Albumin in Advanced Liver Diseases: The Good and Bad of a Drug!
TL;DR: The role of albumin solutions in the treatment of decompensated cirrhosis was discussed in this article, where it was shown that long-term administration of HAS can modify the course of patients with liver failure by reducing the onset of new complications, improving the quality of life, and providing survival benefits.
About: This article is published in Hepatology.The article was published on 2021-11-01. It has received 27 citations till now. The article focuses on the topics: Serum albumin & Hepatorenal syndrome.
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TL;DR: In this article, the correlation of the deletion spanning the pre-S2 gene segment with clinicopathological factors and the association of such correlation with HCC recurrence after curative surgical resection were examined in HBV-related HCC patients.
Abstract: Hepatocellular carcinoma (HCC) is, globally, one of the most prevalent and deadly human cancers; despite curative surgical resection, its high recurrence rate after surgery remains a large threat, resulting in poor patient survival. The hepatitis B virus (HBV) pre-S2 mutant that harbors deletions spanning the pre-S2 gene segment has emerged as an important oncoprotein for HCC development and a valuable prognostic biomarker for HCC recurrence; however, its relationship with clinicopathological factors is largely unexplored. In this study, the correlation of the deletion spanning the pre-S2 gene segment with clinicopathological factors and the association of such correlation with HCC recurrence after curative surgical resection were examined in HBV-related HCC patients. Inverse correlation between serum albumin level and the deletion spanning the pre-S2 gene segment was identified. HCC patients with the presence of the deletion spanning the pre-S2 gene segment and a low serum albumin level were associated with higher HCC recurrence than patients with either factor alone or neither factor were. Moreover, a combination of the serum albumin level and the deletion spanning the pre-S2 gene segment exhibited better performance than that of either factor alone in predicting HCC recurrence. Collectively, this study shows an association of low serum albumin level with pre-S2 mutant-positive HCC patients, and validates the prognostic value of this association in identifying patients with higher HCC recurrence after curative surgical resection.
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TL;DR: Asrani et al. as discussed by the authors identified hepatic protein signatures of alcohol-associated cirrhosis (AC) and AH as well as AH severity, which may facilitate the development of therapeutic strategies.
Abstract: Alcohol-associated liver disease is a global health care burden, with alcohol-associated cirrhosis (AC) and alcohol-associated hepatitis (AH) being two clinical manifestations with poor prognosis. The limited efficacy of standard of care for AC and AH highlights a need for therapeutic targets and strategies. The current study aimed to address this need through the identification of hepatic proteome and phosphoproteome signatures of AC and AH. Proteomic and phosphoproteomic analyses were conducted on explant liver tissue (test cohort) and liver biopsies (validation cohort) from patients with AH. Changes in protein expression across AH severity and similarities and differences in AH and AC hepatic proteome were analyzed. Significant alterations in multiple proteins involved in various biological processes were observed in both AC and AH, including elevated expression of transcription factors involved in fibrogenesis (eg, Yes1-associated transcriptional regulator). Another finding was elevated levels of hepatic albumin (ALBU) concomitant with diminished ALBU phosphorylation, which may prevent ALBU release, leading to hypoalbuminemia. Furthermore, altered expression of proteins related to neutrophil function and chemotaxis, including elevated myeloperoxidase, cathelicidin antimicrobial peptide, complement C3, and complement C5 were observed in early AH, which declined at later stages. Finally, a loss in expression of mitochondria proteins, including enzymes responsible for the synthesis of cardiolipin was observed. The current study identified hepatic protein signatures of AC and AH as well as AH severity, which may facilitate the development of therapeutic strategies. Alcohol-associated liver disease is a global health care burden, with alcohol-associated cirrhosis (AC) and alcohol-associated hepatitis (AH) being two clinical manifestations with poor prognosis. The limited efficacy of standard of care for AC and AH highlights a need for therapeutic targets and strategies. The current study aimed to address this need through the identification of hepatic proteome and phosphoproteome signatures of AC and AH. Proteomic and phosphoproteomic analyses were conducted on explant liver tissue (test cohort) and liver biopsies (validation cohort) from patients with AH. Changes in protein expression across AH severity and similarities and differences in AH and AC hepatic proteome were analyzed. Significant alterations in multiple proteins involved in various biological processes were observed in both AC and AH, including elevated expression of transcription factors involved in fibrogenesis (eg, Yes1-associated transcriptional regulator). Another finding was elevated levels of hepatic albumin (ALBU) concomitant with diminished ALBU phosphorylation, which may prevent ALBU release, leading to hypoalbuminemia. Furthermore, altered expression of proteins related to neutrophil function and chemotaxis, including elevated myeloperoxidase, cathelicidin antimicrobial peptide, complement C3, and complement C5 were observed in early AH, which declined at later stages. Finally, a loss in expression of mitochondria proteins, including enzymes responsible for the synthesis of cardiolipin was observed. The current study identified hepatic protein signatures of AC and AH as well as AH severity, which may facilitate the development of therapeutic strategies. Alcohol-associated liver disease (ALD) is a major health care burden, with alcohol-associated cirrhosis (AC) and alcohol-associated hepatitis (AH) being two major clinical manifestations with frequentl unfavorable outcomes. Globally, liver diseases lead to >2 million deaths per year, with cirrhosis due to excessive alcohol consumption accounting for about one-quarter of them.1Asrani S.K. Devarbhavi H. Eaton J. Kamath P.S. Burden of liver diseases in the world.J Hepatol. 2019; 70: 151-171Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar In 2017, cirrhosis was the 11th leading cause of death in the United States, with 50% of deaths attributed to excessive alcohol use.2Young-Hee Yoon C.M.C. Liver Cirrhosis Mortality in the United States: National, State, and Regional Trends, 2000-2017. US Department of Health and Human Services, National Institutes of Health, Hyattsville, MD2019: 1-88Google Scholar Binge drinking superimposed on chronic alcohol consumption can cause acute AH in patients with or without preexisting cirrhosis (50% of patients with AH present with cirrhosis3O'Shea R.S. Dasarathy S. McCullough A.J. Alcoholic liver disease.Hepatology. 2010; 51: 307-328Crossref PubMed Scopus (938) Google Scholar). Severe AH is a life-threatening condition, with a 6-month mortality rate reaching 60%,4Lucey M.R. Mathurin P. Morgan T.R. Alcoholic hepatitis.N Engl J Med. 2009; 360: 2758-2769Crossref PubMed Scopus (690) Google Scholar which manifests as jaundice, liver dysfunction, and systemic inflammatory response syndrome.4Lucey M.R. Mathurin P. Morgan T.R. Alcoholic hepatitis.N Engl J Med. 2009; 360: 2758-2769Crossref PubMed Scopus (690) Google Scholar,5Michelena J. Altamirano J. Abraldes J.G. Affò S. Morales-Ibanez O. Sancho-Bru P. Dominguez M. García-Pagán J.C. Fernández J. Arroyo V. Ginès P. Louvet A. Mathurin P. Mehal W.Z. Caballería J. Bataller R. Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis.Hepatology. 2015; 62: 762-772Crossref PubMed Scopus (184) Google Scholar The coronavirus disease 2019 (COVID-19) pandemic has further exacerbated this problem, as many states have reported an increase in alcohol sales6NIAAAAlcohol Sales During the COVID-19 Pandemic. National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD2021Google Scholar and alcohol-related hospitalizations,7Rutledge S.M. Schiano T.D. Florman S. Im G.Y. COVID-19 aftershocks on alcohol-associated liver disease: an early cross-sectional report from the U.S. epicenter.Hepatol Commun. 2021; 5: 1151-1155Crossref PubMed Scopus (12) Google Scholar as well as worsened outcomes in COVID-19 patients with alcohol-associated multi-organ pathologies, including ALD.8Moon A.M. Curtis B. Mandrekar P. Singal A.K. Verna E.C. Fix O.K. Alcohol-associated liver disease before and after COVID-19-an overview and call for ongoing investigation.Hepatol Commun. 2021; 5: 1616-1621Crossref PubMed Scopus (9) Google Scholar Effective treatment options for severe AH are limited and primarily target the inflammatory response (corticosteroids) or promote liver regeneration and neutrophil production (granulocyte colony-stimulating factor).9Singal A.K. Shah V.H. Current trials and novel therapeutic targets for alcoholic hepatitis.J Hepatol. 2019; 70: 305-313Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Clearly, there is an urgent need to develop new treatment strategies to improve long-term survival and quality of life for patients with AC and AH.Various omic approaches (eg, genomics, transcriptomics, epigenomics, metabolomics/lipidomics, and metagenomics) have been employed to discover novel molecules, pathways, and mechanisms that may facilitate the development of new targeted treatment strategies for ALD. For example, genome-wide association studies found an increased risk of developing AC in patients with the rs150052 variant of the RNA processing gene HNRNPUL1,10Innes H. Buch S. Hutchinson S. Guha I.N. Morling J.R. Barnes E. Irving W. Forrest E. Pedergnana V. Goldberg D. Aspinall E. Barclay S. Hayes P.C. Dillon J. Nischalke H.D. Lutz P. Spengler U. Fischer J. Berg T. Brosch M. Eyer F. Datz C. Mueller S. Peccerella T. Deltenre P. Marot A. Soyka M. McQuillin A. Morgan M.Y. Hampe J. Stickel F. Genome-wide association study for alcohol-related cirrhosis identifies risk loci in MARC1 and HNRNPUL1.Gastroenterology. 2020; 159: 1276-1289.e7Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar and an increased risk of both AC and AH in patients with the rs738409 single-nucleotide polymorphism of the PNPLA3 gene.11Beaudoin J.J. Long N. Liangpunsakul S. Puri P. Kamath P.S. Shah V. Sanyal A.J. Crabb D.W. Chalasani N.P. Urban T.J. An exploratory genome-wide analysis of genetic risk for alcoholic hepatitis.Scand J Gastroenterol. 2017; 52: 1263-1269Crossref PubMed Scopus (11) Google Scholar Transcriptomic analysis revealed compromised hepatocyte nuclear factor 4α target gene expression, which was associated with hepatocellular failure in AH patients, suggesting that modulation of hepatocyte nuclear factor 4α signaling may improve liver function in AH.12Argemi J. Latasa M.U. Atkinson S.R. Blokhin I.O. Massey V. Gue J.P. et al.Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.Nat Commun. 2019; 10: 3126Crossref PubMed Scopus (62) Google Scholar Another study using a coupled hepatic transcriptomic and metabolomic analysis revealed an important role of elevated hexokinase domain containing 1 in reprogramming of glucose metabolism in patients with AH and proposed this protein as a potential therapeutic target and biomarker for AH.13Massey V. Parrish A. Argemi J. Moreno M. Mello A. García-Rocha M. Altamirano J. Odena G. Dubuquoy L. Louvet A. Martinez C. Adrover A. Affò S. Morales-Ibanez O. Sancho-Bru P. Millán C. Alvarado-Tapias E. Morales-Arraez D. Caballería J. Mann J. Cao S. Sun Z. Shah V. Cameron A. Mathurin P. Snider N. Villanueva C. Morgan T.R. Guinovart J. Vadigepalli R. Bataller R. Integrated multiomics reveals glucose use reprogramming and identifies a novel hexokinase in alcoholic hepatitis.Gastroenterology. 2021; 160: 1725-1740.e2Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar A lipidomics approach was recently used to discriminate ALD stages based on changes in plasma levels of lipid species (eg, elevated levels of 13-hydroxyoctadecadienoic acid, 9,10-dihydroxyoctadecenoate, and 12,13-dihydroxyoctadecenoate in moderate AH versus heavy drinkers with mild ALD).14Warner D. Vatsalya V. Zirnheld K.H. Warner J.B. Hardesty J.E. Umhau J.C. McClain C.J. Maddipati K. Kirpich I.A. Linoleic acid-derived oxylipins differentiate early stage alcoholic hepatitis from mild alcohol-associated liver injury.Hepatol Commun. 2021; 5: 947-960Crossref PubMed Scopus (4) Google Scholar Because the gut-liver axis plays an important role in ALD, metagenomics has been applied to elucidate microbial/bacterial changes contributing to the disease development/progression (eg, cytolysin-positive Enterococcus faecalis was identified as a pathogenic bacterium in clinical AH and successfully targeted in experimental AH15Duan Y. Llorente C. Lang S. Brandl K. Chu H. Jiang L. et al.Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.Nature. 2019; 575: 505-511Crossref PubMed Scopus (283) Google Scholar). Certainly, omic studies have been invaluable for identifying biomarkers, novel mechanisms, and therapeutic targets for AC and AH. However, to the best of our knowledge, there has been limited characterization of liver proteome signatures for these liver pathologies. Therefore, in the present study, a coupled proteomic and phosphoproteomic analysis was performed to identify alterations in hepatic protein and phosphoprotein levels in patients with AC and AH. Additionally, similarities and differences between these two disease stages were analyzed, and specific proteins, pathways, and mechanisms contributing to the progression of AH severity were identified with the collective goal of identifying novel therapeutic targets to treat these manifestations of ALD.Materials and MethodsStudy Populations and Clinical CharacterizationThis study analyzed liver samples from two cohorts of patients, which are referred to as the test and validation cohorts (Supplemental Figure S1). The test cohort consisted of de-identified liver samples that were acquired through the University of Louisville and John Hopkins University Clinical Resources Center for Alcoholic Hepatitis Investigations (1R24AA025017-01) and consisted of non-ALD controls (n = 12 total, with 7 from University of Louisville and 5 from John Hopkins University) and AH patients (n = 6, John Hopkins University) with an average Model for End-Stage Liver Disease (MELD) score of 37.2 ± 1.8. The validation cohort consisted of de-identified liver samples, which were obtained from the Liver Tissue Cell Distribution System at the University of Minnesota (NIH contract HHSN276201200017C; non-ALD controls, n = 10; and AC patients, n = 10) and the biorepository of the National Institute on Alcohol Abuse and Alcoholism–funded Southern California Alcoholic Hepatitis Consortium (U01AA021884-04; AH patients, n = 34). AH patients had an average MELD score of 26 ± 0.8. On the basis of MELD score, AH patients were divided into four groups: AH1 (MELD score, 17 to 20; n = 4), AH2 (MELD score, 21 to 25; n = 14), AH3 (MELD score, 26 to 29; n = 11), and AH4 (MELD score, 30 to 37; n = 5). Demographic and clinical characteristics of the study cohorts are provided in Supplemental Tables S1 and S2. All study protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected by the institutional review board approval for the individual studies and acquisition of informed consent from all participating patients. No liver specimens were acquired from executed prisoners or institutionalized persons.Liver HistologyFormalin-fixed, paraffin-embedded liver samples from AH patients and non-ALD controls were sectioned to a thickness of 5 μm, stained with hematoxylin and eosin, and evaluated by light microscopy at ×200 magnification for gross liver pathology. Liver biopsies were evaluated for AC and AH by expert liver pathologists. Patients were considered to have AH if characteristic features were seen on histology, patients had a history of alcohol abuse, and other liver diseases were excluded. Many patients with AH also had underlying cirrhosis. Patients were diagnosed with AC if they had a history of alcohol abuse that was thought to be the primary cause of their liver disease and no active alcohol-associated steatohepatitis on histology.Liver Proteome and Phosphoproteome AnalysisLiver proteomic and phosphoproteomic analyses were conducted in the Pacific Northwest National Laboratory using standard protocols and procedures.16Diamond D.L. Proll S.C. Jacobs J.M. Chan E.Y. Camp 2nd, D.G. Smith R.D. Katze M.G. HepatoProteomics: applying proteomic technologies to the study of liver function and disease.Hepatology. 2006; 44: 299-308Crossref PubMed Scopus (37) Google Scholar Test cohort liver sample processing: Approximately 20 to 30 mg of frozen liver tissues were homogenized with a hand-held Tissue-Tearor homogenizer (BioSpec Products, Bartlesville, OK) in 300 μL of lysis buffer (8 mol/L urea, 75 mmol/L NaCl, 50 mmol/L Tris, pH 8.0, and 1 mmol/L EDTA, supplemented with protease and phosphatase inhibitors). Lysates were incubated on ice for 15 minutes and then cleared by centrifugation at 20,000 × g for 10 minutes at 4°C. Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA). Disulfide bonds were reduced with 5 mmol/L dithiothreitol for 1 hour at 37°C and subsequently alkylated with 10 mmol/L iodoacetamide for 45 minutes at 25°C in the dark. Samples were diluted fourfold with 50 mmol/L Tris-HCl, pH 8.0, to obtain a final concentration of 2 mol/L urea before digestion with Lys-C (Wako, Richmond, VA) at a 1:50 enzyme/substrate ratio. After 2 hours of digestion at 25°C, sequencing-grade modified trypsin (Promega, Madison, WI) at 1:50 enzyme/substrate ratio was added to the samples, which were further incubated at 25°C for 14 hours. The reaction was stopped by acidifying the samples with a final concentration of 1% formic acid (Sigma-Aldrich, St. Louis, MO), and samples were centrifuged for 15 minutes at 1500 × g. Tryptic peptides were desalted on a C18 SPE cartridge (Waters tC18 SepPak; WAT036820; Milford, MA) and concentrated using a Speed-Vac concentrator. Final peptide concentrations were determined via BCA assay. Peptides (200 μg) were labeled with 10-plex tandem mass tags (TMTs; Thermo Fisher Scientific), according to the manufacturer's recommendations. One of the TMT channels (131) was occupied with a pooled mixture of peptides from all the samples, which serves as a reference to normalize across different sets of samples. Approximately 1.9 mg of 10-plex TMT-labeled sample was separated on a reversed phase Agilent Zorbax 300 Extend-C18 column (250 × 4.6 mm column, containing 3.5-μm particles) using an Agilent 1200 HPLC System (Agilent Technologies, Santa Clara, CA). Solvent A was 4.5 mmol/L ammonium formate, pH 10, and 2% acetonitrile; and solvent B was 4.5 mmol/L ammonium formate, pH 10, and 90% acetonitrile. The flow rate was 1 mL/minute, and the injection volume was 900 μL. TMT-labeled peptides were fractionated into 96 fractions by high-pH reversed-phase chromatography and further concatenated into 24 fractions, as previously described.17Wang Y. Yang F. Gritsenko M.A. Wang Y. Clauss T. Liu T. Shen Y. Monroe M.E. Lopez-Ferrer D. Reno T. Moore R.J. Klemke R.L. Camp 2nd, D.G. Smith R.D. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells.Proteomics. 2011; 11: 2019-2026Crossref PubMed Scopus (386) Google Scholar,18Mertins P. Tang L.C. Krug K. Clark D.J. Gritsenko M.A. Chen L. Clauser K.R. Clauss T.R. Shah P. Gillette M.A. Petyuk V.A. Thomas S.N. Mani D.R. Mundt F. Moore R.J. Hu Y. Zhao R. Schnaubelt M. Keshishian H. Monroe M.E. Zhang Z. Udeshi N.D. Mani D. Davies S.R. Townsend R.R. Chan D.W. Smith R.D. Zhang H. Liu T. Carr S.A. Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry.Nat Protoc. 2018; 13: 1632-1661Crossref PubMed Scopus (178) Google Scholar For proteome analysis, 5% of each concatenated fraction was dried down and resuspended in 3% acetonitrile and 0.1% formic acid to a peptide concentration of 0.1 μg/μL for liquid chromatography–tandem mass spectrometry analysis. The rest of the fractions (95%) were further concatenated into 12 fractions, dried down, and subjected to immobilized metal affinity chromatography for phosphopeptide enrichment.Validation Cohort Liver Sample ProcessingFrozen liver biopsies were homogenized for 30 seconds separately in 120 μL of lysis buffer (8 mol/L urea, 75 mmol/L NaCl, 50 mmol/L Tris, pH 8.0, and 1 mmol/L EDTA, supplemented with protease and phosphatase inhibitors) with a Kontes Pellet Pestle Cordless Motor (DWK Life Sciences, Millville, NJ) equipped with a disposable pestle. Lysates were incubated on ice for 15 minutes and were then precleared by centrifugation at 20,000 × g for 10 minutes at 4°C. Protein concentrations were determined by BCA assay (Thermo Fisher Scientific). Protein disulfide bonds were reduced with 5 mmol/L dithiothreitol for 1 hour at 37°C, and then subsequently alkylated with 10 mmol/L iodoacetamide for 45 minutes at 25°C in the dark. Samples were diluted eightfold with 50 mmol/L Tris-HCl, pH 8, and sequencing-grade modified trypsin (Promega) at a 1:50 enzyme/substrate ratio was added to the samples and digested at 37°C for 4 hours.Phosphopeptide EnrichmentFe3+-NTA-agarose beads were freshly prepared using the Ni-NTA Superflow agarose beads (Qiagen, Hilden, Germany) for phosphopeptide enrichment. For each of the 12 fractions, peptides were reconstituted to 0.5 μg/μL in immobilized metal affinity chromatography binding/wash buffer (80% acetonitrile and 0.1% trifluoroacetic acid) and incubated with 10 μL of the Fe3+-NTA-agarose beads for 30 minutes at room temperature. After incubation, the beads were washed two times each with 50 μL of wash buffer and once with 50 μL of 1% formic acid on the stage tip packed with 2 discs of Empore C18 material (Empore Octadecyl C18, 47 mm; 98-0604-0217-3; CDS Analytical, Northlake, IL). Phosphopeptides were eluted with 70 μL Elution Buffer (500 mmol/L potassium phosphate buffer). Phosphopeptides were then eluted from the C18 stage tips with 50% acetonitrile and 0.1% formic acid. Samples were dried using a Speed-Vac concentrator (Thermo Fisher Scientific), and then reconstituted with 12 μL of 3% acetonitrile and 0.1% formic acid for liquid chromatography–tandem mass spectrometry analysis. The samples were acidified in 1% formic acid (Sigma-Aldrich) and centrifuged for 15 minutes at 1500 × g to clear the digest of precipitated material. Tryptic peptides were desalted on a C18 SPE (Waters tC18 SepPak; WAT036820) and concentrated using a Speed-Vac concentrator. The final peptide concentration was determined via BCA assay.Liquid Chromatography–Tandem Mass SpectrometryGlobal- and phosphopeptide-enriched samples were subjected to a custom high mass accuracy liquid chromatography–tandem mass spectrometry system, as previously described,19Zhang H. Liu T. Zhang Z. Payne S.H. Zhang B. McDermott J.E. et al.Integrated proteogenomic characterization of human high-grade serous ovarian cancer.Cell. 2016; 166: 755-765Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar where the liquid chromatography component consisted of automated reversed-phase columns prepared in-house by slurry packing 3 μm Jupiter C18 (Phenomenex) into 35 cm × 360 μm o.d. × 75 μm i.d. fused silica (Polymicro Technologies Inc., Phoenix, AZ). The mass spectrometry component consisted of a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) outfitted with a custom electrospray ionization interface. Electrospray emitters were custom made using 360 μm o.d. × 20 μm i.d. chemically etched fused silica capillary tubes. Analysis of the phosphoproteome samples applied similar conditions as used in the global proteome sample analysis. All other instrument conditions were set as previously described.19Zhang H. Liu T. Zhang Z. Payne S.H. Zhang B. McDermott J.E. et al.Integrated proteogenomic characterization of human high-grade serous ovarian cancer.Cell. 2016; 166: 755-765Abstract Full Text Full Text PDF PubMed Scopus (533) Google ScholarMass SpectrometryThe key search parameters used were 20 parts per million tolerance for precursor ion masses, 2.5 and −1.5 Da window on fragment ion mass tolerances, no limit on missed cleavages, partial tryptic search, no exclusion of contaminants, dynamic oxidation of methionine (15.9949 Da), static iodoacetamide alkylation on cysteine (57.0215 Da), and static TMT modification of lysine and N-termini (144.1021 Da). The decoy database searching method20Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat Methods. 2007; 4: 207-214Crossref PubMed Scopus (2768) Google Scholar,21Qian W.J. Liu T. Monroe M.E. Strittmatter E.F. Jacobs J.M. Kangas L.J. Petritis K. Camp 2nd, D.G. Smith R.D. Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome.J Proteome Res. 2005; 4: 53-62Crossref PubMed Scopus (294) Google Scholar was used to control the false discovery rate at the unique peptide level to <0.01% and subsequent protein level to <0.1%.22Kim S. Gupta N. Pevzner P.A. Spectral probabilities and generating functions of tandem mass spectra: a strike against decoy databases.J Proteome Res. 2008; 7: 3354-3363Crossref PubMed Scopus (322) Google Scholar Quantification was based on initially summing to the protein level the sample-specific peptide reporter ion intensities captured for each channel across all 12 analytical fractions. Final data for statistical analysis were the ratio of each protein summed value with the pooled reference control within each TMT10 experiment to adjust for experiment-specific variability. All proteomic and phosphoproteomic data sets are deposited in the MassIVE repository (accession number MSV000089168; https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp, last accessed March 31, 2022).Cytoscape Protein Clustering, GO, and STEMHepatic proteins that were significantly differentially expressed between controls and AH in the test cohort (P < 0.05) were analyzed in Cytoscape via Gene Ontology (GO) analysis to identify specific biological processes associated with those proteins.23Hardesty J.E. Warner J.B. Song Y.L. Rouchka E.C. McClain C.J. Warner D.R. Kirpich I.A. Ileum gene expression in response to acute systemic inflammation in mice chronically fed ethanol: beneficial effects of elevated tissue n-3 PUFAs.Int J Mol Sci. 2021; 22: 1582Crossref PubMed Scopus (4) Google Scholar GO processes that met the false discovery rate of 0.05 were used for further analysis. Similarly, proteins that met the previous criteria were analyzed by MCL Cluster Analysis in Cytoscape. Protein String figures of processes were generated in Cytoscape.23Hardesty J.E. Warner J.B. Song Y.L. Rouchka E.C. McClain C.J. Warner D.R. Kirpich I.A. Ileum gene expression in response to acute systemic inflammation in mice chronically fed ethanol: beneficial effects of elevated tissue n-3 PUFAs.Int J Mol Sci. 2021; 22: 1582Crossref PubMed Scopus (4) Google Scholar Short time-series expression miner (STEM) analysis24Ernst J. Bar-Joseph Z. STEM: a tool for the analysis of short time series gene expression data.BMC Bioinformatics. 2006; 7: 191Crossref PubMed Scopus (925) Google Scholar was conducted on the average protein expression for AH1 to AH4 patient groups to identify protein expression patterns that changed with AH severity. Significant STEM protein clusters were used to identify GO Processes associated with proteins within that cluster.Western BlotLiver tissue samples from non-ALD control (n = 5) and AH patients (n = 6; test cohort) were homogenized in radioimmunoprecipitation assay buffer (10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L EDTA, 0.5 mmol/L EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mmol/L NaCl) supplemented with HALT protease and phosphatase inhibitors (Thermo Fisher Scientific), followed by centrifugation for 10 minutes at 16,000 × g. Protein concentrations were determined by the BCA (Pierce BCA protein assay kit; Thermo Fisher Scientific). A total of 30 μg of protein was separated on Criterion TGX Any kDa gels (BioRad, Hercules, CA) and then transferred to polyvinylidene difluoride membranes and blocked in 5% milk Tris-buffered saline + 0.1% Tween-20. Membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution in 5% bovine serum albumin Tris-buffered saline + 0.1% Tween-20), thoroughly washed, and then incubated with secondary antibodies at 1:2000 dilution in 5% milk Tris-buffered saline + 0.1% Tween-20 at room temperature for 1 hour and washed; and signals were developed with enhanced chemiluminescence substrate (Clarity Max; BioRad) and imaged via the ChemiDoc instrument (BioRad). Band densitometry analysis was conducted with ImageLab software version 6.0.1 (BioRad). The primary antibodies used included S61–Yes1-associated transcriptional regulator (YAP1), YAP1, ALBU, and β-actin (Cell Signaling Technology, Danvers, MA), and the secondary antibody used was a horseradish peroxidase–conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific).Liver Cardiolipin AssayA total of 10 μg of liver protein extract was used from non-ALD control liver (n = 15), AC (n = 7), and AH (n = 6). Liver cardiolipin levels were measured using a fluorometric assay kit (Abcam, Cambridge, MA).Statistical AnalysisAll continuous variables are presented as means ± SEM. Data between two groups were compared by unpaired t-test, and data between multiple groups were compared by one-way analysis of variance using InfernoRDN software v1.1.7995 (https://www.pnnl.gov/integrative-omics, last accessed May 14, 2021). Linear correlation analysis was conducted between individual proteins and clinical parameters in R version 4.0.3. (https://www.r-project.org, last accessed March 3, 2021). P < 0.05 was considered significant for all statistical tests. Receiver operating characteristic analysis and principal component analysis were conducted in GraphPad Prism version 9.1.0 (GraphPad Software, San Diego, CA). Principal component scores were then visualized via RStudio Software version 1.3.1093 (RStudio, Boston, MA) using the plot3D function of the rgl package.ResultsAlterations of Hepatic Proteins and Biological Processes in AH Test Cohort Identified by Proteomic AnalysisInitially, explant liver tissue samples from the AH test cohort (characterized by typical histopathologic features of AH) (Figure 1A) were used to identify changes in protein expression, which were investigated further in an independent AH validation cohort. Hepatic proteomic analysis revealed significant differences in numerous proteins (1586 decreased and 1638 increased) and phosphoproteome (2749 decreased phosphopeptides and 2966 increased phopshopeptides) between AH and controls (Figure 1, B and C). The top proteins decreased in AH included the following metabolic enzymes: glutathione S-transferase alpha 1 (GSTA1; −7.8-fold), alcohol dehydrogenase 4 (ADH4; −6.1-fold), alcohol dehydrogenase 1A (ADH1A; −4.6-fold), glutathione S-transferase alpha 2 (GSTA2; −3.9-fold), and alcohol dehydrogenase 6 (ADH6; −3.8-fold); the top proteins increased were: A-kinase anchoring protein 17A (AK17A; 6.9-fold), heat shock 70 KDa protein 1L (HS71L; 4.2-fold), calpain 6 (CAN6; 3.6-fold), keratin 19 (K1C19; 3.3-fold), and zinc finger protein 512 (ZN512; 3.2-fold) (Supplemental Table S3). The changes in the phosphoproteome levels are presented in Supplemental Table S4. GO processes that were significantly increased in AH included mRNA processing, transcription, fibrosis, neutrophils, and extracellular matrix, among others (Supplemental Figure S2A). Clusters of proteins diminished
4 citations
TL;DR: Human albumin infusion is effective for controlling systemic inflammation, thereby probably managing some liver cirrhosis-related complications, such as spontaneous bacterial peritonitis, hepatic encephalopathy, and hepatorenal syndrome.
3 citations
TL;DR: In this article, the impact of albumin use on patient outcomes remains unclear, however, guidelines recommend albumin as the plasma-expander of choice for acute kidney injury (AKI) in cirrhosis.
Abstract: Background & aims Guidelines recommend albumin as the plasma-expander of choice for acute kidney injury (AKI) in cirrhosis. However, the impact of these recommendations on patient outcomes remains unclear. We aimed to determine the practice-patterns and outcomes associated with albumin use in a large, nationwide-US cohort of hospitalized cirrhotics with AKI. Methods A retrospective cohort study was performed in hospitalized cirrhotics with AKI using Cerner-Health-Facts database from January 2009 to March 2018. 6786 were included for analysis on albumin-practice-patterns, and 4126 had available outcomes data. Propensity-score-adjusted model was used to determine the association between albumin use, AKI-recovery and in-hospital survival. Results Median age was 61-years (60% male, 70% white), median serum-creatinine was 1.8 mg/dL and median Model for End-stage Liver Disease Sodium (MELD-Na) score was 24. Albumin was given to 35% of patients, of which 50% received albumin within 48-hours of AKI-onset, and 17% received appropriate weight-based dosing. Albumin was used more frequently in patients with advanced complications of cirrhosis, higher MELD-Na scores and patients admitted to urban-teaching hospitals. After propensity-matching and multivariable adjustment, albumin use was not associated with AKI-recovery (odds ratio [OR] 0.70, 95% confidence-interval [CI]: 0.59-1.07, P = .130) or in-hospital survival (OR 0.76 [95% CI: 0.46-1.25], P = .280), compared with crystalloids. Findings were unchanged in subgroup analyses of patients with varying cirrhosis complications and disease severity. Conclusions USA hospitalized patients with cirrhosis and AKI frequently do not receive intravenous albumin, and albumin use was not associated with improved clinical outcomes. Prospective randomised trials are direly needed to evaluate the impact of albumin in cirrhotics with AKI.
2 citations
TL;DR: The opposite trends in the level of SOD and GPx activities in serum/plasma and erythrocytes of male patients could be used as the biomarker of alcohol-induced OS injury, and the synergistic changes of MDA, vitamin B12, albumin, bilirubin, and homocysteine levels should also be considered.
Abstract: Alcohol-induced oxidative stress (OS) plays a pivotal role in the pathophysiology of alcohol dependence (AD). This meta-analysis was aimed at investigating the changes in the levels of OS biomarkers in AD patients. We included relevant literature published before 1 April 2022, from the PubMed, Web of Science, and EBSCO databases following PRISMA guidelines. Finally, 15 eligible articles were enrolled in this meta-analysis, including 860 patients and 849 controls. Compared with healthy controls, AD patients had lower activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes, and lower levels of albumin, while levels of malondialdehyde (MDA), vitamin B12, homocysteine, and bilirubin were significantly increased in serum/plasma samples of AD subjects (all p < 0.05). In male patients, the activities of SOD and GPx were increased in serum/plasma but decreased in erythrocytes (all p < 0.05). The opposite trends in the level of SOD and GPx activities in serum/plasma and erythrocytes of male patients could be used as the biomarker of alcohol-induced OS injury, and the synergistic changes of MDA, vitamin B12, albumin, bilirubin, and homocysteine levels should also be considered.
2 citations
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TL;DR: A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma or from a Pichia pastoris expression system, was obtained from polyethylene glycol 4000 solution, and three-dimensional structures of pHSA and rHSA were determined.
Abstract: A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.
1,632 citations
TL;DR: In patients with cirrhosis and spontaneous bacterial peritonitis, treatment with intravenous albumin in addition to an antibiotic reduces the incidence of renal impairment and death in comparison with treatment with an antibiotic alone.
Abstract: Background In patients with cirrhosis and spontaneous bacterial peritonitis, renal function frequently becomes impaired. This impairment is probably related to a reduction in effective arterial blood volume and is associated with a high mortality rate. We conducted a study to determine whether plasma volume expansion with intravenous albumin prevents renal impairment and reduces mortality in these patients. Methods We randomly assigned 126 patients with cirrhosis and spontaneous bacterial peritonitis to treatment with intravenous cefotaxime (63 patients) or cefotaxime and intravenous albumin (63 patients). Cefotaxime was given daily in doses that varied according to the serum creatinine level, and albumin was given at a dose of 1.5 g per kilogram of body weight at the time of diagnosis, followed by 1 g per kilogram on day 3. Renal impairment was defined as nonreversible deterioration of renal function during hospitalization. Results The infection resolved in 59 patients in the cefotaxime group (94 percent...
1,448 citations
TL;DR: The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins.
Abstract: Human serum albumin (HSA), the most prominent protein in plasma, binds different classes of ligands at multiple sites HSA provides a depot for many compounds, affects pharmacokinetics of many drugs, holds some ligands in a strained orientation providing their metabolic modification, renders potential toxins harmless transporting them to disposal sites, accounts for most of the antioxidant capacity of human serum, and acts as a NO-carrier The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins Here, structural, functional, biotechnological, and biomedical aspects of ligand binding to HSA are summarized
903 citations
TL;DR: The rate of loss of albumin to the tissue spaces (measured as transcapillary escape rate) rose by more than 300% in patients with septic shock, and the average increase within 7 h of cardiac surgery was 100%.
779 citations
TL;DR: Results indicate that intravenous albumin infusion is important in avoiding renal and electrolyte complications and activation of endogenous vasoactive systems in cirrhotics with ascites who are treated with repeated large-volume paracentesis.
563 citations