Serum proteomic analysis focused on fibrosis in patients with hepatitis C virus infection

Abstract

Background: Despite its widespread use to assess fibrosis, liver biopsy has several important drawbacks, including that is it semi-quantitative, invasive, and limited by sampling and observer variability. Non-invasive serum biomarkers may more accurately reflect the fibrogenetic process. To identify potential biomarkers of fibrosis, we compared serum protein expression profiles in patients with chronic hepatitis C (CHC) virus infection and fibrosis.

Methods: Twenty-one patients with no or mild fibrosis (METAVIR stage F0, F1) and 23 with advanced fibrosis (F3, F4) were retrospectively identified from a pedigreed database of 1600 CHC patients. All samples were carefully phenotyped and matched for age, gender, race, body mass index, genotype, duration of infection, alcohol use, and viral load. Expression profiling was performed in a blinded fashion using a 2D polyacrylamide gel electrophoresis/LC-MS/MS platform. Partial least squares discriminant analysis and likelihood ratio statistics were used to rank individual differences in protein expression between the 2 groups.

Results: Seven individual protein spots were identified as either significantly increased (alpha2-macroglobulin, haptoglobin, albumin) or decreased (complement C-4, serum retinol binding protein, apolipoprotein A-1, and two isoforms of apolipoprotein A-IV) with advanced fibrosis. Three individual proteins, haptoglobin, apolipoprotein A-1, and alpha2-macroglobulin, are included in existing non-invasive serum marker panels.

Conclusion: Biomarkers identified through expression profiling may facilitate the development of more accurate marker algorithms to better quantitate hepatic fibrosis and monitor disease progression.

Figure 1

Principal Components Analysis (PCA) of Two-dimensional Polyacrylamide Gel Electrophoresis Data. Gel images were processed using a commercially available analysis package followed by export into Microsoft Excel, normalization, log transformation, and PCA using Simca P+.

Figure 2

Spot Selection and Two-dimensional Map of Putative biomarkers. (a) Image analysis output from Progenesis showing the relative volume (pixel intensity * mm²) of smaller variant of ApoA4 (spot 846) across all experimental gels. Gel 52 was excluded from analysis due to aberrant isoelectric focusing. (b) Image of 2D PAGE showing positions of putative markers. The direction of change (advanced relative to moderate disease) is indicated by the white arrows, and the asterisk signifies proteins whose behavior is consistent with the commercially available FibroTest. Expression of albumin reflects fragments not adsorbed by affinity-based mimetic ligands.