Increased Serum Levels of Pigment Epithelium-Derived Factor by Excessive Alcohol Consumption-Detection and Identification by a Three-Step Serum Proteome Analysis

Background: The search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel-free proteome analysis methods such as the ProteinChip^® system and the ClinProt™ system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three-step method for identifying potential disease-marker candidates among the low-abundance serum proteins. Methods: Two serum samples-one on admission and one after 8weeks of abstinence-were obtained from 8 patients with alcohol dependency. The samples were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme-linked immunosorbent assays (ELISA). Results: Three-step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2-HS glycoprotein, apolipoprotein A-I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial-derived factor (PEDF), were significantly greater on admission than after 8weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2±7.7μg/ml) than in healthy subjects without a drinking history (5.5±3.0μg/ml) (p<0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects. Conclusion: Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.