Novel Serum Biomarker Candidates for Liver Fibrosis in Hepatitis C Patients Bevin Gangadharan, * Robin Antrobus, Raymond A. Dwek, and Nicole Zitzmann Background: Liver biopsy is currently the gold standard for assessing liver fibrosis, and no reliable noninvasive diagnostic approach is available. Therefore a suitable serologic biomarker of liver fibrosis is urgently needed. Methods: We used a proteomics method based on 2-dimensional gel electrophoresis to identify potential fibrosis biomarkers. Serum samples from patients with varying degrees of hepatic scarring induced by infection with the hepatitis C virus (HCV) were analyzed and compared with serum from healthy controls. Results: We observed the most prominent differences when we compared serum samples from cirrhotic pa- tients with healthy control serum. Inter--trypsin inhib- itor heavy chain H4 (ITIH4) fragments, 1 antichymo- trypsin, apolipoprotein L1 (Apo L1), prealbumin, albumin, paraoxonase/arylesterase 1, and zinc-2-glyco- protein were decreased in cirrhotic serum, whereas CD5 antigen-like protein (CD5L) and 2 glycoprotein I (2GPI) were increased. In general, 2 macroglobulin (a2M) and immunoglobulin components increased with hepatic fibrosis, whereas haptoglobin and complement components (C3, C4, and factor H-related protein 1) decreased. Novel proteins associated with HCV-induced fibrosis included ITIH4 fragments, complement factor H-related protein 1, CD5L, Apo L1, 2GPI, and thio- ester-cleaved products of a2M. Conclusions: Assessment of hepatic scarring may be performed with a combination of these novel fibrosis biomarkers, thus eliminating the need for liver biopsy. Further evaluation of these candidate markers needs to be performed in larger patient populations. Diagnosis of fibrosis during early stages will allow early treat- ment, thereby preventing fibrosis progression. © 2007 American Association for Clinical Chemistry Chronic hepatic disease damages the liver, and the result- ing wound-healing process can lead to liver fibrosis and the subsequent development of cirrhosis. One of the leading causes of hepatic fibrosis and cirrhosis is infection with the hepatitis C virus (HCV) 1 . HCV is a major human pathogen, infecting more than 170 million individuals, approximately 3% of the world’s population (1). Approx- imately 70% of those infected become chronic carriers and are at severe risk of developing liver fibrosis and cirrhosis. Of the patients with HCV-induced cirrhosis, 2%–5% de- velop hepatocellular carcinoma (HCC) (1). Hepatic fibrosis is characterized by the excessive buildup of scar tissue in the liver. This accumulation in extracellular matrix (ECM) changes hepatic morphology. Collagens are the primary components of the ECM in fibrosis. Other ECM components, including laminin, hya- luronan, elastin, and fibronectin, also increase (2). As fibrotic liver diseases advance, cirrhosis occurs and the liver becomes nodular. Expression of collagens can in- crease up to 10-fold in cirrhosis (3). Kupffer cells (liver macrophages) in the blood pass through the hepatic sinusoids and in fibrosis become activated by adverse stimuli such as viruses or toxins. Cytokines such as transforming growth factor 1 (TGF- 1) are released from the Kupffer cells, which activate the cells responsible for ECM production. These hepatic stel- late cells (HSCs) also produce TGF-1 and other cytokines as well as angiotensin II. These profibrogenic factors, along with hepatocyte growth factor (HGF) in the ECM, further activate HSCs in hepatic scarring (2–4). Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom. * Address correspondence to this author at: Oxford Antiviral Drug Dis- covery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Rd., Oxford, OX1 3QU, United Kingdom. Fax 44-1865-275716; e-mail Bevin.Gangadharan@bioch.ox.ac.uk. Received March 19, 2007; accepted July 24, 2007. Previously published online at DOI: 10.1373/clinchem.2007.089144 1 Nonstandard abbreviations: HCV, hepatitis C virus; HCC, hepatocellular carcinoma; ECM, extracellular matrix; TGF-1, transforming growth factor 1; HSC, hepatic stellate cell; HGF, hepatocyte growth factor; 2D, 2 dimensional; MS, mass spectroscopy; MS/MS, tandem MS; ITIH4, inter--trypsin inhibitor heavy chain H4; Apo L1, apolipoprotein L1; CD5L, CD5 antigen-like protein; 2GPI, 2 glycoprotein I; a2M, 2 macroglobulin. Clinical Chemistry 53:10 1792–1799 (2007) Proteomics and Protein Markers 1792