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

Authors

  • Kazuyuki Sogawa,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Yoshio Kodera,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Mamoru Satoh,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Yusuke Kawashima,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Hiroshi Umemura,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Katsuya Maruyama,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Hirotaka Takizawa,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Osamu Yokosuka,

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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  • Fumio Nomura

    1. From the Clinical Proteomics Research Center (KS, YK, MS, YK, FN), Chiba University Hospital, Chiba, Japan; Department of Physics (YK), School of Science, Kitasato University, Kanagawa, Japan; Department of Molecular Diagnosis (HU, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; National Hospital Organization Kurihama Alcoholism Center (KM), Kanagawa, Japan; Kashiwado Clinic in Port-Square (HT), Kashiwado Memorial Foundation, Chiba, Japan; and Division of Gastroenterology (OY), Chiba University Hospital, Chiba, Japan.
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Reprint requests: Fumio Nomura, MD, PhD, Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba 260-8670, Japan; Fax: +81-43-226-2324; E-mail: fnomura@faculty.chiba-u.jp

Abstract

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 8 weeks 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 8 weeks 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 ± 7.7 μg/ml) than in healthy subjects without a drinking history (5.5 ± 3.0 μg/ml) (< 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.

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