Monitoring of Hepatic Fat Metabolism Using Magnetic Resonance Methods
Phase: NA
Conditions: Hepatic Fat Content
Sponsor: Institute for Clinical and Experimental Medicine

 
G285 Study protocol                                                                                                                                   Page 1 
 
Institute for Clinical and Experimental Medicine 
 
 
 
 
 
 
Monitoring of hepatic fat metabolism using magnetic 
resonance methods 
 
Grant no. 16-28427A, Ministry of Health of the Czech Republic 
 
 
 
Study protocol 
 
September 14th, 2018 
(translation of original Study protocol in Czech dated April 2016) 
 
 
 
 

 
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Introduction 
The epidemic of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis is 
becoming a major challenge currently faced by health systems worldwide. Importantly, the 
first stage of the disease is simple steatosis caused by excessive fat accumulation in the liver 
(in the absence of significant alcohol consumption). Hepatic fat comes from three major 
sources – non-esterified fatty acids (NEFA) released from adipose tissue, dietary fat, and 
hepatic de novo lipogenesis (DNL). However, hepatic triglycerides (TG) are subsequently 
metabolized or, more importantly, quickly secreted from the liver in very-low-density 
lipoproteins (VLDL), which explains why TG should not accumulate in the liver. Steatosis 
develops when the delicate balance between these processes is disrupted and hepatic TG 
production outweighs TG export to other tissues.  
In healthy non-steatotic subjects, the daily turnover of hepatic TG might even exceed hepatic 
fat content (HFC). It is estimated that up to 20% of dietary fat is delivered to the liver both as 
TG of chylomicron remnants and spillover fatty acids released from dietary TG by lipoprotein 
lipase in the circulation and not captured by extrahepatic tissues. Therefore, a sufficient 
amount of dietary fat provided to healthy non-steatotic subjects should induce changes in 
HFC detectable using 1H magnetic resonance spectroscopy (1H-MRS) even within hours. 
However, liver fat accumulation may be strongly affected by the coadministration of other 
nutrients, especially carbohydrates, which induce regulatory responses diverting metabolism 
to carbohydrate oxidation and fat storage and, possibly, also affecting HFC.  
The current epidemic of NAFLD seems to be tightly linked to excessive fructose 
consumption. However, it has not been clarified yet whether fructose is just a marker of 
increased caloric intake leading to insulin resistance and ectopic fat accumulation or the 
culprit directly involved in fat accumulation in the liver. It has been documented that 
metabolism of fructose, contrary to that of glucose, can indeed induce metabolic changes 
possibly promoting hepatic fat accumulation but up to now there is no direct proof that 
fructose consumption induces immediate fat accumulation in the liver. 
In this study we therefore aim to determine, using 1H-MRS, the immediate effect of high-fat 
load on HFC and whether such an effect can be modified by fructose and/or glucose 
coadministration. The fat load (150 g, whipping cream) will be administered subjects after 

 
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baseline measurement of HFC. Because of relatively short-term impact of simple sugars on 
regulatory pathways, the glucose and fructose will be administered to subjects in three 
doses in 2-hour intervals. 
 
Objective(s) 
The objectives of the present study are as follows: 
 To determine whether high-fat load (150 g of fat) induces an increase in hepatic fat 
content (HFC) that is detectable by magnetic resonance spectroscopy (MRS) 3 and 6 
hours after administration 
 To determine whether changes in HFC induced by high-fat load can be affected by 
coadministration with glucose at three doses of 50 g throughout the experiment 
 To determine whether changes in HFC induced by high-fat load can be affected by 
coadministration with fructose at three doses of 50 g throughout the experiment 
 
Design 
The present study is a randomised, controlled, crossover design in which 2 groups of 10 
subjects – 10 nonobese healthy males with normal hepatic fat content (HFC) as determined 
by proton magnetic resonance spectroscopy (1H-MRS) and 10 nonobese nondianbetic males 
with hepatosteatosis (HFC more than 5%) will complete total six examinations lasting 
approximately 8 hours. The examinations will differ in the dietary load and will be carried 
out in a randomised order and will consist of three measurements of hepatic fat in (HFC) – 
before and 3 and 6 hours after defined dietary load.  
 
Methods 
Screening 
The subjects will firstly undergo a screening visit to the laboratory in which written informed 
consent will be obtained. Further to this, anthropometric measures (height, weight, hip and 
waist circumferences) and blood sample will be taken for biochemical parameters that are 
used as inclusion/exclusion criteria (cholesterol, glucose, AST, ALT, HbA1c). Moreover, oral 
glucose tolerance test will be carried out to exclude diagnosis of diabetes mellitus. If the 
subjects meet the inclusion criteria, they will be invited to examination in which their 
hepatic fat is measured using 1H-MRS. Based on the results of this examination (Examination 

 
G285 Study protocol                                                                                                                                   Page 4 
A), they will be included into group with normal HFC (less than 5% of fat in the liver) or into 
group with steatosis (more than 5% of fat in the liver). 
Experiments/examinations 
All subjects included into the study will comlete six examinations. They will be asked not to 
change their everyday habits on the day before the study and consume their last meal no 
later than 10 PM. They will come to the MR department at 7:30/8:15, sign the informed 
content for MR examination and pass the first MR examination. After that the cannula for 
blood draws will be inserted into the antecubital vein and the first blood sample taken; the 
volunteers will then receive the first „meal“ (whipping cream and/or fruit tea with 50 g of 
glucose or 50 g of fructose). The time when they will finish the „meal“ will be time 0 of the 
examination. The dietary interventions will be: 
 High-fat load: 150 g of fat – 470 ml of whipping cream  
High-fat load + glucose: 470 ml of whipping cream and fruit tea with 50 g of glucose 
administered to volunteers at the same time as cream (time 0) and then 2 and 4 
hours later  
 High-fat load + fructose: 470 ml of whipping cream and fruit tea with 50 g of fructose 
administered to volunteers at the same time as cream (time 0) and then 2 and 4 
hours later  
 Fasting: no food administered during experiment 
 Glucose: fruit tea with 50 g of glucose administered to volunteers at time 0 and then 
2 and 4 hours later  
 Fructose: fruit tea with 50 g of fructose administered to volunteers at time 0 and 
then 2 and 4 hours later  
During each examination volunteers will not consume any other food and will be allowed to 
drink only water or fruit tea (without sugar).  
Nine more blood samples will be obtained at times 0.5, 1, 2, 2.5, 3, 4, 4.5, 5, and 6 hours, 
two other MR measurements will be carried out at time 3 and 6 hours (immediately after 
blood draws).  
The Sampling protocol that will be used in each experiment is presented (Figure 1). 
During the day of examination the person conducting the study with volunteer will fill in the 
questionnaire on physical activity, previous day's food and possible illnesses or used 

 
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medication. Moreover, during the day of examination, the body fat content and body 
composition will be determined by bioelectrical impedance analysis (BODYSTAT® 1500).  
 
 
Figure 1.  Sampling protocol of the study 
 

 
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Methods 
HFC will be measured by in vivo 1H MRS. Examinations will be performed on a 3T whole body 
scanner (3T Trio Siemens, Germany). Standard PRESS (Point Resolved Spectroscopy 
sequence) single voxel spectroscopy will be used [echo time (TE) 30 ms and repetition time 
(TR) 4500 ms] to measure HFC. Voxel size of our volume of interest (VOI) will be set to have 
40x30x25 mm and the VOI position in V/VIII liver segments will be carefully checked during 
all consecutive examinations. One image acquisition during each breathhold will be obtained 
with the measurement repeated three times. Relaxation times for saturation corrections will 
be measured using the same sequence (PRESS) with echoes: 30, 50, 68, 135, 180 and 270 
ms. Spectra will be evaluated using LCModel version 6.2 (http://s-
provencher.com/lcmodel.shtml). Three lipid signals of aliphatic protons in the range 0.0-3.5 
ppm (-CH2 1.2-1.4 ppm; -CH3 0.8-0.9 ppm; -CH2CH2CH=CH 1.9-2.1 ppm) and water and 
olefinic protons signals (4.7 and 5.3 ppm) will be fitted and the percentage of HFC will be 
calculated according to Longo et al. (J Magn Res Imaging 1995; 5: 281-5). 
Biochemistry 
Blood will be collected into 8ml vacutainers with EDTA and immediately placed on the ice. 
The centrifugation will be carried out for 15 min at 4°C, 3000 g, and plasma divided into 5 
aliquots (min. 0.5 ml) that will be stored at -80°C. One aliquot will be used for determination 
of TG, glucose, and NEFA (Lipid laboratory CEM IKEM), two other aliquots will be used for 
determination of insulin (IRMA, Beckman) and glucagon (RIA, Millipore), and two will serve 
as a reserve for repetition or other analyses.