Pilot Study Fatty Liver and Obesity: MR Fat Quantification and MR Elastography During a Very Low-calorie-ketogenic Diet (Pronokal Method®) Versus a Low Calorie Diet
Phase: NA
Conditions: Liver Fat
Sponsor: Protein Supplies SL

PILOT STUDY FATTY LIVER AND OBESITY: MR FAT QUANTIFICATION 
AND MR ELASTOGRAPHY DURING A VERY LOW-CALORIE-KETOGENIC 
DIET (PRONOKAL METHOD®) VERSUS A LOW CALORIE DIET.  
A multicenter randomized controlled clinical trial 
STUDY PROTOCOL 
Protocol Code: PNK-FATLIV-2016-04 
(Version 2.0 dated, 21 April, 2016) 
Principal investigators  
Dr. Walmir Coutinho 
Dr Guilherme Moura da Cunha 

1 ABSTRACT 
1.1 Type of study 
Prospective, multicenter, comparative, open-label, randomized and controlled pilot 
study  
1.2 Sponsor's identification 
Protein Supplies S.L.   
C/ Roger de Llúria, 58.  
08009 Barcelona. España 
Telf. +0034 934 877 589 
1.3 Title 
Pilot study fatty liver and obesity: MR fat quantification and MR elastography during a 
very low-calorie-ketogenic diet (Pronokal Method®) versus a low calorie diet 
1.4 Protocol Code 
PNK-FATLIV-2016-04 
1.5 Principal investigators 
Dr. Walmir Coutinho  
IEDE - Instituto Estadual de Diabetes e Endocrinologia 
Dr Guilherme Moura da Cunha  
CDPI – Clínica de Diagnóstico por Imagem 
1.6 Coinvestigators 
Dra. Lívia Lugarino 
IEDE - Instituto Estadual de Diabetes e Endocrinologia 
Dra. Luciana Spina 
Dra. Isabela Busade 
1.7 Study locations 
IEDE - Instituto Estadual de Diabetes e Endocrinologia 
Rua Moncorvo Filho, numero 90 
Centro, Rio de Janeiro, Brasil 
CDPI - Clínica de Diagnóstico por imagem, 
Rua Ataulfo de Paiva, numero 699, Leblon, 
Rio de Janeiro, Leblon, Brasil 

 
 
 
1.8  Study design 
Prospective, multicenter, open-label, randomized and controlled study of the effects of 
weight loss and reduced visceral fat on non-alcoholic fat infiltration into liver after a very 
low calorie ketogenic diet (VLCK diet) (Pronokal® Method) versus a hypocaloric diet, 
with a 6-month follow-up. 
1.9 Study objectives 
 
To evaluate the effects of body weight loss after a very low-calorie-ketogenic diet 
(Pronokal Method®) versus low calorie diet, on visceral fat reduction, liver fatty 
infiltration, using a new MRI technique of fat liver quantification (Ideal IQ**). 
 
To correlate the degree of liver fat infiltration and liver stiffness using MR 
Elastography 
 
To investigate the hypothesis of a linear correlation between adrenal gland size 
and variables commonly associated to insulin resistance such as BMI, visceral fat 
and liver steatosis. 
1.10 Condition in study 
Fat infiltration into the liver (nonalcoholic fatty liver disease) 
 
1.11 Study treatment  
A very low-calorie-ketogenic diet (Pronokal Method®)   
 
1.12 Outcome Measures 
 Body weight loss  
 Visceral fat (measured by the area in squared centimeters at the level of the 3rd 
vertebrae, using MRI technique) 
 Porcentaje de contenido graso del hígado 
 Liver stiffness using MR Elastography  
 Linear measurements of the adrenal glands 
 Salivary cortisol levels 
 
1.13 Study population and number of subjects 
Obese patients of either sex with a BMI over 30 kg/m2. 
 

 
 
1.14 Follow-up and duration of the study 
The follow-up will be 6 months.  
 
1.15 Funding 
We were awarded a grant from PronoKal Group® to partially fund this study, where MR 
image acquisition and post-processing costs will be covered. The leading investigators, 
free of charge, will do all data analysis and manuscript writing. 
 
1.16 Schedule 
Study onset: May, 2016. 
Completion of study: May, 2017. 

 
 
2  INDEX 
1 ABSTRACT ............................................................................................................. 2 
1.1 
Type of study .............................................................................................. 2 
1.2 
Sponsor's identification ............................................................................... 2 
1.3 
Title ............................................................................................................ 2 
1.4 
Protocol Code ............................................................................................. 2 
1.5 
Principal investigators ................................................................................. 2 
1.6 
Coinvestigators ........................................................................................... 2 
1.7 
Study locations ........................................................................................... 2 
1.8 
Study design ............................................................................................... 3 
1.9 
Study objectives ......................................................................................... 3 
1.10 
Condition in study ....................................................................................... 3 
1.11 
Study treatment .......................................................................................... 3 
1.12 
Outcome Measures .................................................................................... 3 
1.13 
Study population and number of subjects ................................................... 3 
1.14 
Follow-up and duration of the study ............................................................ 4 
1.15 
Funding ...................................................................................................... 4 
1.16 
Schedule .................................................................................................... 4 
2 INDEX ...................................................................................................................... 5 
3 GENERAL INFORMATION ...................................................................................... 7 
3.1 
Title ............................................................................................................ 7 
3.2 
Protocol code.............................................................................................. 7 
3.3 
Type of study .............................................................................................. 7 
3.4 
Study treatment .......................................................................................... 7 
3.5 
Sponsor ...................................................................................................... 7 
3.6 
Principal investigators ................................................................................. 7 
3.7 
Expected duration ....................................................................................... 7 
4 INTRODUCTION AND RATIONALE OF THE STUDY ............................................. 8 
5 STUDY OBJECTIVES ........................................................................................... 10 
5.1 
Primary aim .............................................................................................. 10 
5.2 
Secondary aims ........................................................................................ 10 
6 STUDY DESIGN .................................................................................................... 10 
6.1 
Type of study ............................................................................................ 10 
6.2 
Randomization process ............................................................................ 10 
6.3 
Type of control .......................................................................................... 10 
6.4 
Masking Techniques ................................................................................. 10 
7 SUBJECT SELECTION ......................................................................................... 10 
7.1 
Study population ....................................................................................... 10 
7.2 
Inclusion criteria: ....................................................................................... 10 
7.3 
Exclusion criteria: ..................................................................................... 11 
7.4 
Number of subjects ................................................................................... 12 
7.5 
Rationale of sample size ........................................................................... 12 
7.6 
Recruitment period ................................................................................... 12 
7.7 
Criteria for withdrawal or dropouts ............................................................ 12 

 
 
8 TREATMENT DESCRIPTION ................................................................................ 12 
8.1 
Weight Loss Program: PronoKal® Method ................................................ 12 
8.2 
Hypocaloric diet ........................................................................................ 13 
9 STUDY PROCEDURES AND OUTCOME MEASURES ........................................ 14 
9.1 
Outcome measures .................................................................................. 14 
9.1.1 
Primary variables ............................................................................................... 14 
9.1.2 
Secondary variable ............................................................................................ 14 
9.1.3 
End points ......................................................................................................... 14 
9.2 
Study procedures ..................................................................................... 14 
9.2.1 
Follow-up of patients ......................................................................................... 14 
9.2.2 
Flow chart .......................................................................................................... 16 
10 STATISTICAL ANALYSIS ..................................................................................... 17 
10.1 
Descriptive statistics ................................................................................. 17 
10.2 
Analysis of objectives ............................................................................... 17 
11 REPORT OF ADVERSE EVENTS ......................................................................... 17 
12 ETHICAL ISSUES.................................................................................................. 18 
12.1 
General considerations ............................................................................. 18 
12.2 
Evaluation by an Authorized Clinical Research Ethics Committee ............ 18 
12.3 
Patient information and informed consent ................................................. 19 
12.4 
Data confidentiality ................................................................................... 19 
13 PRACTICAL CONSIDERATIONS ......................................................................... 19 
13.1 
Workplan .................................................................................................. 19 
13.2 
Disseminating the study results ................................................................ 20 
14 REFERENCES ....................................................................................................... 20 
 
 
 

 
 
3 GENERAL INFORMATION  
3.1 Title 
Fatty Liver and obesity: MR Fat Quantification and MR Elastography during a very low-
calorie-ketogenic diet (Pronokal Method®) versus a low calorie diet. A multicenter 
randomized controlled clinical trial. 
3.2 Protocol code 
PNK-FATLIV-2016-04 
3.3 Type of study 
Prospective, multicenter, open-label, randomized and controlled study of the effects of 
weight loss and reduced visceral fat on non-alcoholic fat infiltration into liver after a very 
low calorie ketogenic diet (VLCK diet) (Pronokal® Method) versus a hypocaloric diet, 
with a 6-month follow-up. 
3.4 Study treatment   
A very low-calorie-ketogenic diet (Pronokal Method®)   
3.5 Sponsor 
Protein Supplies S.L.   
C/ Roger de Llúria, 58. 08009 Barcelona. España  
Telf. +34 934 877 589 
3.6 Principal investigators   
Dr. Walmir Coutinho  
IEDE - Instituto Estadual de Diabetes e Endocrinologia 
Dr Guilherme Moura da Cunha  
CDPI – Clínica de Diagnóstico por Imagem 
3.7 Expected duration 
The expected duration of the study is 10 months (4 months of recruitment and 6 follow-
up months).  
 
 
 
 
 
 
 

 
 
4 INTRODUCTION AND RATIONALE OF THE STUDY  
The liver histology in obese people can be altered by an accumulation of intrahepatic 
triglyceride (IHTG), i.e., steatosis, which is the hallmark feature of nonalcoholic fatty 
liver disease (NAFLD). Nonalcoholic fatty liver disease (NAFLD) is the liver 
manifestation of the complex metabolic derangements associated with obesity, being 
an emerging epidemic in Western countries and affects all ages and ethnicities. 
Individuals with hepatic steatosis alone were thought to have a benign long-term 
prognosis. However, up to 25% of these patients may develop nonalcoholic 
steatohepatitis (NASH), which may progress to cirrhosis in susceptible individual (5). 
Because early NAFLD may be reversible, screening and early intervention may be 
indicated (1-2).  
Weight loss alone by dietary changes has been shown to lead to histological 
improvement in fatty liver making nutrition therapy to become a cornerstone of 
treatment for NAFLD. Studies show that those who achieved a minimum of 7% weight 
loss had improvements in their liver histology (3). In recent years the VLCD diets, 
especially in combination with intensive life interventions (ILI), are gaining recognition 
as a suitable alternative to conventional low-calorie diets for weight loss (6-8), with 
greater weight loss at short and long term (9-11). PronoKal® method is a commercially 
available weight loss program, which includes a dietary regime (very low-calorie-
ketogenic diet based on a high-biological-value protein preparations diet and natural 
foods followed by low calorie diet), lifestyle and behavioral modification support. This 
method has proved more effective for weight loss compared with a standard low-calorie 
diet, achieving a reduction of more than 10 % of the initial weight at 2 months in 96 % 
of the patients, and in 100% of the patients at 4 months (11).  
Liver biopsy is the gold standard in diagnosing NAFLD and the most accurate tool for 
grading fibrosis. However, is invasive and carries the risk of complications (3). Also, 
histological examination of biopsy samples is subject to sampling error due to 
histological heterogeneity. Scoring is semi-quantitative, limiting its ability to detect 
modest changes, and scoring results vary between reports precluding direct 
comparisons. Various imaging techniques are proposed for liver fat quantification. Liver 
fat fraction (FF) can be calculated using fat and water MRI signal intensities in 
chemical-shift images (in-phase [IP] and out of phase [OP] sequences). Typically, the 
fatty liver shows increase in signal intensity (SI) on IP images and shows diffuse signal 
drop in OP image. The fat fraction can then be determined by calculating the loss of SI 
in OP images as compared to IP images. From congruent sets of IP and OP images, 

 
 
acquired within the same breath-hold sequence, the fat fraction can be calculated pixel-
wise, and misregistration errors can be avoided (4). MRI multiecho method estimates 
T2* from six different echo times (TE). This method resulted in improved diagnostic 
specificity compared with that of the triple-echo method which caused systematic 
underestimation at high FFs (1).  New noninvasive methods including MR elastography 
have been developed for o distinguish necroinflammation and mild fibrosis from simple 
steatosis (4). Given that the spectrum of fatty liver disease ranges from simple 
steatosis, through stages of liver cell injury (steatohepatitis), to fibrosis, and eventually 
to cirrhosis, it is appropriate to ask whether hepatic stiffness as a biomarker can be 
used to identify the presence of liver cell injury prior to the onset of fibrosis.  
One of the leading extra-hepatic causes of liver steatosis is metabolic syndrome (MS). 
Although, multiple definitions of MS exists; the essential components of MS include 
insulin resistance, increased levels of stress hormones, such as cortisol, and visceral 
obesity. In the obese population, the hallmarks of a metabolic syndrome are highly 
prevalent, being peripheral insulin resistance and increase levels of stress hormones 
the leading causes of organ injury. In such individuals higher levels of cortisol are 
expected, secondary to an over activated hypothalamic-pituitary-adrenal (HPA) (13, 
14). A linear correlation between adrenal gland volume and metabolic syndrome has 
been demonstrated, with increased adrenal volume in patients under a metabolic 
syndrome status. However, once these patients are engaged on a treatment regime, 
leading to weight loss and diminished insulin resistance, the increase in adrenal 
volume tends to be reversible. Adrenal volume measurements can be achieved using 
cross-sectional imaging and semi-automated softwares. In clinical practice, adrenal 
gland dimensions are generally assessed using limb linear measurements on 
multidetector row computed tomography or magnetic resonance imaging (15,16). 
We conducted a prospective longitudinal clinical study to correlate the effects of a very 
low-calorie-ketogenic diet (PRONOKAL method*), body weight loss and visceral fat 
reduction, with liver fat fraction, using a new MRI model of fat liver quantification (Ideal 
IQ**). Additionally, the degree of liver fat and its evolution, during the diet program, will 
be correlated with liver stiffness using MR Elastography. Adrenal gland measurements 
will also be made using the linear technique described by Carsin-Vu A. (16) to address 
its correlation with weight loss, visceral fat and liver steatosis.  

 
 
5 STUDY OBJECTIVES   
5.1 Primary aim  
 
To evaluate the effects of body weight loss after a very low-calorie-ketogenic diet 
(Pronokal Method®) versus low calorie diet, on visceral fat reduction, liver fatty 
infiltration, using a new MRI technique of fat liver quantification (Ideal IQ**). 
 
5.2 Secondary aims  
 
To correlate the degree of liver fat infiltration and liver stiffness using MR 
Elastography 
 
To investigate the hypothesis of a linear correlation between adrenal gland size 
and variables commonly associated to insulin resistance such as BMI, visceral fat 
and liver steatosis. 
 
6 STUDY DESIGN 
6.1 Type of study 
Prospective, multicenter, comparative, open-label, randomized and controlled pilot 
study 
6.2  Randomization process 
Patients will be randomized at the time of inclusion, in accordance with an 
automatically generated randomization list. One group will start treatment with a very 
low calorie and ketogenic diet (VLCK diet group) while the other group will start a low 
calorie diet (control group). The patient ratio in both groups will be 1: 1.  
6.3 Type of control 
The control group will consist of patients who follow the hypocaloric diet.  
6.4 Masking Techniques 
Not applicable. This is an open-label study  
7 SUBJECT SELECTION 
7.1 Study population 
Obese patients of either sex with BMI over 30 kg/m2, who agree to participate in the 
study.  
7.2 Inclusion criteria: 
 
Patients of either sex, older than 18 years 

 
 
 
Obese patients with BMI equal or over 30kg/m2  
 
Patients who agree to participate and sign the Informed Consent 
7.3 Exclusion criteria: 
 
Pregnant or lactating women. 
 
Patients weighing over 140 kg (by limitation of the MRI device) 
 
Patients on anti-obesity drugs (eg, sibutramine, orlistat and liraglutide) or 
weight-interfering medications (eg, topiramate). In such cases a wash out 
period of 3 months is required.  
 
Patients with alcohol intake > 20 g/day in women and > 30 g/day in men. 
 
Patients with liver failure or with pathologies that may affect the liver 
 
Patients with other causes of liver steatosis: chronic viral hepatitis and/or drug 
intake (corticosteroids, estrogens, NSAIDs, calcium antagonists, amiodarone, 
tamoxifen, tetracyclines, chloroquine, antiretrovirals, perhexiline). 
 
Patients with eating disorders. 
 
Patients with any psychiatric disorder (eg schizophrenia, bipolar disorder, 
substance abuse, severe depression). 
 
Patients receiving dicumarinic anticoagulants (Sintrom®).  
 
Patients taking corticosteroids. 
 
Patients with severe kidney failure (gfr <30). 
 
Patients with type 1 and type 2 diabetes mellitus. 
 
Patients with hemopathies, including coagulation disorders 
 
Patients with cardiovascular or cerebrovascular disease (of heart rate disorders, 
recent infarction [<6m], unstable angina, decompensated heart failure, recent 
stroke [<6m]). 
 
Patients in acute attack of gout. 
 
Patients with renal lithiasis verified by ultrasound. 
 
Patients with cholelithiasis verified by ultrasound.  
 
Patients with electrolyte imbalance, according to medical criteria.  
 
Patients with orthostatic hypotension.  
 
Patients with cancer or history of cancer who have not been discharged from 
oncology 
 
Patients who are not expected to attend follow-up visits 
 
 
 
 
 

 
 
7.4 Number of subjects 
A total of 40 patients will be included: 20 patients randomly assigned to a very low-
calorie-ketogenic diet (VLCK diet group) and 20 patients randomly assigned to low 
calorie diet (control group). 
7.5 Rationale of sample size 
Exploratory pilot study to test a concept, where it has been estimated that 20 patients 
per group will be sufficient to analyze results. 
7.6 Recruitment period 
We expect a 4-month recruitment period  
 
7.7 Criteria for withdrawal or dropouts 
Patients who do not attend the control visits and/or have not undergone at least two 
MRIs (baseline and control after the end of ketosis or at month 6) will be considered as 
dropouts.  
 
8 TREATMENT DESCRIPTION  
8.1 Weight Loss Program: PronoKal® Method 
All patients in the study group will undergo the same standardized weight loss program 
(PronoKal® Method) with progressive diet based on commercial high-biological-value 
protein preparations, dietary supplements, physical exercise plan and emotional 
support, structured in 3 stages: active stage, food re-education stage and maintenance 
stage  
Long-term mantenance of 
weight loss
Step 1
Step 2
Step 3
Step 4
Step 5
Controlled diet 
adapted
DHA VITA: 500 DHA/100 EPA
Weight Loss Moment
My New Lifestyle Moment
Low-fat ketogenic diet
(very low-calorie-ketogenic diet)
600-730 kcal/day
Low calorie diet
(800-1500 kcal/day)
Supplementation
Vitamins, trace elements
OMEGA BALANCE: 700 DHA/500 EPA
Supplementation
Vitamins, trace elements
DHA VITA: 500 DHA/100 EPA
80% of target weight loss
20% of target weight loss
Multidisciplinary team  (Dietary counseling/physical activity/psychological support)
 
 
a) Treatment schedule 
Active stage: The diet will consist of a protein diet, ie a very low-calorie ketogenic diet 
(VLCK diet) (between 650 and 730 kcal/day) based on PronoKal® commercial 
preparations and low-glycemic-indexvegetables. Patients will start with 5 commercial 

 
 
preparations a day. Two of them will be replaced progressively by natural proteins 
(meat or fish). This active stage will be maintained for 2 months.  
 Phase 1: Protein diet of 600 kcal/ day (with 5 PronoKal® products and low-
glycemic-index vegetables) + Nutritional supplements (vitamins and trace 
elements) + Cardiovascular physical exercise + Emotional support. 
 Phase 2a: Protein diet of 680 kcal/day (with 4 PronoKal® products, a high-
protein food [meat or fish] and low-glycemic-index vegetables) + Nutritional 
supplements (vitamins and trace elements) + Cardiovascular physical exercise 
+ Emotional support.+ Soporte emocional. 
 Phase 2b: Protein diet of 780 kcal / day (with 3 PronoKal® products, two high-
protein foods [meat or fish] and low-glycemic-index vegetables) + Nutritional 
supplements (vitamins and trace elements) + Cardiovascular physical exercise 
+ Emotional support. 
Patients will remain in Phase 1 until they have lost 40% of the excess weight or the 
target weight to be lost. Subsequently, they go on to Phase 2a until they have lost 60% 
of the excess weight and then to Phase 2b until they have lost 80% of the excess 
weight.  
For this study, patients will leave the active Ketogenic Stage within 2 months of 
treatment, even if they have failed to achieve 80% of the weight loss goal.  
Phase of physiological adaptation: Progressive introduction of natural foods and 
maintaining the intake of 2 commercial preparations per day, with a progressive 
increase in calorie intake up to 1300-1500 kcal/day and an increased supply of 
carbohydrates. The diet will be accompanied by with cardiovascular physical exercise 
and emotional support. The length of the food re-education stage will be 4 months. 
Maintenance stage: Upon completion of dietary re-education, the patient will go on a 
balanced diet of 1500-2200 kcal/day, along with cardiovascular physical exercise and 
emotional support, until completing the 2-year follow-up. 
 
8.2 Hypocaloric diet 
Balanced hypocaloric diet (caloric intake 10% below basal metabolic rate).  
The diet will also be accompanied by physical exercise.  
 

 
 
9 STUDY PROCEDURES AND OUTCOME MEASURES  
9.1 Outcome measures 
9.1.1 Primary variables  
 
Liver fatty infiltration, measured by a MRI technique of fat liver quantification 
(Ideal IQ**). 
 
Visceral fat, measured by the area in squared centimeters at the level of the 3rd 
vertebrae, using MRI technique.  
 
9.1.2 Secondary variable 
 
Liver stiffness detected by MR Elastography  
 
Adrenal gland linear measurement 
 
Body weight  
 
Salivary cortisol levels 
 
9.1.3 End points 
 
Reduction of fat infiltration into the liver between the baseline visit and 2 months 
(or end of the ketosis stage) and between the baseline visit and the control visit at 
month 6 of treatment.  
 
Visceral fat reduction between the baseline visit and 2 months (or end of the 
ketosis stage) and between the baseline visit and the control visit at month 6 of 
treatment.  
 
Correlation between degree of liver fat infiltration and liver stiffness 
 
Correlation between adrenal gland size and BMI, visceral fat and liver steatosis. 
 
Correlation between salivary cortisol levels and BMI, visceral fat and liver 
steatosis. 
 
Weight loss between the baseline visit and 2 months (or end of the ketosis stage) 
and between the baseline visit and the control visit at month 6 of treatment. 
 
9.2 Study procedures 
9.2.1 Follow-up of patients 
The patient follow-up will be 6 months. The patients will be visited monthly and data will 
be recorded on 4 occasions: pre-inclusion, baseline visit, final visit Active stage (end of 
ketosis) and visit at month 6.  

 
 
Patients will be included after verifying the inclusion and exclusion criteria and after 
obtaining the signed informed consent.  
Screening visit (day -15). In this visit, the patient's medical history will be assessed and 
a complete blood test will be required to verify the inclusion/exclusion criteria: complete 
blood count (red blood cells, leukocytes, neutrophils, hematocrit, hemoglobin), fasting 
blood glucose, Hb1c, insulinemia, lipid profile (total cholesterol, c-HDL, c-LDL, 
triglycerides), kidney function (serum creatinine, creatinine clearence, glomerular 
filtration), nitrogen balance (urea, uric acid), thyroid profile (TSH, T4) and ionogram 
(Na, K, Cl, Ca, Mg) 
The pre-inclusion blood test will also include liver function tests as the first screening of 
hepatic steatosis:  
 GOT 
 GPT 
 Gamma-GT 
 Bilirubin 
 Albumin 
 Prothrombin time 
 Ferritin 
 Alkaline phosphatase.  
In addition, the patients will be informed about the study through the patient information 
sheet (Attachment 1) and they will be provided with an informed consent (Attachment 
2). 
At the baseline visit (day 0). The results of the additional tests (blood tests) will be 
verified to complete the inclusion/exclusion criteria.  
Liver function data will be reviewed. Patients with one or more of the following 
conditions will be considered as suspicious of hepatic steatosis:  
 GPT/GOT ratio over 1 
 Gamma-GT and alkaline phosphatase increased twice to three times the normal 
levels 
 High ferritin  
 Normal bilirubin and albumin in the presence of any of the above changes 
The informed consent will be signed and randomized. The corresponding diet will be 
scheduled depending on the treatment group (VLCK diet group or control group) and 
the physical exercise program. Patient demographics (sex, age, weight, height, 
comorbidities and medication) will be recorded. Anthropometric data (weight, BMI, 
waist circumference) will be recorded. Fat infiltration into liver and visceral fat will be 
scanned by MRI and hepatic stiffness will be assessed by MR Elastography. The 
salivary cortisol levels will also be tested. 
Control visit at month 2 or at the end of the ketogenic stage: For patients in the VLCK 
diet group, as soon as the patient loses 80% of the excess weight or within 2 months of 
treatment, just before moving to phase 3, the patient will undergo a new control. In the 
case of the control group, this visit will take place at month 2 of the hypocaloric diet. 

 
 
The patient will undergo a new MRI and MR Elastography. The salivary cortisol levels 
will also be tested. Anthropometric data are recorded, the patient undergoes a 
complete blood test including liver function (GOT, GPT, GammaGT, bilirubin, albumin, 
prothrombin time, ferritin, alkaline phosphatase) and the potential occurrence of 
adverse effects will be recorded.  
Visit at month 6 At month 6 of the study, a new control will be performed, evaluating 
hepatic steatosis and visceral fat using MRI and liver stiffness using MR Elastography. 
The salivary cortisol levels will also be tested. A complete blood test including liver 
function (GOT, GPT, GammaGT, bilirubin, albumin, prothrombin time, ferritin, alkaline 
phosphatase) will be required and the anthropometric data, the treatment phase, the 
blood test results and the potential occurrence of adverse effects will be recorded.  
9.2.2 Flow chart  
  
Pre-study 
Onset 
Visit at month 2  
 (end of ketosis) 
Visit at month 6 
(Final visit) 
Review of inclusion and 
exclusion criteria. 
X 
X 
  
  
General blood test with liver 
function 
X 
  
X 
X 
Patient information 
X 
  
  
  
Informed  
consent 
  
X 
  
  
Comorbidities and medication 
  
X 
X 
X 
Anthropometric data  
 
(weight, BMI, WC) 
X 
X 
X 
X 
MRI + MR Elastography 
  
X 
X 
X 
Salivary cortisol test 
 
X 
X 
X 
Adverse reactions 
  
  
X 
X 
Drop outs and end 
of study  
  
  
X 
X 
 
 
 

 
 
10 STATISTICAL ANALYSIS 
10.1 Descriptive statistics 
A descriptive statistics will include all variables collected in the Data Collection 
Notebook. Frequency tables will include the nominal variables and measures of central 
tendency and dispersion for the continuous variables. 95% confidence intervals (95% 
CI) will be estimated for the latter.  
10.2 Analysis of objectives 
For the analysis of the primary objective and the secondary objectives, the differences 
between visits and between groups will be studied for the changes in the ratio between 
liver fat and the degree of steatosis (measured by MRI), changes in the degree of liver 
stiffness, changes in cortisol levels in saliva, weight changes, BMI and waist 
circumference, and changes in the amount of visceral fat among visits. The qualitative 
variables will be compared using Fisher's exact test, and the quantitative variables will 
be compared by means of the Student's t test for paired data. If the conditions to apply 
the Student's t test are not met, we will use the non-parametric Mann-Whitney U test. In 
the case of comparing paired data, we will use the tests described above, in their 
version for comparing this type of data.  
The Pearson correlation coefficient or Spearman's correlation coefficient if the 
application conditions are not met will be used to study the following: the association 
between reduction of hepatic steatosis and weight loss and loss of visceral fat; the 
association between hepatic steatosis and liver stiffness; and the association between 
adrenal gland size and salivary cortisol levels with weight changes, BMI, hepatic 
steatosis and visceral fat. 
For all tests, the significance level will be set at p <0.05.  
 
11 REPORT OF ADVERSE EVENTS 
All adverse events shown in patients during the time they are in the study will be 
recorded in the Data Collection Notebook, specifying:  
 
Type of reaction  
 
Intensity: mild (no limitation of daily living activities, patient may have mild 
discomfort), moderate (cause some limitation of daily living activities, patient 
may experience increased discomfort), severe (inability to perform daily living 
activities; patient may report intolerable pain or discomfort). 
 
Length  
 
Causality with treatment and with QEEG scans: definitive, likely, possible, 
unlikely, unrelated, unknown. 
 
Therapy administered 
 
Resolution 

 
 
 
For any serious adverse reaction (see box below), the physician should complete a 
specific form (Attachment 6) with all available data and report it immediately (if possible 
within a maximum period of 24 hours) by fax, telephone or e-mail to the sponsor, who 
will implement the corresponding Pharmacovigilance procedures in accordance with 
current legislation: 
 
 
Dr. Ignacio Sajoux 
International Medical Director de PronokalGroup® 
Roger de Llúria 58 
08009 Barcelona 
Fax : +34 931 833 310 
Tel +34 931 833 310 or +34 666 988 021. 
 
 
 
 
12 ETHICAL ISSUES 
12.1 General considerations 
This study will be conducted in accordance with current regulations, international 
accepted ethical standards of Good Clinical Practice (CPMP/ICH/135/95) and the 
principles provided in the latest version of the Declaration of Helsinki. 
 
12.2 Evaluation by an Authorized Clinical Research Ethics Committee 
Prior to its inception, this study will be submitted to the Ethics Committee of the IEDE - 
Instituto Estadual de Diabetes e Endocrinologia 
 to obtain their approval. 
 
SERIOUS ADVERSE REACTION: is any adverse reaction that can be classified in one or 
more of the following categories: 
- 
Mortal 
- 
Life threatening 
- 
It causes significant or persistent discapacity/ disability 
- 
It causes hospitalization or prolongs hospitalization 
Congenital anomalies/birth defects and serious adverse clinical consequences associated 
with use under conditions other than those set out in the Summary of Product Characteristics 
(SPC), overdose or abuse are also included. 
Medical judgment should be applied when deciding whether an event or reaction is serious in 
other situations. Major adverse reactions or events that do not pose immediate threat to life, 
or do not cause death or hospitalization but may endanger the patient, should be deemed 
serious. 
 

 
 
12.3 Patient information and informed consent  
The patient will be informed about the study issues for their knowledge and written 
informed consent will be requested for participation in the study. 
 
12.4 Data confidentiality 
The patient's identity will appear coded in the study forms. The clinical investigator of 
the clinical trial will maintain a separate confidential record listing the patient 
identification codes and patient record number. The data of the subjects participating in 
the study will be saved in an automated file, property of Protein Supplies S.L .  
  
 
13 PRACTICAL CONSIDERATIONS 
13.1 Workplan 
The investigators will be in charge of recruiting patients. Once the patients have 
confirmed their willingness to participate in the study, the investigator should verify if 
they meet the inclusion/exclusion criteria of this study. If they are eligible, they will be 
requested the additional tests (blood test with liver function) and will be informed on 
every issue of the study through the Patient Information Sheet (Attachment 1).  
At the baseline visit, once the complementary tests have been reviewed and verified to 
meet inclusion criteria, the physician must confirm that the patient agrees to participate 
in the study and obtain the patient's signature of the Informed Consent (Attachment 2). 
Upon signing, the investigator will verify the assigned group according to the 
randomization list.  
At this baseline visit, the physician should request MRI and MR Elastography to assess 
the degree of hepatic steatosis, the amount of visceral fat and liver stiffness. The 
physician will also perform the first salivary cortisol test on the patient. Also, he should 
record the clinical data requested in the Data Collection Notebook and schedule the 
treatment.  
During the weight loss treatment, patients in the study group will be monitored by the 
investigator fortnightly during the protein diet (Ketogenic Active Stage), although at 
these additional visits no data are expected to be collected for the study. Upon 
completion of the first two months of follow-up, the study group will end the 
ketogenic stage and from that moment on, the patients will be controlled outside 
the center by dietitians specialized in the Pronokal® Method. Patients in the 
control group (hypocaloric diet) will undergo controls at the center monthly, although 
data will only be recorded in the visits arranged in this protocol (screening, baseline, 
month2 and month 6).  

 
 
At the moment the patient completes the two months of follow-up, the data requested 
in the DCN will be recorded, the salivary cortisol test will be performed and an MRI and 
a MR Elastography will be requested.  
The investigator, apart from monthly visits or any additional visits deemed necessary, 
will have an appointment with the patient at month 6 of treatment for a new clinical 
assessment, will record the anthropometric data, and request a Control MRI and MR 
Elastography.  
At the end of the 6-month follow-up, the study will be completed.  
In case of drop out or discontinuation of the study, the investigator will submit the DCN 
visit sheets and complete the study completion sheet, specifying the reason for 
discontinuation.  
 
13.2 Disseminating the study results 
The overall data will be used by the whole project, not individually and will be owned by 
Protein Supplies S.L. 
With the results obtained, we will issue the final study report.  
With the overall data we will try to maximize the exploitation of the study results in the 
form of publications or reports in conferences, always mentioning the study.  
 
14 REFERENCES 
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Flip-Angle Multiecho Gradient-Recalled-Echo MR Imaging at 1.5 T Radiology: 
Volume 251: Number 1—April 2009 ▪ radiology.rsnajnls.org  
2. Hepatic Steatosis as a Marker of Metabolic Dysfunction. Nutrients. 2015 Jun 
19;7(6):4995-5019.  
3. Role of diet on non-alcoholic fatty liver disease: An updated narrative review. 
World J Hepatol. 2015 Mar 27;7(3):575-82. doi: 10.4254/wjh.v7.i3.575.. 
4. Imaging of non-alcoholic fatty liver disease: A road less travelled. Indian J 
Endocrinol Metab. 2013 Nov-Dec; 17(6): 990–995. 
5. Early detection of nonalcoholic steatohepatitis in patients with nonalcoholic fatty 
liver disease by using MR elastography. Radiology. 2011 Jun; 259(3):749-56. 
doi: 10.1148/radiol.11101942. Epub 2011 Apr 1.  
6. Seger JC, Horn DB, Westman EC, Lindquist R, ScintaW, Richardson LA et al. 
Obesity Algorithm, presented by the American Society of Bariatric Physicians 
2013-2014. Disponible en: www.obesityalgorithm.org.  
7. Blackburn GL.Weight of the nation: moving forward, reversing the trend using 
medical care. Am J Clin Nutr. 2012;96(5):949-50. 

 
 
8. Hemmingsson E1, Johansson K, Eriksson J, Sundström J, Neovius M, Marcus 
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a very-low-calorie diet, a low-calorie diet, or restricted normal food: 
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9. Handjieva-Darlenska T1, Holst C, Grau K, Blaak E, Martinez JA et al. Clinical 
correlates of weight loss and attrition during a 10-week dietary intervention 
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10. Rolland C, Johnston KL, Lula S, Macdonald I, Broom J. Long-term weight loss 
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11. Look AHEAD Research Group. Eight-year weight losses with an intensive 
lifestyle intervention: the look AHEAD study. Obesity (Silver Spring). 2014 
Jan;22(1):5-13.  
12. Moreno B, Bellido D, Sajoux I, Goday A, Saavedra D, Crujeiras AB et al. 
Comparison of a very low-calorie-ketogenic diet with a standard low-calorie diet 
in the treatment of obesity. Endocrine. 2014;47(3):793-805.