Increased Serum Levels of Pigment Epithelium-Derived Factor by Excessive Alcohol Consumption-Detection and Identification by a Three-Step Serum Proteome Analysis
Increased Serum Levels of Pigment Epithelium-Derived Factor by Excessive Alcohol Consumption-Detection and Identification by a Three-Step Serum Proteome Analysis
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DOI：
10.1111/j.1530-0277.2010.01336.x
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发表时间：
2011-02-01
影响因子：
3.2
通讯作者：
Nomura, Fumio
中科院分区：
文献类型：
--
作者：
Sogawa, Kazuyuki;Kodera, Yoshio;Nomura, Fumio
背景：在酒精实验和临床研究中，寻找酒精滥用的生物学标志物一直是人们感兴趣的问题。我们以前使用无凝胶蛋白质组分析方法，如ProteinChip (R)系统和ClinProt （TM）系统来寻找新的酒精中毒血清标记物，并发现了几个新的候选标记物。由于血清含有成千上万的蛋白质和多肽，这些蛋白质和多肽以大的动态浓度存在，因此需要耗尽丰富的蛋白质并进一步分离剩余的蛋白质，以进入深层蛋白质组。我们最近描述了一种简单且高度可重复性的三步方法，用于在低丰度血清蛋白中识别潜在的疾病标志物候选物。方法：对8例酒精依赖患者进行入院时和戒酒8周后的血清采集。对样品进行三步血清蛋白质组分析。步骤如下：首先，免疫消耗6个最丰富的蛋白；二是采用反相高效液相色谱法进行分馏；采用一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离。蛋白质染色显示的差异通过Western blotting和酶联免疫吸附试验（ELISA）进一步证实。结果：三步血清蛋白质组学分析显示，入院时血清5种蛋白水平，α 2- hs糖蛋白、载脂蛋白A-I、谷胱甘肽过氧化物酶3、肝素辅助因子II和色素上皮衍生因子（PEDF）明显高于戒断8周后。我们之所以关注PEDF，是因为我们还不清楚酒精受试者中PEDF水平的变化。Western blotting和ELISA证实PEDF表达上调。中度至重度饮酒者血清PEDF水平（14.2 +/- 7.7 μ g/ml）显著高于无饮酒史的健康受试者（5.5 +/- 3.0 μ g/ml） （p < 0.001）。非酒精性慢性肝病患者的血清PEDF水平与健康受试者的PEDF水平相当。结论：三步血清蛋白质组分析显示过量饮酒可使PEDF水平升高。
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 (R) system and the ClinProt (TM) 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 mu g/ml) than in healthy subjects without a drinking history (5.5 +/- 3.0 mu g/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.