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Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis.

ABSTRACT: 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 ?/? 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.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.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 GST? 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.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.

SUBMITTER: Shearn CT

PROVIDER: S-EPMC4762037 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis.

Shearn C T CT Orlicky D J DJ Saba L M LM Shearn A H AH Petersen Dennis R DR

Free radical biology & medicine 20151027

<h4>Objective</h4>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 p ...[more]

PMID: 26518673

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Project description:OBJECTIVE:In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease is oxidative stress leading to the accumulation of highly reactive electrophilic ?/? unsaturated aldehydes. The objective of this study was to determine if significant differences were evident when evaluating carbonylation in human end-stage fatty nonalcoholic steatohepatitis (fNASH) compared to end-stage nonfatty NASH (nfNASH). METHODS:Using hepatic tissue obtained from healthy humans and patients diagnosed with end stage nfNASH or fNASH, overall carbonylation was assessed by immunohistochemistry (IHC) and LC-MS/MS followed by bioinformatics. RESULTS:Picrosirius red staining revealed extensive fibrosis in both fNASH and nfNASH which corresponded with increased reactive aldehyde staining. Although significantly elevated when compared to normal hepatic tissue, no significant differences in overall carbonylation and fibrosis were evident when comparing fNASH with nfNASH. Examining proteins that are critical for anti-oxidant defense revealed elevated expression of thioredoxin, thioredoxin interacting protein, glutathione S-transferase p1 and mitochondrial superoxide dismutase in human NASH. As important, using immunohistochemistry, significant colocalization of the aforementioned proteins occurred in cytokeratin 7 positive cells indicating that they are part of the ductular reaction. Expression of catalase and Hsp70 decreased in both groups when compared to normal human liver. Mass spectrometric analysis revealed a total of 778 carbonylated proteins. Of these, 194 were common to all groups, 124 unique to tissue prepared from healthy individuals, 357 proteins exclusive to NASH, 124 proteins distinct to samples from patients with fNASH and 178 unique to nfNASH. Using functional enrichment analysis of hepatic carbonylated proteins revealed a propensity for increased carbonylation of proteins regulating cholesterol and Huntington's disease related pathways occurred in nfNASH. Examining fNASH, increased carbonylation was evident in proteins regulating Rho cytoskeletal pathways, nicotinic acetylcholine receptor signaling and chemokine/cytokine inflammatory pathways. Using LC-MS/MS analysis and trypsin digests, sites of carbonylation were identified on peptides isolated from vimentin, endoplasmin and serum albumin in nfNASH and fNASH respectively. CONCLUSIONS:These results indicate that cellular factors regulating mechanisms of protein carbonylation may be different depending on pathological diagnosis of NASH. Furthermore these studies are the first to use LC-MS/MS analysis of carbonylated proteins in human NAFLD and explore possible mechanistic links with end stage cirrhosis due to fatty liver disease and the generation of reactive aldehydes.

Project description:Background & aimsHepatocellular carcinoma (HCC) screening of patients with cirrhosis is recommended by professional societies to increase detection of early stage tumors and survival, but is underused in clinical practice.MethodsWe conducted a retrospective cohort study of 13,714 patients diagnosed with HCC from 2003 through 2013 included in the Surveillance, Epidemiology, and End Results Program-Medicare database. We characterized receipt of HCC screening in the 3 years before HCC diagnosis using mutually exclusive categories (consistent vs inconsistent vs no screening) and the proportion of time covered with screening. Correlates for screening receipt were assessed using a multivariable 2-part regression model. We examined the association between screening receipt and early detection of tumors using multivariable logistic regression. We evaluated associations between screening receipt and overall survival using a Cox proportional hazards model, after adjustments for effects of lead-time bias and length-time bias on survival rate estimators.ResultsMost patients with cirrhosis (51.1%) did not receive any screening in the 3 years before a diagnosis of HCC, and only 6.8% of patients underwent consistent annual screening. The proportion with consistent screening increased from 5.4% in 2003 to 2006 to 8.8% in 2011 to 2013 (P < .001). The mean proportion of time covered was 13.4% overall, which increased from 11.7% in 2003 to 2006 to 15.2% in 2011 to 2013. Receipt of consistent screening was associated with detection of early stage tumors (odds ratio, 1.98; 95% CI, 1.68-2.33) and a reduced risk of death after correction for lead-time bias (hazard ratio, 0.76; 95% CI, 0.70-0.83). Inconsistent screening was associated with a slightly smaller increase in early detection of HCC (odds ratio, 1.31; 95% CI, 1.20-1.43) and a reduced risk of death (hazard ratio, 0.86; 95% CI, 0.83-0.90). After correction for lead- and length-time biases, higher proportions of patients with consistent (23%; 95% CI, 21%-25%) and inconsistent screening (19%; 95% CI, 19%-20%) survived for 3 years compared with patients without screening (13%; 95% CI, 12%-14%).ConclusionsIn an analysis of the Surveillance, Epidemiology, and End Results Program-Medicare database, we found HCC screening to be underused for patients with cirrhosis. This contributes to detection of liver tumors at later stages and shorter times of survival. However, the proportion of patients screened for HCC has increased over time.

Dataset Information

Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis.

Publications

Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis.

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