CBX788417 1..9 Research Article A proteomic approach of biomarker candidate discovery for alcoholic liver cirrhosis Krishna Sumanth Nallagangula 1 , V Lakshmaiah 2 , C Muninarayana 3 , KV Deepa 4 , and KN Shashidhar 1 Abstract Alcoholic liver disease (ALD) progresses from steatosis to alcoholic hepatitis to fibrosis and cirrhosis. Liver biopsy is considered as the gold standard method for diagnosis of liver cirrhosis and provides useful information about damaging process which is an invasive procedure with complications. Existing biomarkers in clinical practice have narrow applicability due to lack of specificity and lack of sensitivity. The objective of this article is to identify proteomic biomarker candidates for alcoholic liver cirrhosis by differential expression analysis between alcoholic liver cirrhotic and healthy subjects. Blood samples were collected from 20 subjects (10 alcoholic liver cirrhosis and 10 healthy) from R. L. Jalapa Hospital and Research Centre, Kolar, Karnataka, India. Differential protein analysis was carried out by two-dimensional electrophoresis after albumin depletion, followed by liquid chromatography–mass spectrometry. The image analysis found 46 spots in cirrhotic gel and 69 spots in healthy gel, of which 14 spots were identified with significant altered expression levels. Based on the protein score and clinical significance, among 14 spots, a total of 28 protein biomarker candidates were identified: 13 with increased expression and 15 with decreased expression were categorized in alcoholic liver cirrhosis compared to healthy subjects. Protein biomarker candidates identified by “-omics” approach based on differential expression between alcoholic liver cirrhotic subjects and healthy subjects may give better insights for diagnosis of ALD. Prioritization of candidates identified is a prerequisite for validation regimen. Biomarker candidates require verification that demonstrates the differential expression will remain detectable by assay to be used for validation. Keywords Alcoholic liver cirrhosis, protein biomarker candidates, albumin depletion, two-dimensional electrophoresis, liquid chromatography–mass spectrometry Date received: 13 March 2018; accepted: 15 June 2018 Introduction Cirrhosis of the liver is the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury, leading to portal hyperten- sion and end-stage liver disease. Causes of cirrhosis of the liver are multifactorial. Despite varied etiology, the patho- logical characteristics which result in liver dysfunction are common. 1 In recent years, alcohol consumption has corre- lated with deaths from asymptomatic and self-limited fatty liver to cirrhosis of the liver. Alcoholic liver disease (ALD) progresses from steatosis to alcoholic hepatitis to fibrosis 1 Department of Biochemistry, Sri Devaraj Urs Medical College, SDUAHER, Kolar, Karnataka, India 2 Department of Medicine, Sri Devaraj Urs Medical College, SDUAHER, Kolar, Karnataka, India 3 Department of Community Medicine, Sri Devaraj Urs Medical College, SDUAHER, Kolar, Karnataka, India 4 Centre for Cellular and Molecular Platforms, GKVK Campus, Bengaluru, Karnataka, India Corresponding Author: KN Shashidhar, Department of Biochemistry, Sri Devaraj Urs Medical College, SDUAHER, Tamaka, Kolar, Karnataka, India. Email: drshashikn1971@yahoo.co.in Journal of Circulating Biomarkers Volume 7: 1–9 ª The Author(s) 2018 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1849454418788417 journals.sagepub.com/home/cbx Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:drshashikn1971@yahoo.co.in https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418788417 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage and frank cirrhosis (micronodular, occasionally mixed micronodular, and macronodular) and often occurs acutely against background chronic liver disease. 2 Multifactorial pathogenesis plays a role in progression of the disease. Accumulation of triacylglycerol in liver is an early and reversible effect of alcohol which increases peripheral lipo- lysis and alters liver redox potential leading to fatty acid synthesis. In ALD, generated prooxidants enhance antiox- idant system results in lipid peroxidation. 3 Acetaldehyde generated from alcohol is highly reactive and toxic; it binds to phospholipids and amino acids, result- ing in the formation of abnormal folding of proteins in endoplasmic reticulum (ER) leading to ER stress. 3 Prolif- eration and activation of hepatic stellate cells (HSCs) in ALD are induced by Kupffer cells and hepatocytes. Chief mitogen for the activation of HSCs is platelet-derived growth factor, which is produced by Kupffer cells. Kupffer cells induce collagen synthesis through the production of transforming growth factor b, tumor necrotic factor a (TNF-a), and reactive oxygen species (ROS). Activated HSCs migrate and accumulate at tissue repair sites and secrete large amounts of extracellular matrix (ECM). HSCs collagen synthesis is regulated at transcription and post- transcriptional levels. Dysregulation of cytokine metabo- lism and activity is vital for alcohol-induced liver damage. TNF-a, a pro-inflammatory cytokine, is one of the key factors for pathophysiology of ALD. Hepatocytes induce fibrosis through the production of ROS or apoptosis fol- lowed by regenerative nodular formation. 3,4 Diagnosing the degree of disease is a crucial step for successful management of ALD. Despite the development of potential diagnostic tests for the past 50 years, liver biopsy is considered as the gold standard method for diag- nosis of liver cirrhosis and provides useful information about damaging process, namely, steatosis, lobular inflam- mation, periportal fibrosis, Mallory bodies, nuclear vacuo- lation, bile ductal proliferation, and fibrosis/cirrhosis. 2,5 Limitation of liver biopsy is highly invasive; poor sample quality and tissue size make biopsy nonreproducible, and it depends on the experience of the pathologist leading to interobserver variations. Risk allied for liver biopsy range from pain (84%) and hypertension, bleeding (0.5%), and damage to biliary system with approximately 0.01% mor- tality rate. 6 No single biomarker can establish alcohol to be the etiology of liver disease; existing biomarkers in clinical practice have narrow applicability due to lack of specificity and lack of sensitivity (distinguish intermediate stages) which prevent reliance on any single biomarker. 2,7 An ideal biomarker should be organ specific, a sensitive indicator for active damage, easily accessible in peripheral tissue, and cost-effective and should give insights for diag- nosis, monitor the activity of disease, and assess therapeu- tic response. The determination of biomarkers could be an easy, noninvasive, and inexpensive method to monitor the progression of liver disease. This leads to urgency in the progression of biomarker discovery for cirrhosis of the liver with the help of technological advancement in “-omics” approach. Discovery of biomarker candidates should be a simplified, unbiased, semiquantitative binary comparison between diseased and normal. 8 Although individual sample analysis is recommended, pooling strategy with definite selection criteria from multiple individuals reduces sample number and cost. 9 Alterations in protein domain due to ALD which enters into circulation hold good for discovery of biomarker candidates. In the present study, we tried to discover protein biomarker candidates for alcoholic liver cirrhosis whose concentration may be altered due to changes in translation, posttranslational modifications, and/or degradation using two-dimensional electrophoresis (2DE) after albumin depletion, followed by liquid chroma- tography–mass spectrometry (LC-MS). Materials and methods Samples Blood samples were collected from 20 subjects: 10 clini- cally and diagnostically proven alcoholic liver cirrhotic subjects with varying degree and age- and gender- matched 10 healthy subjects (Table 1) from R. L. Jalappa Hospital and Research Centre, attached to Sri Devaraj Urs Medical College, a constituent of Sri Devaraj Urs Acad- emy of Higher Education, Kolar, Karnataka, India. Indi- viduals diagnosed with cirrhosis of the liver caused by ALD based on clinical history and symptoms, namely, ascites, encephalopathy, jaundice, and altered biochem- ical parameters, were included in the study. Individuals with diabetes and/or its complications, myocardial infarc- tion, acute and chronic renal failure, pneumonia, and can- cer were excluded from the study. Collection of blood samples from cirrhotic liver subjects and healthy subjects was carried out after obtaining informed consent, and the study is approved by Institutional Ethical Committee (DMC/KLR/IEC/61/2016-17). Table 1. Details of 20 blood samples (10 alcoholic liver cirrhotic subjects and age- and gender-matched 10 healthy subjects) used for discovery of biomarker candidates by proteomic approach. Sample ID Gender Age Etiology Sample ID Gender Age Etiology C1 M 36 NA D1 M 36 ALD C2 M 28 NA D2 M 28 ALD C3 M 62 NA D3 M 62 ALD C4 M 36 NA D4 M 36 ALD C5 M 35 NA D5 M 35 ALD C6 M 40 NA D6 M 40 ALD C7 M 70 NA D7 M 70 ALD C8 M 30 NA D8 M 30 ALD C9 M 62 NA D9 M 62 ALD C10 M 30 NA D10 M 30 ALD C: control; D: diseased (alcoholic liver cirrhosis); M: male; NA: not appli- cable; ALD: alcoholic liver disease. 2 Journal of Circulating Biomarkers Serum separation Serum was collected from clotted blood using serum separator tubes centrifuged at 4000 r/min for 10 min. Serum was stored at �20�C till further analysis. All sam- ples were used for discovery of protein biomarker candi- dates after depletion of abundant albumin. Desalting was carried out by acetone precipitation. 2DE was carried out to find differentially expressed proteins between cirrhotic and healthy subjects. Identified spots were characterized by LC-MS after in-gel trypsin digestion. Reagents Dye-based (Cibacron blue) prefractionation albumin depletion kit was procured from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Precast gels and other chemicals of analytical grade for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2DE, in- gel trypsin digestion, and LC-MS were procured from Bio- Rad (Hercules, California, USA) and Sigma Aldrich (St Louis, Missouri, USA). Depletion of albumin Resuspended resin (200 mL aliquot of resin) was transferred into spin column (column volume: 900 mL; 10-mm pore- size polyethylene filter). Bottom of the column was twisted off and placed in a 1.5-mL collecting tube. Then, the sam- ple is centrifuged at 12,000 � g for 1 min, the flow-through is discarded, and finally the spin column back is placed into the same collection tube. Around 200 mL of binding/wash buffer was added to the spin column. Then, the sample is centrifuged at 12,000 � g for 1 min, the flow-through is discarded, and, finally, the spin column back is placed into the new collection tube. About 50 mL of pooled serum sam- ple (cirrhotic and healthy in separate columns) was added into resin and incubated for 2 min at room temperature. Then, the sample is centrifuged at 12,000 � g for 1 min, flow-through is reapplied to spin column, and incubated for 2 min at room temperature. Again, the sample is centrifuged at 12,000 � g for 1 min and flow-through is retained. Spin column was placed in a new collection tube. Resin was washed to release unbound proteins by adding 50 mL of bind- ing/wash buffer for each 200 mL of the resin used. Retained fractions of cirrhotic and healthy samples were run in SDS- PAGE for confirmation of albumin depletion. 10 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS gel was prepared according to the standard protocol. Samples from cirrhotic liver subjects and healthy subjects after depletion of albumin along with un-depleted samples were loaded in gel, and SDS-PAGE was carried out at 25 mA in 1� SDS running buffer for confirmation of depletion along with molecular weight marker. After electrophoresis, gel was incubated in a fixing solution (40% methanol, 10% acetic acid) at room temperature for 20 min. Gel was subjected for staining with sliver stain (0.1% silver nitrate, 36% formaldehyde) at room tempera- ture for 20 min. Excess staining solution was removed, and the gel was washed with 5% acetic acid.11–13 Acetone precipitation Acetone precipitation was carried out to remove excess salts which interferes electrophoretic run. Protein samples after depletion of albumin (cirrhotic and healthy sepa- rately) were placed in acetone-compatible tubes. Four times the sample volume of cold acetone (�20�C) was added into both tubes. The tubes were vortexed and incu- bated for 60 min at �20�C, followed by centrifugation at 13,000 � g for 10 min. Then the supernatant was disposed carefully for the retention of protein pellet. 14 Two-dimensional polyacrylamide gel electrophoresis Albumin depleted and desalted protein pellet (200 mg) from cirrhotic and healthy subjects were diluted with rehydration buffer (8 M urea, 2% 3-((3-cholamidopropyl) dimethylam- monio)-1-propanesulfonate, 50 mM dithiothreitol (DTT), 0.2% w/v Bio-Lyte 3/10 ampholyte, bromophenol blue) and used separately for 2DE with 7 cm (pH 4–7) nonlinear immobilized pH gradient dry strips (Bio-Rad). Samples were left overnight for rehydration on 7 cm (pH 4–7) dry strips. Isoelectric focusing was carried out at 250 V for 20 min, followed by 4000 V for 5 h at 20�C. Proteins were separated by 8–16% precast polyacrylamide gels at 200 V for 40 min. After electrophoresis, gels were subjected to staining with silver stain (0.1% silver nitrate, 36% formal- dehyde) at room temperature for 20 min. Excess staining solution was removed, and the gel was washed with 5% acetic acid. 15 Image analysis For image analysis, scanned gels were processed using PDQuest 2-D analysis software (Bio-Rad). For differential analysis, the cirrhotic gel was compared with that of the healthy gel. Differential expression of proteins present in both cirrhotic and healthy gels was considered significant when the fold change was least 2 and p � 0.05 with 95% confidence interval with the application of rank-sum test. In-gel digestion and peptide extraction Excised spots were cut into cubes and transferred into a microcentrifuge tube, and 100 mL of destaining solution (100 mM ammonium bicarbonate/acetonitrile (1:1 vol/ vol)) was added and incubated for 30 min; 500 mL of neat acetonitrile was added and the tubes were incubated for 10 min until gel pieces shrink; 50 mL of DTT solution (10 mM DTT in 100 mM ammonium bicarbonate buffer) Nallagangula et al. 3 was added to cover the gel pieces and incubated in 56�C thermostat for 30 min; and 500 mL of acetonitrile was added to the tubes and further incubated for 10 min. All the liquid was removed from the tube. Following DTT treatment, to get reduction and alkylation of cystines and cysteines in the protein, 50 mL of iodoacetamide solution (55 mM iodoacetamide in 100 mM ammonium bicarbonate solution) was added to the tubes and incubated for 20 min at room temperature in dark. The gel pieces were again treated with acetonitrile for 10 min, and the entire liquid was removed from the tube. The gel pieces were saturated with trypsin buffer (13 ng/mL of trypsin in 10 mM ammo- nium bicarbonate in 10% acetonitrile) for 30 min. Cold trypsin (20 mg of trypsin in 1.5 mL of ice-cold 1 mM hydrochloric acid) was added to the tubes and incubated overnight at 37�C. Tubes were cooled to room temperature, gel pieces were centrifuged at 10,000 r/min for 1 min, and peptides were extracted in 100 mL of extraction buffer (1:2, 5% formic acid/acetonitrile) by incubating for 15 min at 37�C shaker, and the supernatant was withdrawn directly for LC-MS analysis. 16 Mass spectrometric analysis Mass spectrometric analysis of the extracted peptides was performed using Nano LCMS-LTQ-Orbitrap Discovery (Thermo Scientific) coupled to Nano LC (Agilent 1200). The samples were reconstituted in 0.1% formic acid prior to injec- tion; 70-min gradient run was set up using acetonitrile and water with formic acid as the mobile phase. LTQ Orbitrap Discovery is a hybrid-type MS system with the ability to determine accurate m/z of intact precursors. The raw files post-MS run was analyzed using Proteome Discover software and MASCOT as search engine against human database. 17 Results SDS-PAGE analysis for depletion of albumin Immobilized resin form of Cibacron blue was effective in binding abundant albumin from plasma/serum samples for depletion of significant amount. Human serum albumin (HSA) from pooled serum samples of cirrhotic and healthy subjects was depleted using Cibacron blue dye-loaded resin columns. Proteins in the flow-through were analyzed by SDS-PAGE (Figure 1) along with prestained molecular weight marker to investigate efficient depletion of HSA. Sensitive staining, silver stain, was helpful for the detection of low nanogram proteins when compared with Coomassie Brilliant Blue. Silver-stained gel demonstrated significant amount of abundant albumin depletion from serum samples of both cirrhotic and healthy subjects. Identification of biomarker candidates Synthetic gel image representative of all features in the differential analysis comparing samples from cirrhotic and healthy is shown in Figure 2. The image analysis software and statistical analysis found 46 spots in cirrhotic gel and 69 spots in control gel, of which 14 spots were identified with significant altered expression levels between cirrhotic and healthy based on quantitative ratio. These spots were Figure 1. SDS-PAGE analysis for confirmation of albumin depletion (silver-stained gel). 1: Normal pooled albumin depleted serum. 2: Normal pooled serum. 3: Cirrhotic liver pooled albumin depleted serum. 4: Cirrhotic liver pooled serum. M: pre-stained molecular weight marker. SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis. Figure 2. Comparison of 2DE gel images representative of all features in differential analysis from alcoholic cirrhotic and healthy subjects. (a) 2DE gel image of alcoholic liver cirrhotic subjects. (b) 2DE gel image of healthy subjects. 2DE: two-dimensional electrophoresis. 4 Journal of Circulating Biomarkers excised, digested with trypsin, analyzed by LC-MS, and identified by MASCOT database. These spots contain more than one protein; among 14 spots, a total of 68 proteins were identified. Many of the proteins were identified as the same protein in different locations on the gels and so among 68 proteins, we identified 46 candidate biomarkers for alcoholic liver cirrhosis. Among 46 candidate biomar- kers, 28 were identified based on protein score and clinical significance (Table 2). Among 28 protein biomarker candidates, 13 with increased expression and 15 with decreased expression were identified in alcoholic liver cirrhotic when compared to healthy subjects. Serum concentrations of keratin isoforms were found to increase in alcoholic cirrhosis. Immunoglobu- lins (Igs), namely, polymeric immunoglobulin receptor iso- form X1 and IgGFc-binding protein precursor, were increased in cirrhotic liver when compared to healthy sub- jects but with low protein scores. Along with 13 features whose expression is increased in cirrhosis of the liver, angio- tensinogen preproprotein, a2-macroglobulin isoform X1, were found to increase compared to healthy subjects. Serum albumin preproprotein, a-1-antitrypsin precursor and a-1-antichymotrypsin precursor showed decreased expres- sion in alcoholic liver cirrhosis. Concentrations of glycerol kinase isoform X1 and kininogen-1 isoform 1 precursor were decreased in cirrhotic liver compared to healthy subjects. Discussion Invasive liver biopsy is the gold standard diagnostic tool for liver fibrosis/cirrhosis with varied etiology and to distin- guish between intermediate stages. Reliable noninvasive biomarker with sensitivity and specificity is needed for diagnosis/prognosis and effective management of the dis- ease. 8 In the present study, we used 2DE followed by LC- MS for identification of biomarker candidates for alcoholic liver cirrhosis. For maximal detection of meaningful pro- tein expression difference, cases and controls should differ absolutely in terms of disease of interest. Simplified, unbiased binary comparison between diseased and healthy avoids contamination by other diseases and confounding Table 2. Proteomic biomarker candidates identified by 2DE followed by LC-MS for alcoholic liver cirrhosis. Biomarker candidate Expression in ALD Mol. Wt Cal. pI Physiological role Keratin isoforms Protects epithelial cells from damage or stress Keratin type II cuticular Hb6 isoform X1 Increases 62 6.37 Keratin type I cuticular Ha1 Increases 47.2 4.88 Keratin type II cuticular Hb5 isoform 1 Increases 55.8 6.55 Keratin type II cytoskeletal 6C Increases 60 8 Keratin type II cytoskeletal 2 epidermal Increases 65.4 8 Keratin type I cytoskeletal 9 Increases 62 5.24 Lumican precursor Increases 38.4 6.61 Collagen binding proteoglycan pIgR isoform X1 Increases 83.2 5.74 Mediates transcellular transport of Igs Serotransferrin precursor Increases 77 7.12 Ferric ion binding protein Ig lambda like polypeptide 5 isoform 1 Increases 23 8.84 Not known Vitamin D binding protein isoform 3 precursor Increases 55 5.63 Vitamin D transport and storage Haptoglobin isoform 1 preproprotein Increases 45.2 6.58 Binds free plasma hemoglobin Transmembrane protein 201 isoform 1 Increases 72.2 9.22 Involved in nuclear movement during fibroblast polarization and migration a-1-Antitrypsin precursor Decreases 46.7 5.59 Protease inhibitor Hemopexin precursor Decreases 51.6 7.02 Scavenging heme Apolipoprotein A-IV precursor Decreases 45.3 5.38 Chylomicrons and VLDL secretion and catabolism CD5 antigen like isoform X1 Decreases 38.7 5.66 Key regulator of lipid synthesis Zinc-a2-glycoprotein precursor Decreases 34.2 6.05 Lipid mobilization and fertilization Dermcidin isoform 1 preproprotein Decreases 11.3 6.54 Antimicrobial activity a1-B-glycoprotein precursor Decreases 54.2 5.86 Not known Glycerol kinase isoform X1 Decreases 63.6 6.54 Transfer of phosphate from ATP to glycerol a2-HS-glycoprotein preproprotein Decreases 39.3 5.72 Role in endocytosis Kininogen-1 isoform 1 precursor Decreases 71.9 6.81 Role in blood coagulation Sex hormone binding globulin isoform1 precursor Decreases 43.8 6.71 Androgen transport protein a1-Acid glycoprotein 1 precursor Decreases 23.5 5.11 Acute phase protein Leucine-rich a2-glycoprotein precursor Decreases 38.2 6.95 Protein-protein interactions, signal transduction and cell adhesion a2-Antiplasmin isoform X1 Decreases 56.6 6.89 Inactivating plasmin Antithrombin-III precursor Decreases 52.6 6.71 Serine protease inhibitor pIgR: polymeric Ig receptor; 2DE: two-dimensional electrophoresis; LC-MS: liquid chromatography–mass spectrometry; ALD: alcoholic liver disease; Mol. Wt: molecular weight; Calc. pI: calculated isoelectric pH; Igs: immunoglobulins. Nallagangula et al. 5 factors which may alter the expression of protein results in false discovery of biomarker candidates. 18 Discovery of biomarker candidates by proteomic approach is difficult, especially when the pH range is between 3 and 7, as abundant albumin interferes in identi- fication and characterization of low abundant proteins by mass spectral and electrophoretic analysis. Accurate pro- tein biomarker candidate discovery was achieved after depletion of albumin using dye-based affinity columns. Antibody-based immunoprecipitation is more robust for depletion of abundant proteins from plasma/serum and is suitable for identification of novel biomarker candi- dates. 19–21 Depletion dilemma can be rectified using nar- row pH (3–5.6) range and avoids interference of abundant proteins (albumin, transferrin, and Igs) but chance to miss proteins whose isoelectric pH is in the alkaline range. 22 Technological advancement in biomarker candidate dis- covery resulted in identification of protein biomarker can- didates for chronic liver diseases (CLDs) with varied etiology (Table 3). Biomarker candidates identified require verification which demonstrates that the differential expression should remain detectable by assay to be used for validation. 18 Despite numerous biomarker candidates identified, verification may be done only for few qualified candidates in terms of marker performance and reagent availability. 27 Biomarker candidates that show significant expressional differences between diseased and healthy in discovery phase are prioritized. Proteins that are secreted and/or present on cell surface and that act in cellular path- ways and deregulated in ALD should be considered for further validation. 9 In the present study, keratin isoforms showed upregula- tion in alcoholic liver cirrhosis. Keratin is a fibrous struc- tural protein that protects epithelial cells from damage and stress and regulates key cellular activities, namely, cell growth and protein synthesis. 28 Lumican, leucine-rich repeat proteoglycan, constitute an important fraction of noncollagenous ECM proteins. It plays a major role in tissue homeostasis and modulates cellular functions, namely, cell proliferation, migration, and differentiation. 29 Polymeric Ig receptor (pIgR) isoform X1 is a type-I trans- membrane protein expressed from glandular epithelial cells of liver and breast. It mediates transcellular transport of polymeric Igs. Pro-inflammatory cytokines, namely, Table 3. Protein biomarker candidates identified by proteomic analysis for liver fibrosis. Authors Etiology of liver fibrosis Type of sample Proteomic techniques Protein biomarker candidates identified White et al.23 HCV Serum 2DE, LC-MS a2 Macroglobulin Haptoglobin Complement C4 Serum retinol binding protein Apolipoprotein A1 Apolipoprotein A-IV Bevin et al.24 HCV Serum 2DE, LC-MS a2 Macroglobulin Inter-a-trypsin inhibitor heavy chain H4 a-1-Antichymotrypsin Apolipoprotein L1 Paraoxonase/aryleserase 1 Zinc-a2-glycoprotein CD5 antigen-like protein b2 Glycoprotein I Bevin et al.25 HCV Serum 2DE, LC-MS, in-solution isoelectric focusing Beta chains of C3 and C4 Bevin et al.22 HCV Serum 2DE, LC-MS Adiponectin, sex hormone binding protein 14-3-3 protein zeta/delta, complement C3dg Immunoglobulin J chain Apolipoprotein CIII Corticosteroid binding globulin a2-HS-glycoprotein Lipid transfer inhibitor protein Haptoglobin-related protein Katrinli et al.26 HBV Liver tissue 2DE, LC-MS Apolipoprotein A1 Pyruvate kinase Glyceraldehyde 3-phosphate dehydrogenase Glutamate dehydrogenase Alcohol dehydrogenase Transferrin, peroxiredoxin 3 Keratin 5, Annexin HCV: hepatitis C virus; HBV: hepatitis B virus; 2DE: two-dimensional electrophoresis; LC-MS: liquid chromatography–mass spectrometry. 6 Journal of Circulating Biomarkers interferon-g, TNF, and interleukin 1, which are the key regulators of pIgR expression, upregulate in ALD. 8,30,31 Vitamin D-binding protein, a multifunctional protein that belongs to the albumin gene family can bind various forms of vitamin D (ergocalciferol, cholecalciferol, and calcife- diol) for the transport. It is synthesized by hepatic parench- ymal cells. 32 Haptoglobin which is included in the existing noninvasive marker panel has showed increased expression in the present study as it is an acute phase protein. Liver is the major site for the synthesis of haptoglobin; hepatic expression will be stimulated by upregulated IL-6 in ALD. 23,33,34 Transmembrane protein 201 is involved in nuclear movement during fibroblast polarization and migra- tion; actin-dependent nuclear movement is through associa- tion with transmembrane actin-associated nuclear lines. 35 Serine protease inhibitors, a-1-antitrypsin (SERPINA1) and a-1-antichymotrypsin (SERPINA3), produced primar- ily in liver hepatocytes and released directly into the blood stream showed downregulation in alcoholic liver cirrhosis compared to healthy subjects. 36–38 Hemopexin which showed decreased expression in ALD is a single polypep- tide chain of 439 amino acid residues with a molecular weight of 63 kDa is expressed from liver, and it acts as a heme-scavenging protein. 39 Apolipoprotein A-IV, even though not evident from liver, its expression, was also decreased in ALD. Downregulation of Apolipoprotein A- IV was reported in hepatic fibrosis in rat models. 23,40 CD5 antigen-like isoform X1, a key regulator of lipid synthesis, was downregulated in alcoholic liver cirrhosis, whereas upregulation was noted in liver cirrhotic patients in hepa- titis C virus infection. 24 Zinc-a2-glycoprotein, adipokine, which plays an important role in fat catabolism and which reduces insulin resistance, was downregulated in ALD. 24,41 Glycerol kinase, a phosphotransferase and a key enzyme in the regulation of glycerol uptake and metabo- lism, is involved in triglyceride and glycerophospholipid synthesis. Glycerol kinase converts glycerol, a product of lipolysis to glucose in the liver, and shows downregula- tion in alcoholic liver cirrhosis. In the present study, a2-HS-glycoprotein has shown decreased expression in ALD. It is a secretory protein expressed from liver and key regulator in inhibition of vascular calcification, bone metabolism regulation, control of protease activity, insu- lin resistance, and breast tumor cell proliferative signal- ing. 42 a-1-Acid glycoprotein, an acute phase synthesized primarily in hepatocytes, which acts as a carrier of lipo- philic compounds was down regulated in ALD. 43 An SERPINF2, a2-antiplasmin, synthesized in the liver as a single-chain glycoprotein with a molecular weight of 51 kDa inhibits plasmin and was decreased in liver cirrhotic patients. 44 Antithrombin III, a member of the serpin family and an inhibitor of proteinases, namely, thrombin and factor Xa, is primarily synthesized by hepatocytes and downregulated in ALD. 45 Our studies are also corroborated with the downregulation of a2-antiplasmin and antithrombin III in ALD. Newly identified proteomic biomarker candidates for ALD need validation and clinical assay optimization which require measurement of thousands of patient samples with narrow measurement coefficient of variation values. 18 Concentration of proteins in serum/plasma ranges from picograms to nanograms per milliliter. Enzyme-linked immunosorbent assay is the best alternative for quantifica- tion of these protein candidates for ALD with high speci- ficity and sensitivity capture and detection antibodies. Newly developed assay requires analytical validation before evaluating clinical utility in terms of performance characteristics, namely, outcome studies, clinical require- ment, proficiency testing, and goals set by regulatory agen- cies. 46 Indicators of accuracy, precision, analytical measurement range, and reference intervals should be defined for newly discovered biomarker candidates. 47 After analytical validation of new methodology for protein of interest, biomarker candidate should confirm the perfor- mance characteristics in terms of consistency and accuracy in clinical evaluation to diagnose or predict the clinical outcome of ALD. The newly identified biomarker candi- date should satisfy specificity and sensitivity. Evidence- based biomarker should fulfill regulatory requirements before introduced into clinical practice for ALD. 18,48 Conclusion With the help of technological advancement in “-omics” approach, we identified 28 protein biomarker candidates (13 with increased expression and 15 with decreased expression) for alcoholic liver cirrhosis. Despite numer- ous biomarker candidates identified, verification may be done only for few qualified candidates that act in cel- lular pathways and deregulated in ALD. These differen- tially expressed proteins between alcoholic cirrhosis and healthy subjects need to be validated to get the same differential expression detectable by assay to be used for validation. Acknowledgements The authors thank Dr Kiranmayee P, SDUAHER for her timely suggestions to carry out this research work. Declaration of Conflicting Interests The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. References 1. Zhou WC, Zhang QB, and Qiao L. Pathogenesis of liver cirrhosis. World J Gastroenterol 2014; 20(23): 7312–7324. Nallagangula et al. 7 2. O’Shea RS, Dasarathy S, and McCullough AJ. Practice guideline committee of the American association for the study of liver diseases; Practice parameters committee of the American college of gastroenterology alcoholic liver disease. Hepatology 2010; 51(1): 307–328. 3. Stewart S and Day C. Alcohol and liver. 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