Publikationsdatenbank

Identification of metabolic prognostic markers in liver diseases after carbon tetrachloride intoxication (2018)

Art

Hochschulschrift

Autor

Hofney Othman, Amnah (WE 2)

Quelle

Berlin: Mensch und Buch Verlag, 2018 — iv, 87 Seiten

ISBN: 978-3-86387-912-9

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/22755

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Chronic liver disease is a common clinical problem. Currently, the diagnosis is based on non-invasive techniques like e.g. fibroscan, MRI, elastography, on minimal-invasive techniques like e.g. liver transaminases, and on the invasive biopsy method. However, these aforementioned methods have several drawbacks. Briefly, non-invasive methods are usually not-sensitive to early chronic liver disease; whereas blood based liver enzymes are not specific for liver. Therefore, the gold standard for the diagnosis of hepatic diseases is the histological assessment of liver biopsies. The use of liver biopsies is, however, subject to sampling- as well as inter- and intra-observer variability. These limitations affect the diagnostic accuracy, reliability, and responsiveness of treatment end-points. Moreover, liver biopsies are risky for the patient because of potential bleeding, organ perforation and even death. Thus, an urgent medical need exists for reliable and highly accurate treatment end-point indicators that can replace liver biopsies. In this thesis the use of metabolite screening for diagnosis and staging is suggested to be used. It is well accepted that acute and chronic intoxications alter the regulation of liver metabolism at different levels. The blood or urine metabolome should therefore represent the final outcome of liver cellular regulation and the phenotype of a disease. The scope of this thesis is to establish and to validate marker(s) based on metabolite levels, both for the early diagnosis of liver diseases and for the prediction of liver disease progression. To achieve this goal, a metabolite screening for diagnosis and staging was performed to identify the presence and severity of inflammation and fibrosis in murine livers and blood. To characterize the metabolic alterations upon acute and chronic liver intoxications, nuclear magnetic resonance (NMR) spectroscopy was employed. NMR is fully quantitative, highly reproducible, detects all metabolites simultaneously in one non selective measurment, it is non-invasive, non-destructive and allows for a complete recovery of the sample. Moreover, just a minimum of sample preparation with no need for derivatization is required. In order to induce acute toxic liver injuries, male C57BL/6N mice exposed to a single dose of CCl4 were used. Moreover, chronic liver damage was induced by repeated injections of CCl4 for 6 weeks. At specific time points both blood and livers of the mice were harvested. A control group received the same dose of just olive oil. Three to four mice were used for each group. Further, MDR2-/- mice (6 months old) were treated with Ly2157299 (Galunisertib; ALK5 inhibitor). ALK5 (TGFβ type I receptor kinase) was used to interfere with TGFβ signalling in MDR2-/- mice. Two groups of mice were used. Ly2157299 treated mice for 14 consecutive days (150 mg/kg) orally. The vehicle treated group received the same amount for 14 days orally. Two days after the last injection, both blood and livers were harvested. Biochemical parameters e.g. ALT and AST increased during disease progression upon CCl4 injection. This alteration was correlated with accumulation of extracellular matrix (ECM) and ECM-producing cells as indicated by picro-sirius red and alpha-SMA staining, respectively. These data give us a deep insight into the test models, where both acute and chronic liver damage were confirmed. Blood plasma-based metabolic profiling was performed. Surprisingly, some metabolites e.g. acetate, alanine, formate, glucose, glutamine, lactate and phenylalanine are considered very good predictors for chronic liver diseases - regardless to spatial (septal or biliary fibrosis) extracellular matrix- as indicated by AUC approximately 1.000. However, in the septal CCl4-induced fibrosis model betaine, citrate, glutamine and phenylalanine, however, in biliary fibrosis 3-Hydroxyisobutyrate, formate, glutamate, glycine, methionine and tyrosine can be used to stage the disease. These differences in metabolite levels can be explained by hepatic zonation of extracellular matrix deposition. It is generally accepted that in CCl4 induced injury, glutamine synthesis, glycolysis, tricarboxylic acid cycle and phase I as well as phase II drug metabolism are principally altered. Whereas urea formation, gluconeogenesis and oxidative phosphorylation are mainly affected in case of biliary fibrosis, further validation in clinical cohorts is absolutely mandatory and will be a followup story. My study shed light on the importance of the metabolites identified in septal and biliary fibrosis: i) Validation in clinical cohorts of circulatory based liver disorders (septal fibrosis) and cholestasis (biliary fibrosis); this provides the scientists working in the field of liver diseases with a list of biomarkers; ii) Molecular analysis of chronic liver disease progression and comparing this blood based metabolic profiling with liver tissue to mechanistically understand disease dynamics; and iii) Providing metabolism scientists with quantitative data for single metabolites in a time-dependent manner for further functional analysis.