Combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats.
BACKGROUND
Proteomic approaches may provide new insights into pathological conditions associated with alcoholism. The aim of this study was to conduct a proteomic analysis of liver tissue and serum in chronically alcohol-fed rats using agarose 2-dimensional gel electrophoresis (2-DE) and 3-step serum proteome analysis.


METHODS
A total of 12 rats were pair-fed nutritionally adequate liquid diet containing ethanol as 36% of the total energy or an isocaloric control diet for 2 months. Rat liver homogenates and cytosol fractions were subjected to agarose 2-DE. Serum samples were subjected to 3-step serum proteome analysis involving immunodepletion of abundant proteins followed by fractionation using reverse-phase high-performance liquid chromatography and 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Candidate proteins were digested with trypsin and identified using mass spectrometry. Observed differences in protein expression levels were confirmed using Western blotting.


RESULTS
A total of 46 protein spots were found to be differentially expressed in the liver homogenates and cytosol fractions of alcohol-fed rats relative to pair-fed controls. The most notable change was down-regulation of a 29-kDa protein, which was subsequently identified as carbonic anhydrase III (CA III). Down-regulation of this protein in alcohol-fed rats was confirmed by Western blotting. The messenger RNA level of CA III was decreased as well. In rat serum, a total of 41 proteins were differentially expressed. Of these proteins, only betaine-homocysteine methyltransferase (BHMT) was also found to be differentially expressed in the liver.


CONCLUSIONS
A combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats revealed that the expression of CA III is significantly down-regulated in the liver of alcohol-fed rats. Our results also showed that BHMT expression is up-regulated in both the liver and serum of alcohol-fed rats.
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
Mako
M
Yamada
Mamoru
M
Satoh
Masanori
M
Seimiya
Kazuyuki
K
Sogawa
Sakae
S
Itoga
Takeshi
T
Tomonaga
Fumio
F
Nomura
D000818:Animals; D050940:Betaine-Homocysteine S-Methyltransferase / Q000096:biosynthesis / Q000097:blood; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D024403:Carbonic Anhydrase III / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000097:blood; D015536:Down-Regulation / Q000187:drug effects; D000431:Ethanol / Q000008:administration & dosage*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation / Q000187:drug effects
D015415:Biomarkers; D000431:Ethanol; D050940:Betaine-Homocysteine S-Methyltransferase; C494550:Bhmt protein, rat; D024403:Carbonic Anhydrase III