Abstract
Steatosis is the accumulation of triglycerides in hepatic cells wherein fats exceed 5% of the entire liver weight. Although steatotic liver damage is reversible due to the liver's regenerative capability, protracted damage often and typically leads to irreversible conditions such as cirrhosis and hepatocellular carcinoma (HCC). Therefore, early steatotic detection is critical for preventing progression to advanced liver diseases. This also becomes particularly important given the higher prevalence of drug usage, as drugs are a frequent cause of liver damage. Currently, the recommendation to diagnose steatosis is using liver enzymes and performing a liver biopsy. Liver biopsy remains the gold standard method of detection, but the procedure is invasive and an unreliable diagnostic tool. Non-invasive, specific and sensitive diagnostic solutions such as biomarkers are therefore needed for the early detection of steatosis. Our aim is to identify changes in urinary metabolites in tetracycline-induced hepatic steatotic rats at different stages of the diseases using metabolomic-based techniques. Sprague Dawley male rats are treated by intraperitoneal injection (I.P.) with either 62.5 mg kg−1 or 125 mg kg−1 tetracycline, an antibiotic previously known to induce steatosis. We analyse the metabolic profile of the urinary tetracycline induced hepatic steatotic rats using 1H nuclear magnetic resonance (NMR), 2D 1H–1H TOCSY (total correlation spectroscopy) and electrospray liquid chromatography-mass spectrometry (ESI-LC-MS/MS) based metabolomics. The combined analysis of haematoxylin & eosin (H&E), oil red O (ORO) and direct measurement of triglyceride content in the liver tissues of the control samples against 125 mg kg−1 and 62.5 mg kg−1 treated samples, reveals that 125 mg kg−1 tetracycline exposure potentially induces steatosis. The combination of 1H NMR, 2D 1H–1H TOCSY and ESI-LC-MS/MS alongside multivariate statistical analysis, detected a total of 6 urinary metabolites changes, across 6 metabolic pathways. Furthermore, lysine concentration correlates with liver damage as tetracycline dose concentration increases, whilst both H&E and ORO fail to detect hepatocellular damage at the lowest dose concentration. We conclude that the combination of 1H NMR and ESI-LC-MS/MS suggests that these are suitable platforms for studying the pathogenesis of steatosis development, prior to morphological alterations observed in staining techniques and offer a more detailed description of the severity of the steatotic disease.
Highlights
Ultrasound is commonly used in detecting hepatic steatosis and can provide grading of the hepatic fat content based on the visual assessment.[13]
In the livers of rats treated with 125 mg kgÀ1 tetracycline haematoxylin & eosin (H&E) stained photomicrographs revealed changes in the lobular architecture, whilst rats treated with a lower dose (62.5 mg kgÀ1) displayed architecture similar those of control animals
Data from our 1H nuclear magnetic resonance (NMR) and Electronic supplementary information (ESI)-LC-MS/MS was able to distinguishing between controls, low dose and high dose treated animals
Summary
Non-alcoholic fatty liver disease (NAFLD) has become one of the leading causes of chronic liver disease globally.[1,2,3] NAFLD represents a spectrum of diseases ranging from simple steatosis progressing to nonalcoholic steatohepatitis (NASH), brosis, cirrhosis and hepatocellular carcinoma (HCC).[4,5]Steatosis, known as fatty liver, is caused by hepatic cells retaining abnormal levels of triglycerides, wherein fats exceed 5% of the entire liver weight.[2,5,6,7,8] Whilst damage here is typically reversible given the liver's high regenerative capability,[9] early detection remains critical for preventing progression towards irreversible conditions, which include cirrhosis and HCC.[4,5,10]Abnormal enzyme liver function tests (serum aminotransferase enzymes) and non-invasive methods (ultrasound or 1Hmagnetic-resonance spectroscopy) are used to diagnose liver disease such as steatosis.[11]. Non-alcoholic fatty liver disease (NAFLD) has become one of the leading causes of chronic liver disease globally.[1,2,3] NAFLD represents a spectrum of diseases ranging from simple steatosis progressing to nonalcoholic steatohepatitis (NASH), brosis, cirrhosis and hepatocellular carcinoma (HCC).[4,5]. Known as fatty liver, is caused by hepatic cells retaining abnormal levels of triglycerides, wherein fats exceed 5% of the entire liver weight.[2,5,6,7,8] Whilst damage here is typically reversible given the liver's high regenerative capability,[9] early detection remains critical for preventing progression towards irreversible conditions, which include cirrhosis and HCC.[4,5,10]. Ultrasound is commonly used in detecting hepatic steatosis and can provide grading of the hepatic fat content based on the visual assessment.[13] it is difficult to
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