The liver is very patient

We need better methods to detect liver disease earlier

LiSyM-Pillar IV develops and improves methods for diagnosing liver disease as early as possible. Pillar IV adopts an integrative research approach. It combines mathematical models of liver metabolism with data from established clinical trials as well as from cell biological and molecular biological analyses of hepatocytes. Computer simulations result, which represent the functional status of individual liver zones. The aim is to distinguish sub-pathological changes in time: there is no clear evidence of disease symptoms on account of the liver’s ability to counterbalance them. Nevertheless, the adaptation of the liver indicates that early damage has already occurred.

Kinetic metabolism model – a cell’s metabolism depends on thousands of chemical reactions, which are catalyzed by specialized proteins (enzymes). The chemical conversion rate determines how many molecules of each substance a chemical reaction creates or consumes per time unit. This can be described mathematically in differential equations. The solutions of the equations, obtained with the aid of a computer, calculates the concentration of the substance at a specific point in time.
Hepatocytes – epithelial liver cells make up approx. 80 % of the liver volume. They carry out most of the organ’s metabolic tasks.
Proteomics – the proteome comprises all proteins present in cells, in tissue or in a complete organism. Proteomics is the term experts use to describe the research of proteomes using biochemical methods. Mass spectrometry is commonly used to quantify the abundance of proteins. First the proteins are broken down into small fragments (peptides). The peptides are then separated in an electric field by mass and charge. From the abundance of peptides determined using this procedure, the abundance of the underlying initial protein is deduced.
Elastography – a method for measuring stiffness of soft tissue. This measurement helps determine the extent of spread of fibrosis, that is the changes in the connective tissue, in the liver. Liver stiffness increases when the proportion of connective tissue increases. Dynamic elastography uses external pressure waves to produce vibrations in the tissue being assessed. The stiffer the tissue, the faster and more widespread the waves disperse.
NAFLD – non-alcoholic fatty liver disease. Chronic liver disease due to high fat accumulation, in which alcohol consumption plays no considerable role.

Our Research Approach

The basis of Pillar IV research is a complex metabolism model, which has been developed by the research group under Project Coordinator Prof. Hermann Georg Holzhütter at the Institute for Biochemistry at the Charité, in Berlin, over several years. The model simulates the metabolism of hepatocytes. The computer program simulates how liver cells chemically convert hundreds of different substances and how fast the closely interconnected reactions take place. With this information, the model can calculate how quickly the liver absorbs various substances from the bloodstream, and subsequently converts and releases them to the bloodstream.

Every chemical reaction in the liver is catalyzed, that is accelerated, by an enzyme. However, the amount of these enzymes changes in damaged liver cells. Correspondingly, the rate of reactions changes. The metabolism model from Pillar IV can represent these changes. Together with his research group, Dr David Meierhofer from the Max Planck Institute for Molecular Genetics (MPIMG) have determined the abundance of enzymes in normal and damaged liver tissue. The data from this so-called proteomic study have been entered into the model. Now the model can predict how damage in the liver impacts metabolic dynamics. Pillar IV uses this new method for predicting liver cell function to reveal the changes on the liver metabolism in children and youth with NAFLD.

Associate Professor Dr. Susanna Wiegand, and Senior Physician Dr. Christian Hudert, at the Social Pediatric Center, Charité Hospital, in Berlin, supervise this pediatric cohort study. A comprehensive characterization of the liver cell histology was undertaken on tissue samples from young patients. In addition, Prof. Ingolf Sack’s research group at the Institute of Radiology and Pediatric Radiology at the Charité Hospital, Berlin, analyzed the tissue samples using elastography. With this method, one can determine how much fat has accumulated in the liver, and how much of it has been converted to connective tissue. This way Pillar IV could establish the link between structural changes in the liver tissue and the functional changes in liver metabolism calculated by the model.

NAFLD in children and youth

Today NAFLD is the most widespread chronic liver disease in children and youth. Between five and ten percent of this group is affected. Obesity (adipositas) is the most significant risk factor for NAFLD. In Germany the percentage of overweight children and youth has increased by 50 percent compared to the 1980s and 1990s. Dr. Hudert, Senior Physician at the Charité Hospital, Berlin, supervises about 80 young NAFLD patients between ten and eighteen years old in his study: “The liver of these patients often reveals a high degree of fat accumulation together with changes in the connective tissue.” However, Hudert explains that the structural pattern of the observed changes differs from that observed in adult patients, where the liver usually accumulates fat in the area of the central vein – the site where blood exits the liver. “In contrast, in young patients, the fat accumulation usually begins in the region of the liver where the blood flows through the artery and portal vein into the liver,” Hudert explains. “One possible explanation for the difference maybe that the liver of young patients is still growing, and the growth hormones have a strong effect on their metabolism.”

The Liver –the central metabolic organ in humans

The liver monitors and regulates the composition of nutrients as well as potentially harmful substances in the blood. Part of the glucose humans ingest in their food is transformed into glycogen and stored in the liver. In the state of hunger, the liver can release it as glucose again and, make it available to the other internal organs. When the capacity of our fatty tissue to store fat is exhausted, the liver can take over and store substantial quantities of fat. Once a certain level of fat storage has been reached in the liver, experts speak of a steatosis, fatty liver or metabolic liver disease. The liver also absorbs almost all drugs and chemically converts them into a form the body can expel. This is part of the detoxification function of the liver. It also includes the ability of the liver to convert toxic ammoniac – produced during the breakdown of proteins – into urea, which is then expelled when urinating. The liver can still fulfil its many functions even if severely damaged as a result of inflammation. This ability makes it difficult to diagnose chronic liver disease early on.

Small changes in the protein configuration of liver cells – large functional consequences

“With the help of high-resolution mass spectrometry, we can classify and quantify many thousands of different proteins – far more than through traditional methods”, Dr. David Meierhofer from MPIMG confirms. When analyzing proteins from liver cells of young NAFLD patients at different stages of the disease using mass spectrometry, only very few proteins show considerable changes in frequency. However, when Meierhofer enters his proteomic data into the Pillar IV metabolism model, clear distinctions in liver function can be found.
Dr Nikolaus Berndt, Leading Scientist in the development of the model, and Prof. Holzhütter’s colleague, explains how these deviations come about: “Our model reveals how a combination of many minor changes at the molecular level can severely affect the metabolic network functions of the liver.” Berndt provides an example: “In children with slight to moderate liver fibrosis we see no changes in ammoniac detoxification. Those with an advanced stage of fibrosis show a significant loss in liver detoxification capacity.”

Clear evidence of functional changes in a very early phase of NAFLD

“Our study is the first to provide clear evidence of functional changes in the liver occur in a very early stage of non-alcoholic fatty liver disease,” Pillar IV Coordinator, Prof. Holzhütter, summarizes. “Based on the mathematical predictions of functional changes our model makes, we can predict the degree of severity of liver fibrosis with the exact same accuracy as the histologic cell analysis conducted in the laboratory.” Up until now, the results have based on data from tissue samples taken during liver biopsies, which involve a minor surgical procedure. Therefore, Pillar IV’s next aims is, through computer simulations, to assess if these early functional changes can be detected using a capacity test – a simple procedure. One such test could involve administering a suitable ‘cocktail’ of various substances, which can be broken down by the liver, and monitoring the elimination of these substances from the patient's bloodstream over time.

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