title: Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis.
abstract: OBJECTIVE
  Oxidative stress is a significant contributing factor in the pathogenesis of alcoholic liver disease (ALD). In the murine models of chronic alcohol consumption, induction of oxidative stress results in increased peroxidation of polyunsaturated fatty acids to form highly reactive electrophilic α/β unsaturated aldehydes that post-translationally modify proteins altering activity. Data are presented here suggesting that oxidative stress and the resulting carbonylation of hepatic proteins is an ongoing process involved in alcohol-induced cirrhosis.
  
  
  METHODS
  Using age-matched pooled hepatic tissue obtained from healthy humans and patients with end stage cirrhotic ALD, overall carbonylation was assessed by immunohistochemistry and LC-MS/MS of streptavidin purified hepatic whole cell extracts treated with biotin hydrazide. Identified carbonylated proteins were further evaluated using bioinformatics analyses.
  
  
  RESULTS
  Using immunohistochemistry and Western blotting, protein carbonylation was increased in end stage ALD occurring primarily in hepatocytes. Mass spectrometric analysis revealed a total of 1224 carbonylated proteins in normal hepatic and end-stage alcoholic cirrhosis tissue. Of these, 411 were unique to cirrhotic ALD, 261 unique to normal hepatic tissue and 552 common to both groups. Bioinformatic pathway analysis of hepatic carbonylated proteins revealed a propensity of long term EtOH consumption to increase post-translational carbonylation of proteins involved in glutathione homeostatic, glycolytic and cytoskeletal pathways. Western analysis revealed increased expression of GSTA4 and GSTπ in human ALD. Using LC-MS/MS analysis, a nonenaldehyde post-translational modification was identified on Lysine 235 of the cytoskeletal protein vimentin in whole cell extracts prepared from human end stage ALD hepatic tissue.
  
  
  CONCLUSIONS
  These studies are the first to use LC-MS/MS analysis of carbonylated proteins in human ALD and begin exploring possible mechanistic links with end-stage alcoholic cirrhosis and oxidative stress.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver Anschutz Medical Campus, 12850 East Montview Blvd Box C238, Building V20 Room 2131, Aurora, CO 80045, United States. Electronic address: Colin.Shearn@ucdenver.edu.
authors.forename: C T
authors.initials: CT
authors.lastname: Shearn
authors.affiliation: Department of Pathology, School of Medicine, University of Colorado Denver Anschutz Medical Campus, Aurora, CO 80045, United States.
authors.forename: D J
authors.initials: DJ
authors.lastname: Orlicky
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver Anschutz Medical Campus, 12850 East Montview Blvd Box C238, Building V20 Room 2131, Aurora, CO 80045, United States.
authors.forename: L M
authors.initials: LM
authors.lastname: Saba
authors.affiliation: Alpine Achievement Systems, Inc., 9635 Maroon Circle, Suite 120, Englewood, CO 80112, United States.
authors.forename: A H
authors.initials: AH
authors.lastname: Shearn
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver Anschutz Medical Campus, 12850 East Montview Blvd Box C238, Building V20 Room 2131, Aurora, CO 80045, United States. Electronic address: Dennis.Petersen@ucdenver.edu.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Petersen
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000737:chemistry* / Q000378:metabolism*; D015153:Blotting, Western; D016022:Case-Control Studies; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006801:Humans; D008104:Liver Cirrhosis, Alcoholic / Q000175:diagnosis* / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D050050:Protein Carbonylation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D015415:Biomarkers
keywords: 4-HNE; Alcoholic liver disease; Liver; Oxidative stress; Reactive aldehydes