Identification by NMR spectroscopy of urinary biomarkers of HCV cirrhosis and of systemic metabolic changes due to anti-viral HCV therapy

Hepatitis C is a hepatic infection caused by the hepatitis C virus (HCV). Globally, an estimated 71 million people have chronic hepatitis C infection and approximately 399,000 people die each year from hepatitis C, mostly from cirrhosis and hepatocellular carcinoma. Recent NMR-based metabolomics studies have been conducted on the serum of patients affected by hepatitis B related cirrhosis [1], and hepatitis C related fibrosis [2]. In both works the NMR technique proved to be crucial in differentiating the metabolic patterns of healthy individuals from those of cirrhotic and fibrotic patients. NMR-based metabolomics was also applied to urine of patients with HBV (hepatitis B), who developed hepatocellular carcinoma, successfully highlighting the differences between the urinary metabolic profile of cirrhotic patients and that of healthy controls [3]. In the present study the urinary metabolic profile of subjects with HCV-induced cirrhosis was analyzed by using NMR spectroscopy. The aim of this work was to define the characteristic metabolic profile of HCV-induced cirrhosis and to evaluate the metabolic changes due to viral replication inhibitor drugs. For this purpose, study on the urines of 43 fasting male subjects, aged between 43 and 82 years, was conducted, including 20 healthy subjects (Ctrl) and 23 affected by HCV cirrhosis. Patients were examined at the BT0 stage (Baseline Time 0) before treatment, at the EOT stage (End Of Treatment) at the end of the eight-week therapy, and at the FU1 and FU3 stage (Follow Up 1 and Follow Up 3 one month and three months after the end of treatment, respectively). The qualitative and quantitative determination of metabolites was carried out by mono- and bidimensional 1H-NMR analysis of urine. The comparison between the urinary metabolic profile of patients with liver cirrhosis HCV (genotype 1a and 1b) and healthy subjects was performed on 45 metabolites, 38 of which were identified, while 6 were classified as unassigned signals. Data from the Ctrl, BT0, EOT and FU experimental groups were analyzed by multivariate PLS-DA and univariate statistical analysis, that allowed to define the metabolic changes induced by cirrhosis from those related to the active phase of HCV infection. In addition, urinary metabolic biomarkers, linked to the systemic effect of the anti-HCV drugs (side effects) and long-term drug metabolic effects, were identified. This study represents the first investigation by NMR-based metabolomics on the metabolic changes occurring in cirrhosis patients with HCV over time during and after anti-viral therapy.