.
Exclusion criteria:
- BMI <30 or BMI >40.
- Presence of evolved chronic liver disease into cirrhosis (histological F4 or elastometric
LSM >14 kPa).
- Type 1 diabetes mellitus.
14
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
- Model for End-stage Liver Disease (MELD) score >12, AST or ALT ≥5× ULN, HbA1c
>9.5%, INR≥1.4, creatinine >1.5 mg/dl, platelets <100,000/mm3, and total bilirubin >1.5
mg/dl.
- Concomitance of any other known chronic liver disease beyond MAFLD/NAFLD, such as
alcoholic liver disease, viral (HCV/HBV), cholestatic-autoimmune (PBC/PSC/AIH), Wilson's
disease, hemochromatosis, alpha-1-antitrypsin deficiency, drug-induced liver injury (DILI),
or the presence or suspicion of hepatocellular carcinoma (HCC).
- Average alcohol consumption exceeding 4/2 units/day (males/females) in the 6 months
preceding and a history of excessive alcohol consumption in the last 5 years.
- Previous or planned liver transplant, bariatric surgery, ileal resection, or biliary diversion.
- History of acute cholecystitis and biliary obstructions (cholangitis).
- Recent (within the last 12 months) or concurrent use of agents known to cause hepatic
steatosis  (long-term  systemic  corticosteroids  [>10  days],  amiodarone,  methotrexate,
tamoxifen, tetracyclines, high-dose estrogens, valproic acid).
- Recent (within the last 3 months) change in dose/regimen or introduction of Vitamin E (at
doses  ≥400  IU/day),  ursodeoxycholic  acid  (UDCA),  betaine,  s-adenosyl  methionine,
silymarin, or pentoxifylline.
- Presence of psychiatric disorders and/or a diagnosis of any eating disorder.
- Life expectancy <6 months.
Withdrawal criteria from the study:
Subjects previously enrolled can withdraw at any time upon their request or at the request
of their legal representative. The principal investigator may withdraw a subject from the
study at any time for the following reasons:
- Serious adverse events related to the study intervention.
- Presence or appearance of exclusion criteria.
- Development of pathological conditions that make it impossible to continue the study.
- Non-compliance of the participant with study visits and/or procedures.
- A serious protocol violation determined by the PI or investigators.
STUDY DESIGN
The KETONASH study is a multicenter, open-label, randomised, controlled clinical trial that
will be consecutively proposed to all patients with histological diagnosis of non-alcoholic
15
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
steatohepatitis (NASH) and hepatic fibrosis (F1-F2-F3) in the context of chronic metabolic
liver disease (MAFLD/NAFLD).
Once the inclusion criteria are confirmed and the exclusion criteria are ruled out, patients
will be subsequently randomly assigned (randomisation) with a 2:1 ratio to one of the two
study arms:
-
VLCKD Study Arm → will receive experimental diet therapy with very low-calorie
ketogenic meals (VLCKD) consisting of 5 successive phases (600 – 1500 kcal/day).
-
LCD Control Arm → will receive standard low-calorie diet therapy, a Mediterranean-
type  diet  in  accordance  with  the  most  recent  guidelines  [2,  6,  30,  34]  on
MAFLD/NAFLD (1200-1500 kcal/day).
The KETONASH study consists of an initial 4-month diet intervention phase (Visits 1-8),
followed by a second 8-month weight maintenance phase (Visits 9-15). This structure,
particularly in the intervention arm, has been validated in previously published studies [43–
46].  In  both  study  arms,  the  intervention  will  be  conducted  through  a  standardised
multidisciplinary approach (Physician/Dietitian/Nurse/Psychologist) aimed at weight loss
through changes in dietary regimen, exercise program, and emotional support techniques.
The two study arms differ in nutritional composition, types of foods, and caloric intake.
IDENTIFICATION, SCREENING, AND RANDOMIZATION (Visit 1)
Potential participants will be identified during routine outpatient visits or scheduled hospital
appointments at the study centre. Eligible participants will be invited to join the study by
their specialist or a member of the clinical and/or research team, who will also explain the
study. A specially created information sheet detailing the study will be provided, and the
patient will have sufficient time to review it and make their considerations. If new scientific
evidence  that  could  affect  a  subject's decision  to  continue  participating  in  the  study
becomes available, it will be discussed with the enrolled patient.
16
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
A screening register will be established to retain details of patients invited to participate in
the study. This is in accordance with CONSORT recommendations to ensure that available
data are not influenced by unexpected biases from those who refuse to participate in the
study. For subjects who decline participation, the register will keep track of each reason
and also ensure that potential participants are involved only once. The screening register
will store pseudonymised data containing the presence/absence of inclusion/exclusion
criteria for the study.
After assessing the patient's eligibility for the study, using a dedicated application on the
REDCap UNIBO platform and stratifying the patient by the presence of type 2 diabetes
and gender, the patient will be randomised into one of the two study arms. The following
assessments will be collected and recorded on the study's electronic case report form
(eCRF):
-
Age
-
Average alcohol intake (units/week) in the last six months
-
Alcohol intake – history of abuse?
-
Smoking status – Yes/No/Ex
-
Tea/Coffee consumption – cups/day
-
Co-morbidities:
-
Hypertension, dyslipidemia, type 2 diabetes
-
Obstructive sleep apnea
-
Malignant neoplasms
-
Cardiovascular diseases/Stroke
17
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
-
Other relevant diseases
-
Recent/ongoing  therapy  (including  over-the-counter  medications,  herbs,
homoeopathic remedies)
-
Results  of  recent  haematological/biochemical/immunological/virological  analyses
such as:
-
Haematological (complete blood count, coagulation)
-
Clinical biochemistry (urea and electrolytes, liver function [Alb, Bili, ALP, ALT, AST,
gGT], Ferritin/transferrin saturation, A1AT, copper/ceruloplasmin, glucose, HbA1C,
Insulin, C-peptide, TSH, lipid profile, etc.)
-
Autoimmunity screening, Immunoglobulins (IgG, IgA, IgM)
-
Viral serology (HBV, HCV)
-
Liver biopsy outcomes reported within six months
-
Electrocardiogram (ECG)
-
Report of abdominal ultrasound evaluation
DIETARY INTERVENTION (Visits 2-8)
VLCKD Study Arm:
The  VLCKD  dietary  intervention  consists  of  five  successive  phases  involving  the
consumption  of  meal  replacements  and  nutritional  supplementation  (vitamins,  trace
elements, and omega-3 fatty acids), organised as follows:
Ketogenic Low-Calorie Period (2 months):
-
Phase 1 (30 days – Visits 1-3): Ketogenic diet with low-fat content, 600 kcal/day
(with low glycemic index products and vegetables).
-
Phase 2 (15 days – Visit 5): Ketogenic diet with low-fat content, 660 kcal/day (with
four products as a high-protein food source [meat or fish] and vegetables with a low
glycemic index).
-
Phase 3 (15 days – Visit 5): Ketogenic diet with low-fat content, 730 kcal/day (with
two products as high-protein food sources [meat or fish] and vegetables with a low
glycemic index).
Low-Calorie Period (2 months):
18
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
-
Phase 4 (30 days – Visits 6-7): Hypocaloric diet with the reintroduction of different
foods, 1,050 kcal/day (with products).
-
Phase 5 (30 days – Visits 7-8): Hypocaloric diet with the reintroduction of different
foods, 1,400 kcal/day (with products).
During these phases, the patient will receive nutritional supplementation with vitamins,
trace elements, and omega-3 fatty acids with a DHA: EPA ratio of 700:500 during phases
1-3 and 500:100 during phases 4 and 5. Throughout the very low-calorie ketogenic period,
the patient will have three interim dietetic consultations (Visits 2-4) and a medical visit at
the end of the ketosis period (Visit 5). During the low-calorie period, the patient will receive
alternating two dietetic consultations (Visits 6 and 7) and one medical visit every 30 days
(Visit 8).
Control LCD Study Arm:
The control LCD arm consists of a diet with natural low-calorie foods (1200-1500 kcal/day
or a reduction of 500-1000 kcal/day compared to baseline) and a low glycemic index
based  on  the  "Mediterranean  Diet"  model,  following  the  most  recent  guidelines  on
MAFLD/NAFLD [2, 6, 30, 34]. Similar to the VLCKD arm, for the entire duration of the
dietetic treatment, the patient will alternately receive dietetic consultations and medical
visits.
19
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
Maintenance Phase (Visits 9-13):
At the end of the dietary intervention, patients from both study arms will continue with a
controlled, low glycemic index diet tailored to the patient's basal metabolic rate (BMR)
(estimated with bioimpedance assessment) for an additional six months. According to the
current clinical practice of the study centre, all patients will receive alternating dietetic
consultations and/or medical visits every 30 days (Visits 9-15). Depending on patient
availability,  some  visits/consultations  during  the  maintenance  phase  will  occur  via
teleconsultation.
End of Study (Visit 15):
At the end of the 12-month study, the patient will undergo an evaluation of the primary and
secondary study objectives. Following the end of the study, the patient, based on the
achieved BMI and existing comorbidities, will resume the diagnostic and care pathway
planned by the Investigator Center in accordance with current clinical practice.
Physical Activity:
Throughout the study, regardless of the patient's randomisation arm, the physician will
prescribe  structured  and  standardised  physical  exercises  specifically  tailored  to  the
patient's  abilities  and  physical  conditions,  progressively  increasing  in  frequency  and
intensity, following the latest evidence on physical activity in patients with MAFLD/NAFLD
[34, 47]. During the dietary intervention phases, the prescribed physical activity will include
muscle  toning  exercises (resistance);  conversely, during  the  maintenance  period, the
patient will alternate muscle toning exercises with cardiovascular training.
Psychological-Motivational Support:
Throughout the study, regardless of the patient's randomisation arm, the multidisciplinary
team,  including  a  clinical  psychologist  and  a  pedagogue  with  specific  motivational
coaching skills, will support the study participants. They will utilise techniques currently
used at the centre for psycho-emotional support and provide the patient with a set of easily
applicable tools to help make lasting lifestyle changes, following the experience of the
Investigator Center [48] and the latest evidence in MAFLD/NAFLD patients [47].
STUDY PROCEDURES
20
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
Medical Visits and Dietetic Consultations:
Throughout  the  study,  there  will  be  a  total  of  15  alternating  visits  between  medical
assessments and dietetic consultations. These evaluations will occur at variable intervals
during different study phases and can be conducted in person or via teleconsultation
(phone/online).
During the in-person visits, an anthropometric assessment will be conducted, and in
particular, the following variables will be evaluated:
Anthropometric Assessment:
-
Height (cm)
-
Current weight (kg)
-
BMI (m/kg²)
-
Waist circumference (cm)
-
Hip circumference (cm)
-
Neck circumference (cm)
-
Arm circumference (MAC)
-
Hand grip test
-
Skinfold thickness measurements (triceps, suprailiac, abdominal, anterior thigh)
-
Blood pressure
-
Heart rate
These assessments will follow the National Health and Nutrition Examination Survey
(NHANES) 2017-2018 guidelines.
The visits V1, V5, V8, V12, and V15 will be of medical relevance; consequently, the others
will be of a dietary nature.
During these visits, patients will complete translated transcultural versions of the following
validated questionnaires:
-
Chronic
 
Liver
 
Disease
 
Questionnaire
 
for
 
MAFLD/NAFLD/NASH,
CLDQ-MAFLD/NAFLD-NASH (HRQQL)
-
Food Frequency Questionnaire (FFQ)
-
Mediterranean Diet Score (MDS)
-
International Physical Activity Questionnaire (IPAQ)
21
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
-
SIO-Obesity correlated Disability Test (TSD-OC)
-
STOP-Bang questionnaire
-
Binge Eating Scale (BES)
-
Beck Depression Inventory (BDI)
-
State-Trait Anxiety Inventory Forma Y (STAY-Y)
Body Composition Assessment via Bioimpedance (BIA):
At visits V1, V5, V8, and V15, body composition will be evaluated using Bioelectrical
Impedance Vector Analysis (BIVA) following European Society for Clinical Nutrition and
Metabolism (ESPEN) guidelines. Bioelectrical impedance analysis (BIA) is one of the most
useful  techniques  for  assessing  body  composition  because  of  its  simplicity,  quick
execution, non-invasiveness, and cost-effectiveness, and requires minimal participation
from the subject. Vectorial BIA (BIVA) is based on the principle that biological tissues
behave  as  conductors,  semiconductors,  or  dielectrics.  It  involves  administering  an
alternating current at a fixed frequency of 50Hz through the skin, using two surface
electrodes as injectors. A second pair of electrodes (sensors) are tasked with recording the
body's  resistance  to  the  applied  current.  For  the  KETONASH  study,  a  BIA  101
bioimpedance  analyser  (Akern  s.r.l.,  Florence,  Italy)  will  be  used.  By  measuring
Resistance (Rz, representing the ability of all biological structures to resist the passage of
electrical current) and Reactance (Xc, also known as capacitive resistance - the force
opposing the passage of an electric current due to capacitance, i.e., a capacitor), the
following estimated parameters will be obtained:
-
Total Body Water (TBW)
-
Extracellular Water (ECW)
-
Intracellular Water (ICW)
-
Body Cell Mass (BCM)
-
Fat-Free Mass (FFM)
-
Fat Mass (FM)
-
Muscle Mass (MM)
-
Basal Metabolism related to cell mass
Liver Biopsy:
22
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
The transcutaneous liver biopsy with a needle (using, for example, a Trucut or Menghini
needle) will be performed as standard clinical practice and under ultrasound guidance [62,
63] for the diagnostic purposes of non-alcoholic steatohepatitis (NASH) and repeated
solely for study purposes to assess the effectiveness of the intervention after 12 months
from randomisation. For an accurate histological evaluation/diagnosis, it will be necessary
to obtain tissue of a minimum length of 1-1.5 cm. If the total length of the biopsy is less
than indicated, the operator may decide to take a second sample to ensure there is
sufficient material for diagnostic purposes. Given the nature of the procedure, commonly, a
tissue longer than necessary for diagnostic purposes is collected at the time of the biopsy.
The excess tissue beyond the length necessary for diagnostic purposes of 1-1.5 cm will be
considered surplus for diagnostic requirements.
Before performing the liver biopsy, the patient will be explained the indications for the
biopsy, and it will be emphasised that the risks to the patient do not outweigh the potential
benefits.  The  procedure  will  be  performed  after  checking  the  platelet  count  and
coagulation indices according to the standards and procedures of the IRCCS Azienda
Ospedaliera Universitaria Policlinico di Sant'Orsola, Bologna.
Samples that meet diagnostic requirements can be collected at the time of the procedure
for research purposes. The samples will be processed for histological evaluation and/or
frozen in liquid nitrogen (or fixed in a suitable preservative) for subsequent analysis and
stored  at  -80°C.  After  histological  analysis,  the  excess  tissue,  fixed  in  formalin  and
mounted in wax blocks, is kept indefinitely in the archive.
Histological  evaluations  will  be  centralised  and  carried  out  by  a  single  pathologist
experienced in liver diseases, following the NASH CRN criteria[1] and blinded to the
patient's clinical, laboratory, and imaging characteristics, as well as randomisation for
study purposes.
Abdominal ultrasound with Transient Elastography (FibroScan®) and Controlled
Attenuation Parameter (CAP™)
In  accordance  with  clinical  practice  and  the  European  Federation  of  Societies  for
Ultrasound in Medicine and Biology (EFSUMB) guidelines [65, 66], the study-specific
abdominal ultrasound will be performed concurrently during visits V1, V8, and V15 under
fasting conditions of at least 12 hours. Specifically, the following variables will be assessed
and recorded:
23
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
-
Hepatomegaly (yes, no)
-
Echostructure (homogeneous, bright, granular, nodular)
-
Surface (regular, irregular, nodular)
-
Hepatic steatosis (absent, mild/Grade I, moderate/Grade II, severe/Grade III)
-
Gallbladder (alithiasic, lithiasis, ectopic)
-
Focal lesions (type)
-
Vascular diameters (PV, SV, UMV)
-
Portal flow (hepatopetal/hepatofugal)
-
Portal vein velocity
-
Suprahepatic vein flow (triphasic, biphasic, monophasic, reversed)
-
Splenic diameter/area
-
Presence of collateral circulation
-
Free abdominal fluid (mild, moderate, severe)
Simultaneously, measurement of liver elasticity will be performed using the Transient
Elastography method with FibroScan® (Echosens, Paris, France), and quantification of
hepatic fat using Controlled Attenuation Parameter (CAP™).
The  physical  principle,  known  as  the  "Young's  Modulus,"  on  which  these  indirect
techniques  for  quantifying  liver  fibrosis  and  steatosis  are  based,  is  the  ability  of  a
mechanical  wave  to  propagate  at  different  speeds  through  a  propagation  medium,
depending on the elasticity of the medium itself.
The probe of the ultrasound device used for elastography is placed on the skin at the
anatomical  location  of  the  liver.  It  consists  of  a  small  transducer  that  produces  a
mechanical wave of constant frequency, amplitude, and shape, emitting a minimal amount
of energy. The propagation speed of the wave in the medium, which in this case is the liver
parenchyma, is studied by emitting ultrasound from the probe itself. The acoustic energy
required to follow the propagation of the mechanical wave is also very low and not harmful.
Data related to the propagation of the wave in the liver parenchyma are analysed by the
software  included  in  the  device  through  sophisticated  algorithms,  leading  to  the
determination  of  the  liver  elasticity  value.  The  execution  procedures  of  elastometric
methods using FibroScan® will be in accordance with the operators' experience [67, 68],
the manufacturer's recommendations, and the EFSUMB European guidelines [65, 66].
24
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
Laboratory Tests and Study-Specific Biomarkers
During visits V1, V5, V8, and V15, the patient will undergo a blood draw and chemical-
physical analysis of urine, where the following tests will be assessed:
25
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
Concomitantly, the following biomarkers based on laboratory, elastometric, and clinical
data of the patient will be evaluated for the risk stratification of liver fibrosis (FIB4 & NFS),
liver steatosis (FLI), and NASH with significant fibrosis (FAST SCORE - Fibrotic-NASH)
[64, 69]. The formulas for the mentioned scores are summarised in the table.
26
KETONASH;
Versione 4.0 23.11.2023
Procedure
VLCKD
LCD
Maintenance
Standard – LCD (Mediterranean Diet) 
Phase
B
F2
EOT
EOS
Week
0
4-6
16-18
40-44
Visit
V1
V4
V8
V15
BLOOD COUNT
x
x
x
x
PROTHROMBIN TIME (PT)
x
x
x
CREATININE
x
x
x
x
SODIUM
x
x
x
x
POTASSIUM
x
x
x
x
TOTAL CALCIUM
x
x
x
x
CHLORIDE
x
x
x
x
TOTAL MAGNESIUM
x
x
x
x
PHOSPHORUS
x
x
x
x
TOTAL AND FRACTIONATED BILIRUBIN
x
x
x
x
TOTAL PROTEINS 
x
x
x
ALBUMIN
x
x
x
x
ALANINE AMINOTRANSFERASE (ALT) (GPT)
x
x
x
x
ASPARTATE AMINOTRANSFERASE  (AST) (GOT)
x
x
x
x
GAMMA GLUTAMYL TRANSPEPTIDASE (gamma GT) 
x
x
x
x
ALKALINE PHOSPHATASE
x
x
x
GLUCOSE
x
x
x
x
GLYCATED HEMOGLOBIN (Hb)
x
x
x
INSULIN
x
x
x
x
URATE
x
x
x
UREA
x
x
x
x
 HDL CHOLESTEROL
x
x
x
 LDL CHOLESTEROL
x
x
x
TOTAL CHOLESTEROL 
x
x
x
TRIGLYCERIDES
x
x
x
x
 C REACTIVE PROTEIN
x
x
x
URINE CHEMICAL, PHYSICAL AND MICROSCOPIC EXAMINATION
x
x
x
x

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
The table below summarises the various study procedures in different phases of the
protocol.
MEASUREMENT OF STUDY OBJECTIVES
The measurement of primary objectives will occur at the end of the study (EOS - Visit 15)
12 months after enrollment and will be based on the evaluation of liver biopsy using NASH
CRN criteria[1]. Specifically, the following will be assessed:
-
Reduction  ≥1  stage  of  hepatic  fibrosis  without  an  increase  in  hepatocellular
ballooning, lobular inflammation, or steatosis compared to baseline biopsy.
-
Improvement  in  NASH  through  histopathological  assessment  defined  as  a)
"absence of fatty liver," or b) "simple or isolated hepatic steatosis in the absence of
steatohepatitis" and NAS 0 for ballooning and 0-1 for inflammation without an
increase in fibrosis stage compared to baseline biopsy.
The measurement of secondary objectives will take place at the end of treatment (EOT -
Visit 8) and at the end of the study (EOS - Visit 15) 12 months after enrollment, through the
comparison of biochemical, biomarker, and imaging parameters compared to baseline
evaluation.
Expected Results
27
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
At the end of the study, it is expected that the group of patients who underwent VLCKD
intervention will  show  an improvement of at least one  stage of hepatic fibrosis with
improvement in NASH compared to the group of patients treated with LCD. Additionally,
alongside  weight  reduction  and  improvement  in  anthropometric  measurements,  a
simultaneous improvement in other histological, biochemical, and biomarker parameters
and  an  improvement  in  fibrosis  and  steatosis  grade  through  non-invasive  imaging
evaluation (TE, CAP, RM-PDFF) is expected, especially in the VLCKD study arm. Patients
treated with VLCKD will demonstrate a better quality of life at the end of the study
compared to the control group. Finally, greater tolerance and compliance are expected in
the VLCKD study arm.
Study Duration
Scheduled start date of the study (enrollment of the first patient): 01/05/2021
Protocol-specified last date for the end of enrollment: 31/12/2024
Data access will, however, be possible for ten years from the conclusion of the study.
STATISTICAL ANALYSIS PLAN
Sample Size Determination 
The study is designed to demonstrate that the effect of VLCKD is superior to LCD after 12
months from randomisation. It has been calculated that, to have a 90% probability of
finding, with a significance level of 0.05%, a difference between nutritional treatments of 1
stage of fibrosis (assessed by liver biopsy) with a statistical power of 80% and an effect
size (Cohen's d) equal to 80%, a total of 42 patients need to be randomised, with a 2:1
ratio in the two study arms (VLCKD vs LCD).
Statistical Analysis
Parametric variables will be compared using the Student's T-test, while non-parametric
categorical variables will be analysed using Fisher's test, Chi-square; continuous non-
parametric variables will be analysed using the Mann-Whitney test. Continuous variables
will be reported as median ± interquartile range (IQR), while categorical variables as
numbers and percentages; predictors of events occurring in a specific time interval will be
tested  using  logistic  regression  or  Cox  methods.  Subsequently,  to  confirm  their
independent predictive value, variables with p < 0.1 will be studied in a multivariate logistic
28
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
or Cox model; comparison groups will be studied using Student's t-test and the Chi-square
test. Associations will be considered statistically significant if they present a p-value < 0.05.
The incidence of events will be calculated using the Kaplan-Meier method and compared
through the log-rank test. The aforementioned analyses will be performed using STATA15
or another statistical analysis software.
Interim Analysis
There is no planned interim analysis of primary objectives.
DATA COLLECTION
Electronic Data Capture Form (eCRF)
Structured  data  collection  necessary  for  evaluating  objectives  will  occur  through  an
electronic data capture form. Designated personnel will collect information obtained from
clinical records, outpatient visits, and instrumental diagnostics within the normal care
pathway. The collected data will be recorded with the help of the REDCap UNIBO platform.
Data processing will be carried out through procedures for collection, recording, storage,
and processing using electronic tools suitable for processing them according to the law
and  in  compliance  with  security  and  confidentiality  standards,  respecting  applicable
regulations.
Data Management and Data Quality
The study sponsor and the experimentation centre are autonomous data controllers, each
for their competence. Data will be collected through the REDCap UNIBO platform. Access
to the REDCap platform occurs via the https protocol, ensuring a secure connection
protected by SSL algorithms and a certificate guaranteeing encrypted data transmission.
Access to the archive on the REDCap platform will be granted only to registered users
(researchers  belonging  to  the  KETONASH  study  research  group)  who  will  access it
through their credentials (self-chosen username and password): each researcher user will
be  able  to  view,  and  modify,  and  update  the  data  of  enrolled  patients  from  the
experimenting centre.
Server-level  management  of  the  REDCap  platform,  such  as  installation,  periodic
monitoring of hardware resources, resolution of any blocking events, and issues related to
direct server access, is guaranteed by CESIA - AREA SISTEMI E SERVIZI INFORMATICI,
29
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
University of Bologna. Access to the hardware and software resources of the REDCap
platform is regulated by the Personal Data Protection Authority regulations.
Regarding  administrative  operations  at  the  application  level,  such  as  creating  and
configuring the study and creating users authorised to participate in the study, these are
the responsibilities of the sponsor.
To protect the identity of individuals involved in the study, the REDCap platform generates
a unique identification code for each subject associated with their data collection forms,
allowing  each  centre  to  maintain  the  association  with  the  patient's  personal  data.
Additionally,  the  REDCap  platform  requires  an  external  list  outside  the  application
containing the association of these codes with the nominative data of patients, kept
exclusively at the experimentation centre as a confidential document, carefully preserved,
and concerning only their patients.
The patient data required for the conduct of this research study, while not directly or
indirectly  identifying  the  patient,  are  recorded  within  the  REDCap  database  with
asymmetric SSL double-key encryption combined with AES 256 encryption.
As outlined in the Personal Data Protection Authority regulations "Code on the protection
of personal data," the REDCap platform provider guarantees the execution of data saving
with a weekly frequency on archive files protected by passwords.
The main experimenter and their collaborators, who will be bound by an obligation of
confidentiality for this data, will have access to the data in pseudonymised form in Excel or
another data processing format (exported from the REDCap platform after the end of
active enrollment). However, access to pseudonymised data will be possible for ten years
beyond the final enrollment deadline for patients.
The  data  resulting  from  the  analysis  will  be  processed  with  statistical  methods  in
pseudonymised form to obtain information constituting the purpose of the study. The study
results may be material for scientific publication, where the data will be presented in an
equally anonymous form.
STUDY COSTS
The KETONASH study is a non-profit study aimed at improving clinical practice according
to Article 1 of the Ministerial Decree of 17.12.2004. The ketogenic dietary regimen with
substitute foods will be provided at no additional cost by companies producing commercial
VLCKD products.
30
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
REGULATORY AND ETHICAL ASPECTS
Statements of Compliance 
The conduct of this study will be in accordance with the recommendations for physicians
involved in research on human subjects adopted by the 18th World Medical Assembly in
Helsinki in 1964 and its subsequent revisions. The process of obtaining informed consent
at the recruiting centres involved is conducted according to the Declaration of the World
Medical Association of Helsinki 2000, respecting the Charter of Fundamental Rights of the
European Union (2000/C 364/01) and the Belmont Report. Before commencing the study,
favourable  ethical  opinions  will  be  obtained  from  an  appropriate  Ethics  Committee.
Information sheets and written informed consent will be provided to all eligible subjects.
Ethics Committee
The  study  protocol,  any  protocol  amendments,  informed  consent,  and  any  other
information for patients must be approved by the relevant Ethics Committee. Any protocol
amendments can be immediately implemented by the Principal Investigator, in agreement
with the Study Scientific Committee, without waiting for approval if patient safety is at
stake. If, for safety reasons, an immediate protocol modification is deemed necessary by
the Principal Investigator in agreement with the Study Scientific Committee, the Ethics
Committee must be informed within ten working days.
Informed Consent Management
For enrolled patients, Informed Consent and adherence to the study will be obtained
during  a  visit  as  part  of  the  normal  care  pathway  at  the  centre.  Informed  consent
discussions will be conducted by appropriate personnel involved in the study (as indicated
in the delegation log) at the reference centre, including medical and research nursing staff,
with the opportunity for participants to ask any questions. Once provided with information
about the study, candidates will be given adequate time to decide on their participation.
Those wishing to participate will provide a signed and dated written informed consent,
which will be counter-signed and dated by a research team member with documented
responsibility for this task. Written informed consent will always be required before any
study-related  procedures.  The  right  to  refuse  participation  without  providing  any
explanation will be respected. At the time of consent or any subsequent time, each
individual may decide to discontinue their participation without any consequences. Non-
31
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
participation in this study will not prejudice the patient's therapeutic course. The original
signed copy of the informed consent will be kept in the patient's medical record, a copy will
be kept in the investigator's study file, and a copy will be provided to the patient.
The data resulting from the analysis will be processed using statistical methods in an
anonymous form to extract the information constituting the purpose of the study. The
Principal Investigator and their collaborators, bound by confidentiality obligations, will have
access to the data. Access to the data will be possible for ten years after the completion of
the study. The study results may be used for scientific publication, where the data will be
presented in an equally anonymous form.
Confidentiality and Data Protection
The operations of collecting, recording, storing, and modifying personal data will take place
through manual and computerised tools with logic strictly related to the above purposes.
The data will be processed to ensure the confidentiality and security of information, per
Articles 25 and 32 of the GDPR, only by the Principal Investigator or their collaborators.
Insurance
A liability insurance policy for trial-related risks and the ethics committee will be taken out,
with a limit of €1,500,000 per event for the entire duration of the study.
Data Use and Results Publication
Information about the study and study data will be made available through publication on
clinicaltrials.gov.
REFERENCES
1. 
Kleiner DE, Brunt EM, Van Natta M, et al (2005) Design and validation of a histological scoring system for non-
alcoholic fatty liver disease. Hepatology 41:1313–1321
2. 
(2016) EASL–EASD–EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
J Hepatol 64:1388–1402
3. 
Eslam M, Newsome PN, Sarin SK, et al (2020) A new definition for metabolic dysfunction-associated fatty liver
disease: An international expert consensus statement. J Hepatol 73:202–209
4. 
Adams LA, Anstee QM, Tilg H, Targher G (2017) Non-Alcoholic fatty liver disease and its relationship with
cardiovascular disease and other extrahepatic diseases. Gut 66:1138–1153
32
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
5. 
Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, George J, Bugianesi E (2018) Global burden of
NAFLD and NASH: Trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 15:11–20
6. 
Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, Harrison SA, Brunt EM, Sanyal AJ (2018)
The diagnosis and management of non-alcoholic fatty liver disease: Practice guidance from the American
Association for the Study of Liver Diseases. Hepatology 67:328–357
7. 
Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ (2018) Mechanisms of NAFLD development and
therapeutic strategies. Nat Med 24:908–922
8. 
Geisler  CE,  Renquist  BJ  (2017)  Hepatic  lipid  accumulation:  cause  and  consequence  of  dysregulated
glucoregulatory hormones. J Endocrinol 234:R1–R21
9. 
Trovato FM, Martines GF, Brischetto D, Trovato G, Catalano D (2016) Neglected features of lifestyle: Their
relevance in non-alcoholic fatty liver disease. World J Hepatol 8:1459
10. 
Loomba R, Abraham M, Unalp A, Wilson L, Lavine J, Doo E, Bass NM (2012) Association between diabetes,
family history of diabetes, and risk of non-alcoholic steatohepatitis and fibrosis. Hepatology 56:943–951
11. 
Williams CD, Stengel J, Asike MI, Torres DM, Shaw J, Contreras M, Landt CL, Harrison SA (2011) Prevalence of
Non-alcoholic Fatty Liver Disease and Non-alcoholic Steatohepatitis Among a Largely Middle-Aged Population
Utilising Ultrasound and Liver Biopsy: A Prospective Study. Gastroenterology 140:124–131
12. 
Miller RA, Chu Q, Le Lay J, Scherer PE, Ahima RS, Kaestner KH, Foretz M, Viollet B, Birnbaum MJ (2011)
Adiponectin suppresses gluconeogenic gene expression in mouse hepatocytes independent of LKB1-AMPK
signaling. J Clin Invest 121:2518–2528
13. 
Kitade H, Chen G, Ni Y, Ota T (2017) Non-alcoholic Fatty Liver Disease and Insulin Resistance: New Insights
and Potential New Treatments. Nutrients 9:387
14. 
Abenavoli L, Milic N, Di Renzo L, Preveden T, Medić-Stojanoska M, De Lorenzo A (2016) Metabolic aspects of
adult patients with non-alcoholic fatty liver disease. World J Gastroenterol 22:7006
15. 
Almeda-Valdes P, Aguilar-Olivos N, Uribe M, Mendez-Sanchez N (2015) Common Features of the Metabolic
Syndrome and Non-alcoholic Fatty Liver Disease. Rev Recent Clin Trials 9:148–158
16. 
Buzzetti E, Pinzani M, Tsochatzis EA (2016) The multiple-hit pathogenesis of non-alcoholic fatty liver disease
(NAFLD). Metabolism 65:1038–1048
17. 
Rinella ME (2015) Non-alcoholic fatty liver disease a systematic review. JAMA - J Am Med Assoc 313:2263–2273
18. 
Machado M, Cortez-Pinto H (2016) Diet, Microbiota, Obesity, and NAFLD: A Dangerous Quartet. Int J Mol Sci
17:481
19. 
Asrih M, Jornayvaz FR (2014) Diets and non-alcoholic fatty liver disease: The good and the bad. Clin Nutr
33:186–190
20. 
Kosinski C, Jornayvaz F (2017) Effects of Ketogenic Diets on Cardiovascular Risk Factors: Evidence from
Animal and Human Studies. Nutrients 9:517
21. 
Yki-Järvinen H (2015) Nutritional Modulation of Non-Alcoholic Fatty Liver Disease and Insulin Resistance.
Nutrients 7:9127–9138
22. 
Paoli A, Rubini A, Volek JS, Grimaldi KA (2013) Beyond weight loss: a review of the therapeutic uses of very-low-
carbohydrate (ketogenic) diets. Eur J Clin Nutr 67:789–796
23. 
Gershuni VM, Yan SL, Medici V (2018) Nutritional Ketosis for Weight Management and Reversal of Metabolic
Syndrome. Curr Nutr Rep 7:97–106
24. 
Vilar-Gomez E, Athinarayanan SJ, Adams RN, et al (2019) Post hoc analyses of surrogate markers of non-
alcoholic fatty liver disease (NAFLD) and liver fibrosis in patients with type 2 diabetes in a digitally supported
continuous care intervention: an open-label, non-randomised controlled study. BMJ Open 9:e023597
33
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
25. 
Li R, Liu Y, Liu H, Li J (2020) Ketogenic diets and protective mechanisms in epilepsy, metabolic disorders,
cancer, neuronal loss, and muscle and nerve degeneration. J Food Biochem. doi: 10.1111/jfbc.13140
26. 
Castellana M, Conte E, Cignarelli A, Perrini S, Giustina A, Giovanella L, Giorgino F, Trimboli P (2019) Efficacy
and safety of very low calorie ketogenic diet (VLCKD) in patients with overweight and obesity: A systematic
review and meta-analysis. Rev Endocr Metab Disord. doi: 10.1007/s11154-019-09514-y
27. 
Bueno NB, de Melo ISV, de Oliveira SL, da Rocha Ataide T (2013) Very-low-carbohydrate ketogenic diet v. low-
fat diet for long-term weight loss: a meta-analysis of randomised controlled trials. Br J Nutr 110:1178–1187
28. 
Weiner RA (2010) Surgical Treatment of Non-Alcoholic Steatohepatitis and Non-Alcoholic Fatty Liver Disease.
Dig Dis 28:274–279
29. 
Muscogiuri G, Barrea L, Laudisio D, Pugliese G, Salzano C, Savastano S, Colao A (2019) The management of
very low-calorie ketogenic diet in obesity outpatient clinic: a practical guide. J Transl Med 17:356
30. 
Merli M, Berzigotti A, Zelber-Sagi S, Dasarathy S, Montagnese S, Genton L, Plauth M, Parés A (2019) EASL
Clinical Practice Guidelines on nutrition in chronic liver disease. J Hepatol 70:172–193
31. 
Caprio M, Infante M, Moriconi E, et al (2019) Very-low-calorie ketogenic diet (VLCKD) in the management of
metabolic diseases: systematic review and consensus statement from the Italian Society of Endocrinology (SIE).
J Endocrinol Invest 42:1365–1386
32. 
Bischoff SC, Bernal W, Dasarathy S, Merli M, Plank LD, Schütz T, Plauth M (2020) ESPEN practical guideline:
Clinical nutrition in liver disease. Clin Nutr 39:3533–3562
33. 
Plauth M, Bernal W, Dasarathy S, Merli M, Plank LD, Schütz T, Bischoff SC (2019) ESPEN guideline on clinical
nutrition in liver disease. Clin Nutr 38:485–521
34. 
Sharma HB, Panigrahi S, Sarmah AK, Dubey BK (2020) AGA Clinical Practice Update on Lifestyle Modification
Using Diet and Exercise to Achieve Weight Loss in the Management of Non-alcoholic Fatty Liver Disease
(NAFLD): Expert Review. Gastroenterology 135907
35. 
Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, Torres-Gonzalez A, Gra-Oramas B, Gonzalez-Fabian L,
Friedman SL, Diago M, Romero-Gomez M (2015) Weight loss through lifestyle modification significantly reduces
features of non-alcoholic steatohepatitis. Gastroenterology 149:367-378.e5
36. 
Mazzotti A, Caletti MT, Brodosi L, Di Domizio S, Forchielli ML, Petta S, Bugianesi E, Bianchi G, Marchesini G
(2018) An internet-based approach for lifestyle changes in patients with NAFLD: Two-year effects on weight loss
and surrogate markers. J Hepatol 69:1155–1163
37. 
Watanabe M, Tozzi R, Risi R, Tuccinardi D, Mariani S, Basciani S, Spera G, Lubrano C, Gnessi L (2020)
Beneficial effects of the ketogenic diet on non-alcoholic fatty liver disease: A comprehensive review of the
literature. Obes Rev. doi: 10.1111/obr.13024
38. 
Younossi  ZM,  Ratziu  V,  Loomba  R,  et  al  (2019)  Obeticholic  acid  for  the  treatment  of  non-alcoholic
steatohepatitis:  interim  analysis  from  a  multicentre,  randomised,  placebo-controlled  phase  3  trial.  Lancet
394:2184–2196
39. 
Harrison SA, Abdelmalek MF, Caldwell S, et al (2018) Simtuzumab Is Ineffective for Patients With Bridging
Fibrosis or Compensated Cirrhosis Caused by Non-alcoholic Steatohepatitis. Gastroenterology 155:1140–1153
40. 
Harrison SA, Wong VWS, Okanoue T, et al (2020) Selonsertib for patients with bridging fibrosis or compensated
cirrhosis due to NASH: Results from randomised phase III STELLAR trials. J Hepatol 73:26–39
41. 
Ampuero J, Romero-Gomez M (2020) Stratification of patients in NASH clinical trials: A pitfall for trial success.
JHEP Reports 2:100148
42. 
Sanyal  AJ,  Brunt  EM,  Kleiner  DE,  Kowdley  K,  Chalasani  N,  Lavine  J,  Ratziu  V,  McCullough  A (2011)
ENDPOINTS AND CLINICAL TRIAL DESIGN FOR NON-ALCOHOLIC STEATOHEPATITIS. Hepatology 54:344
34
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
43. 
de Luis D, Domingo JC, Izaola O, Casanueva FF, Bellido D, Sajoux I (2016) Effect of DHA supplementation in a
very low-calorie ketogenic diet in the treatment of obesity: a randomised clinical trial. Endocrine 54:111–122
44. 
Sajoux I, Lorenzo PM, Gomez-Arbelaez D, et al (2019) Effect of a very-low-calorie ketogenic diet on circulating
myokine levels compared with the effect of bariatric surgery or a low-calorie diet in patients with obesity.
Nutrients. doi: 10.3390/nu11102368
45. 
Gomez-Arbelaez D, Crujeiras AB, Castro AI, et al (2017) Acid–base safety during the course of a very low-
calorie-ketogenic diet. Endocrine 58:81–90
46. 
A G, D B, I S, AB C, B B, PP G-L, A O, B M, FF C (2016) Short-term safety, tolerability and efficacy of a very low-
calorie-ketogenic diet interventional weight loss program versus hypocaloric diet in patients with type 2 diabetes
mellitus. Nutr Diabetes 6:e230
47. 
Hallsworth K, Adams LA (2019) Lifestyle modification in NAFLD/NASH: Facts and figures. JHEP Reports 1:468–
479
48. 
Centis E, Moscatiello S, Bugianesi E, Bellentani S, Fracanzani AL, Calugi S, Petta S, Dalle Grave R, Marchesini
G (2013) Stage of change and motivation to healthier lifestyle in non-alcoholic fatty liver disease. J Hepatol
58:771–777
49. 
(CDC) C for DC and P (2018) NHANES 2017-2018 Procedure Manuals. Natl Heal Nutr Exam Surv. doi:
https://wwwn.cdc.gov/nchs/nhanes/continuousnhanes/manuals.aspx?BeginYear=2017
50. 
P R, G T, L C, et al (2005) Validity and reliability of the Italian version of the Chronic Liver Disease Questionnaire
(CLDQ-I) for the assessment of health-related quality of life. Dig Liver Dis 37:850–860
51. 
Zanini B, Simonetto A, Bertolotti P, et al (2020) A new self-administered semi-quantitative food frequency
questionnaire to estimate nutrient intake among Italian adults: development design and validation process. Nutr
Res 80:18–27
52. 
Van Dongen MCJM, Lentjes MAH, Wijckmans NEG, et al (2011) Validation of a food-frequency questionnaire for
Flemish and Italian-native subjects in Belgium: The IMMIDIET study. Nutrition 27:302–309
53. 
Sofi F, Dinu M, Pagliai G, Marcucci R, Casini A (2017) Validation of a literature-based adherence score to
Mediterranean diet: the MEDI-LITE score. Int J Food Sci Nutr 68:757–762
54. 
A M, D M, D M, AM T, P V, G LT (2018) International Physical Activity Questionnaire for Adolescents (IPAQ A):
reliability of an Italian version. Minerva Pediatr. doi: 10.23736/S0026-4946.16.04727-7
55. 
Donini LM, Brunani A, Sirtori A, et al (2011) Assessing disability in morbidly obese individuals: The Italian Society
of Obesity test for obesity-related disabilities. Disabil Rehabil 33:2509–2518
56. 
Clinical  utility  of  preoperative  screening  with  STOP-Bang  questionnaire  in  elective  surgery  -  Minerva
Anestesiologica
 
2014
 
August;80(8):877-84
 
-
 
Minerva
 
Medica
 
-
 
Journals.
https://www.minervamedica.it/en/journals/minerva-anestesiologica/article.php?cod=R02Y2014N08A0877.
Accessed 7 Jul 2021
57. 
V R, E M, S M, M DB, T Z, PL C, CM R (2000) Screening for binge eating disorder in obese outpatients. Compr
Psychiatry 41:111–115
58. 
Sica C, Ghisi M (2007) The Italian versions of the Beck Anxiety Inventory and the Beck Depression Inventory-II:
Psychometric properties and discriminant power. In: Leading-Edge Psychol. Tests Test. Res. pp 27–50
59. 
Pedrabissi L, Santinello M (1989) Verifica della validità dello STAI forma Y di Spielberger.  [Verification of the
validity of the STAI, Form Y, by Spielberger.]. Giunti Organ Spec 191–192:11–14
60. 
Kyle UG, Bosaeus I, De Lorenzo AD, et al (2004) Bioelectrical impedance analysis - Part I: Review of principles
and methods. Clin Nutr 23:1226–1243
61. 
Kyle UG, Bosaeus I, De Lorenzo AD, et al (2004) Bioelectrical impedance analysis - Part II: Utilisation in clinical
practice. Clin Nutr 23:1430–1453
35
KETONASH;
Versione 4.0 23.11.2023

KETONASH
CONFIDENTIAL
"The Role of Very Low-Calorie Ketogenic Diet (VLCKD) in Patients Affected by
Non-Alcoholic Steatohepatitis (NASH) with Fibrosis"
NCT number:
TBD
Version:
4.0
62. 
Rockey DC, Caldwell SH, Goodman ZD, Nelson RC, Smith AD (2009) Liver biopsy. Hepatology 49:1017–1044
63. 
Neuberger J, Patel J, Caldwell H, et al (2020) Guidelines on the use of liver biopsy in clinical practice from the
British Society of Gastroenterology, the Royal College of Radiologists and the Royal College of Pathology. Gut
69:1382–1403
64. 
Castera L, Friedrich-Rust M, Loomba R (2019) Non-invasive Assessment of Liver Disease in Patients With Non-
alcoholic Fatty Liver Disease. Gastroenterology 156:1264-1281.e4
65. 
Dietrich CF, Bamber J, Berzigotti A, et al (2017) EFSUMB Guidelines and Recommendations on the Clinical Use
of Liver Ultrasound Elastography, Update 2017 (Long Version). Ultraschall der Medizin. doi: 10.1055/s-0043-
103952
66. 
Ferraioli G, Monteiro LBS (2019) Ultrasound-based techniques for the diagnosis of liver steatosis. World J
Gastroenterol 25:6053–6062
67. 
Piscaglia F, Salvatore V, Mulazzani L, et al (2017) Differences in liver stiffness values obtained with new
ultrasound  elastography  machines  and  Fibroscan:  A  comparative  study.  Dig  Liver  Dis.  doi:
10.1016/j.dld.2017.03.001
68. 
Ravaioli F, Montagnani M, Lisotti A, Festi D, Mazzella G, Azzaroli F (2018) Noninvasive Assessment of Portal
Hypertension in Advanced Chronic Liver Disease: An Update. Gastroenterol Res Pract 2018:1–11
69. 
Newsome PN, Sasso M, Deeks JJ, et al (2020) FibroScan-AST (FAST) score for the non-invasive identification
of patients with non-alcoholic steatohepatitis with significant activity and fibrosis: a prospective derivation and
global validation study. Lancet Gastroenterol Hepatol 5:362–373
36
KETONASH;
Versione 4.0 23.11.2023