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 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) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects.


CONCLUSIONS
Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.
Clinical Proteomics Research Center, Chiba University Hospital, Japan.
Kazuyuki
K
Sogawa
Yoshio
Y
Kodera
Mamoru
M
Satoh
Yusuke
Y
Kawashima
Hiroshi
H
Umemura
Katsuya
K
Maruyama
Hirotaka
H
Takizawa
Osamu
O
Yokosuka
Fumio
F
Nomura
D000428:Alcohol Drinking / Q000097:blood*; D000437:Alcoholism / Q000097:blood*; D015415:Biomarkers / Q000097:blood*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000097:blood*; D005260:Female; D006801:Humans; D008297:Male; D009414:Nerve Growth Factors / Q000097:blood*; D020543:Proteome / Q000032:analysis*; D015843:Serpins / Q000097:blood*; D000099174:Pigment Epithelium-Derived Factor
D015415:Biomarkers; D005136:Eye Proteins; D009414:Nerve Growth Factors; D020543:Proteome; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor