Biomolecular map of albumin identifies signatures of severity and early mortality in acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is associated with high mortality. Alterations in albumin structure and function have been shown to correlate with outcomes in cirrhosis. We undertook a biomolecular analysis of albumin to determine its correlation with hepatocellular injury and early mortality in ALF. METHODS: Altogether, 225 participants (200 patients with ALF and 25 healthy controls [HC]) were enrolled. Albumin was purified from the baseline plasma of the training cohort (ALF, n = 40; survivors, n = 8; non-survivors, n = 32; and HC, n = 5); analysed for modifications, functionality, and bound multi-omics signatures; and validated in a test cohort (ALF, n = 160; survivors, n = 53; non-survivors, n = 107; and HC, n = 20). RESULTS: In patients with ALF, albumin is more oxidised and glycosylated with a distinct multi-omics profile than that in HC, more so in non-survivors (p <0.05). In non-survivors, albumin was more often bound (p <0.05, false discovery rate <0.01) to proteins associated with inflammation, advanced glycation end product, metabolites linked to arginine, proline metabolism, bile acid, and mitochondrial breakdown products. Increased bacterial taxa (Listeria, Clostridium, etc.) correlated with lipids (triglycerides [4:0/12:0/12:0] and phosphatidylserine [39:0]) and metabolites (porphobilinogen and nicotinic acid) in non-survivors (r2 >0.7). Multi-omics signature-based probability of detection for non-survival was >90% and showed direct correlation with albumin functionality and clinical parameters (r2 >0.85). Probability-of-detection metabolites built on the top five metabolites, namely, nicotinic acid, l-acetyl carnitine, l-carnitine, pregnenolone sulfate, and N-(3-hydroxybutanoyl)-l-homoserine lactone, showed diagnostic accuracy of 98% (AUC 0.98, 95% CI 0.95-1.0) and distinguish patients with ALF predisposed to early mortality (log-rank <0.05). On validation using high-resolution mass spectrometry and five machine learning algorithms in test cohort 1 (plasma and paired one-drop blood), the metabolome panel showed >92% accuracy/sensitivity and specificity for prediction of mortality. CONCLUSIONS: In ALF, albumin is hyperoxidised and substantially dysfunctional. Our study outlines distinct 'albuminome' signatures capable of distinguishing patients with ALF predisposed to early mortality or requiring emergency liver transplantation. IMPACTS AND IMPLICATIONS: Here, we report that the biomolecular map of albumin is distinct and linked to severity and outcome in patients with acute liver failure (ALF). Detailed structural, functional, and albumin-omics analysis in patients with ALF led to the identification and classification of albumin-bound biomolecules, which could segregate patients with ALF predisposed to early mortality. More importantly, we found albumin-bound metabolites indicative of mitochondrial damage and hyperinflammation as a putative indicator of <30-day mortality in patients with ALF. This preclinical study validates the utility of albuminome analysis for understanding the pathophysiology and development of poor outcome indicators in patients with ALF.

Fecal microbiota transplantation compared with prednisolone in severe alcoholic hepatitis patients: a randomized trial.

BACKGROUND: Severe alcoholic hepatitis (SAH) has high 90-day mortality. Prednisolone therapy has shown modest survival benefits over placebo at 28 but not 90 days. Fecal microbial transplantation (FMT) has shown promise in these patients. We compared the efficacy and safety of the two therapies in SAH patients. METHODS: Steroid eligible SAH patients were randomized in an open-label study to prednisolone (n = 60) 40 mg/day for 28 days (assessed at day-7 for continuation) or healthy donor FMT (n = 60) through naso-duodenal tube, daily for seven days. Primary outcome of study was day-90 survival. RESULTS: Patients in prednisolone and FMT arms were comparable at baseline (discriminant function score 65 ± 16.2 and 68 ± 14, MELD score 17.1 and 16.5, respectively). Of 120 patients, 112 [prednisolone-57; FMT-55] completed trial. As per intention-to-treat analysis, 90-day survival was achieved by 56.6% (34/60) patients in prednisolone and 75% (45/60) in FMT group (p = 0.044, FMT HR = 0.528, 95%CI 0.279-0.998). Secondary outcome of 28-day survival [78.33% (47/60) and 88.33% (53/60) (p = 0.243, FMT HR = 0.535, 95%CI 0.213-1.34)] with comparable severity scores over time between both arms. Infections accounted for 11 (19.3%) and 2 (3.6%) deaths in prednisolone and FMT groups, respectively (p = 0.01). Path-tracing showed a slow establishment of microbiota and alpha diversity (Shannon index) improvement by day-28 (p = 0.029). FMT resulted in 23 new taxa by day-28, reduction from baseline in pathogenic taxa [Campylobacter (19-fold, p = 0.035), anaerobes (Parcubacteria, Weisella and Leuconostocaceae)], and increase of Alphaproteobacteria [~ sevenfold, p = 0.047] and Thaumarcheota (known ammonia oxidizer, p = 0.06). Lachnospiraceae (p = 0.008), Prevotella and Viellonella communities in gut favored survival (p < 0.05). CONCLUSION: In severe alcoholic hepatitis, FMT is safe and improves 90-day survival and reduces infections by favorably modulating microbial communities. It can be a useful alternative to prednisolone therapy.

Defects in energy metabolism are associated with functional exhaustion of bone marrow mesenchymal stem cells in cirrhosis.

OBJECTIVES: Cellular and functional exhaustion of bone marrow mesenchymal stem cells (BM-MSC) is significantly associated with the loss of HSCs and hepatic osteodystrophy in cirrhosis. The molecular mechanisms underlying the dysfunction of BM-MSCs are not well understood. We investigated the underlying mechanisms of cellular and functional exhaustion of BM-MSCs in cirrhosis. METHODS: The MSCs were isolated retrospectively from bone marrow of decompensated alcoholic cirrhosis patients {(Trial registration: ClinicalTrials.gov NCT01902511) (n=10; MELD=16.2±2.3; CTP=8.7±2.3)} and age and gender-matched healthy controls (n=8). Global gene expression profile of healthy bone marrow MSCs (hBM-MSCs) and cirrhosis patients BM-MSCs (cBM-MSCs) were done by mRNA sequencing. XFe24-bioanalyzer analyzed the bioenergetic potential of cells. Level of different cytokines and growth factors in BM-plasma and MSCs secretome were analyzed by Luminex-based bead array. RESULTS: Analysis of differentially expressed genes showed significant (P<0.01) up-regulation of genes associated with ubiquitination and catabolism of proteins; TNF signaling, insulin resistance, and down-regulation of genes associated with DNA repair, protein processing, cell cycle, and mitochondrial respiration in cBM-MSCs in comparison to hBM-MSCs. Compared to hBM-MSCs, cBM-MSCs showed a significant defect in glycolysis due to insulin resistance and poor glucose uptake (P=0.002). This led to compromised self-renewal capacity and cellular loss of MSCs in cirrhosis. cBM-MSCs also showed a significant impairment in Oxidative phosphorylation (OXPHOS) due to mitochondrial dysfunction leading to defects in the osteogenic differentiation with early aging and senescence. CONCLUSION: Compromised energy metabolism due to inflammatory and metabolic stress-induced insulin resistance underlies the cellular and functional exhaustion of BM-MSCs in cirrhosis.

Protocol for global proteome, virome, and metaproteome profiling of respiratory specimen (VTM) in COVID-19 patient by LC-MS/MS-based analysis.

In this protocol, we describe global proteome profiling for the respiratory specimen of COVID-19 patients, patients suspected with COVID-19, and H1N1 patients. In this protocol, details for identifying host, viral, or bacterial proteome (Meta-proteome) are provided. Major steps of the protocol include virus inactivation, protein quantification and digestion, desalting of peptides, high-resolution mass spectrometry (HRMS)-based analysis, and downstream bioinformatics analysis. For complete details on the use and execution of this profile, please refer to Maras et al. (2021).

Global metabolome profiling of COVID-19 respiratory specimen using high-resolution mass spectrometry (HRMS).

Here we describe a protocol for identifying metabolites in respiratory specimens of patients that are SARS-CoV-2 positive, SARS-CoV-2 negative, or H1N1 positive. This protocol provides step-by-step instructions on sample collection from patients, followed by metabolite extraction. We use ultra-high-pressure liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) for data acquisition and describe the steps for data analysis. The protocol was standardized with specific customization for SARS-CoV-2-containing respiratory specimens. For complete details on the use and execution of this protocol, please refer to Maras et al. (2021).

Multi-omics analysis of respiratory specimen characterizes baseline molecular determinants associated with SARS-CoV-2 outcome.

Rapid diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection still remains a major challenge. A multi-omic approach was adopted to analyze the respiratory specimens of 20 SARS-CoV-2-positive, 20 negative and 15 H1N1 pdm 2009 positive cases. Increased basal level of MX1 (MX dynamin-like GTPase 1) and WARS (tryptophan-tRNA ligase) correlated with SARS-CoV-2 infection and its outcome. These markers were further validated in 200 suspects. MX1>30pg/ml and WARS>25ng/ml segregated virus positives [AUC = 94% CI: (0.91-0.97)] and severe patients [AUC>0.85%]. Our results documented significant increase in immune activation; metabolic reprograming and decrease in oxygen transport, wound healing and others linked proteins and metabolites in patients with coronavirus disease 2019 (COVID-19). Multi-omics profiling correlated with viremia and segregated asymptomatic patients with COVID-19. Additionally, we identified increased respiratory pathogens (Burkholderiales, Klebsiella pneumonia) and decreased lactobacillus salivarius (FDR<0.05) in COVID-19 specimens. In conclusion, increased basal MX1 and WARS levels correlates with SARS-CoV-2 infection and could aid in the identification of patient's predisposed to higher severity.

Differential blood transcriptome modules predict response to corticosteroid therapy in alcoholic hepatitis.

BACKGROUND & AIMS: In patients with severe alcoholic hepatitis (SAH), little is known about the profile of peripheral blood mononuclear cells (PBMCs) at baseline and during corticosteroid therapy, among those who can be treated successfully with steroids (steroid-responders [R] and those who cannot (steroid-non-responders [NR]); 2 groups with different outcomes. METHODS: We performed RNA-seq analysis in PBMCs from 32 patients with definite SAH, at baseline and after 7 days of corticosteroids. The data were sorted into R and NR (n = 16, each group) using the Lille model and 346 blood transcription modules (BTMs) were identified. BTMs are predefined modules of highly co-expressed PBMC genes, which can determine specific immune cell types and cellular functions. The activity of each BTM was taken as the mean value of its member genes. RESULTS: At baseline, 345 BTMs had higher activity (i.e. were upregulated) in NR relative to R. The 100 most upregulated BTMs in NR, included several modules related to lymphoid lineage (T, B, and natural killer [NK] cells), modules for cell division and mitochondrial respiratory electron transport chain (ETC, relating to energy production), but only a few modules of myeloid cells. Correlation studies of BTM activities found features of significantly greater activation/proliferation and differentiation for T and B cells in NR relative to R. After 7 days of corticosteroids, NR had no significant changes in BTM activities relative to baseline, whereas R had downregulation of BTMs related to innate and adaptive immunity. CONCLUSIONS: At baseline and during corticosteroid therapy, increased activity in the PBMCs of gene modules related to activation/proliferation and differentiation of T and B cells, NK cells, and mitochondrial ETC, is a hallmark of SAH patients who are steroid-non-responders. LAY SUMMARY: Patients with severe alcoholic hepatitis receive steroid therapy as the main line of treatment; however, this treatment is ineffective in some patients. This only becomes apparent after 7 days of steroid therapy. We have developed an approach where it can be estimated if a patient is going to respond or not to steroid therapy using the gene expression information of blood cells. This method will allow clinicians to assess the response of patients to steroids earlier, and will help them in adopting alternate strategies if the treatment is found to be ineffective in a particular patient.

Gilbert's Syndrome in Children with Unconjugated Hyperbilirubinemia - An Analysis of 170 Cases.

There is limited literature on Gilbert's syndrome (GS) in children with persistent unconjugated hyperbilirubinemia from Indian subcontinent. All patients (< 18 y of age) with genetically confirmed GS were included, and their profile was analysed. A total of 170 subjects were confirmed as having GS as per genetic analysis (133 with homozygous and 37 with heterozygous status). Majority were diagnosed in the adolescent age group (mean age 13.6 y). The median serum total bilirubin (TB) levels were around 3.3 mg/dl with maximum levels reaching upto 18 mg/dl. Around 15% subjects had an associated condition including hematological or hepatobiliary disease amongst others. GS is an important but under-recognised cause of unexplained unconjugated hyperbilirubinemia in Indian pediatric subjects. It may co-exist with other hematological and hepatobiliary disorders, and complicate the clinical/laboratory picture. Extent of hyperbilirubinemia may fluctuate to levels much higher than what is usually described in current world literature.

Predictors of steroid non-response and new approaches in severe alcoholic hepatitis.

Severe alcoholic hepatitis (SAH) remains a disease with high mortality. Steroid is the main stay and has been shown to give modest 28-day survival benefit in carefully selected patients, but no 90-day survival benefit. Since non-responders have high incidence of infections and increased mortality, it would be worthwhile to identify them before starting steroid therapy. A high and rising bilirubin, urinary acetyl carnitine >2,500 ng/mL, high asiloglycoprotein positive microparticles, and specific features in liver biopsy could predict steroid non-response at baseline. There is an ever-growing need to find new and effective therapies for SAH patients. Besides aggressive nutrition, granulocyte colony stimulating factor, fecal microbiota transplantation, and plasma exchange appear promising therapies and provide a hope for steroid ineligible or steroid non-responsive patients. Suppression of hepatic inflammation, preventing new bacterial or fungal infections, and enhancing liver regeneration will remain the key targets for next generation therapies.

Increased Expression of RUNX1 in Liver Correlates with NASH Activity Score in Patients with Non-Alcoholic Steatohepatitis (NASH).

Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF- significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH.

Modification Patterns of Urinary Albumin Correlates With Serum Albumin and Outcome in Severe Alcoholic Hepatitis.

BACKGROUND AND AIMS: Albumin modifications and deranged functions are well documented in serum of severe alcoholic hepatitis (SAH). We investigated whether urinary albumin (u-Alb) can serve as surrogate marker of circulatory albumin phenotype, functionality, and could predict outcome in SAH patients. PATIENTS AND METHODS: Baseline serum and urine samples from 100 SAH, 20 alcoholic cirrhosis, and 20 healthy controls were subjected to u-Alb, ischemia modified albumin (IMA), IMA to albumin ratio (IMAr), advanced oxidation protein products, advanced glycation end-products, albumin-binding capacity determination. In addition, SAH urinary samples were also analyzed at day 4 and day 7 to predict nonresponse to corticosteroid therapy. RESULTS: Urine and serum levels of IMA, advanced oxidation protein products and advanced glycation end-products were higher (P<0.05) in SAH versus alcoholic cirrhosis and healthy controls. IMAr was low in urine but high in serum of SAH (P<0.05). Albumin-binding capacity was lower (P<0.05) in both urinary and serum albumin of SAH. Urinary and serum albumin parameters showed direct correlation, whereas IMAr showed inverse correlation (cc>0.2, P<0.05). Baseline u-Alb level was significantly higher in SAH, and was correlated directly with corticosteroid treatment outcome and 12-month mortality in SAH. Baseline u-Alb showed an area under the receivers operating curve analysis of 0.7 and a hazard ratio of 1.23 for prediction of 12-month mortality in SAH. Baseline u-Alb level >9.0 mg/dL was associated with reduced 12-month survival in SAH (log rank <0.01). CONCLUSIONS: u-Alb modifications are reflective of serum albumin modifications. Further baseline u-Alb levels could be exploited to predict steroid response and mortality in SAH patients.

Suboptimal Level of Bone-Forming Cells in Advanced Cirrhosis are Associated with Hepatic Osteodystrophy.

Bone loss is common in advanced cirrhosis, although the precise mechanisms underlying bone loss in cirrhosis are unknown. We studied the profile and functionality of bone-forming cells and bone-building proteins in bone marrow (BM) of individuals with cirrhosis (n = 61) and individuals without cirrhosis as normal controls (n = 50). We also performed dual energy X-ray absorptiometry for clinical correlation. BM mesenchymal cells (MSCs) were analyzed for colony-forming units-fibroblasts and their osteogenic (fibronectin-1 [FN1], insulin-like growth factor binding protein 3 [IGFBP3], collagen type 1 alpha 1 chain [COL1A1], runt-related transcription factor 2 [RUNX2], and alkaline phosphatase, liver [ALPL]) and adipogenic ( adiponectin, C1Q, and collagen domain containing [ADIPOQ], peroxisome proliferator-activated receptor gamma [PPARγ], and fatty acid binding protein 4 [FABP4]) potentials. Colony-forming units-fibroblasts were lower in patients with cirrhosis (P = 0.002) than in controls. Cirrhotic BM-MSCs showed >2-fold decrease in osteogenic markers. Compared to controls, patients with cirrhosis showed fewer osteocytes (P = 0.05), osteoblasts, chondroblasts, osteocalcin-positive (osteocalcin+) area, clusters of differentiation (CD)169+ macrophages (P < 0.001, each), and nestin+ MSCs (P = 0.001); this was more apparent in Child-Turcotte-Pugh (CTP) class C than A (P < 0.001). Multivariate logistic regression showed low nestin+ MSCs (P = 0.004) as a predictor of bone loss. Bone-resolving osteoclasts were comparable among CTP groups, but >2-fold decreased anti-osteoclastic and increased pro-osteoclastic factors were noted in patients with CTP C compared to CTP A. Bone-building proteins (osteocalcin [P = 0.008], osteonectin [P < 0.001], and bone morphogenic protein 2 [P = 0.001]) were decreased while anti-bone repair factors (fibroblast growth factor 23 [P = 0.015] and dipeptidyl peptidase 4 [P < 0.001]) were increased in BM and peripheral blood; this was more apparent in advanced cirrhosis. The dual energy X-ray absorptiometry scan T score significantly correlated with the population of osteoblasts, osteocytes, MSCs, and CD169+ macrophages. Conclusion: Osteoprogenitor cells are substantially reduced in patients with cirrhosis and more so in advanced disease. Additionally, increased anti-bone repair proteins enhance the ineffective bone repair and development of osteoporosis in cirrhosis. Hepatology Communications 2018;0:0-0).

Study of Family Clustering and PNPLA3 Gene Polymorphism in Pediatric Non Alcoholic Fatty Liver Disease.

OBJECTIVE: To find association of pediatric NAFLD with metabolic risk factors, and Patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene polymorphism. DESIGN: Cross-sectional study. SETTING: Pediatric Hepatology unit of a tertiary care hospital. PARTICIPANTS: Overweight/obese children (<18 years) with (69 patients) or without (30 patients) NAFLD (ultrasonography based), and their parents. INTERVENTION: Metabolic screening, PNPLA3 gene polymorphism, and transient elastography. OUTCOME MEASURE: Association of pediatric NAFLD with parental metabolic risk factors and PNPLA3 gene polymorphism. RESULTS: In the NAFLD group, there was high parental incidence of metabolic diseases, fatty liver (80%) and low high-density lipoproteins levels (84%). Family history of NAFLD (in any parent), higher alanine aminotransferase levels and higher total cholesterol levels in the child independently predicted possibility of NAFLD, but similar results could not be replicated for PNPLA3 gene polymorphism. Controlled attenuation parameter measurement (by transient elastography) had high sensitivity and specificity to diagnose steatosis. CONCLUSIONS: There is high familial incidence of metabolic diseases in children with NAFLD. Controlled attenuation parameter can be useful as a non-invasive modality to screen fatty liver in children.

Iron-Overload triggers ADAM-17 mediated inflammation in Severe Alcoholic Hepatitis.

Severe alcoholic hepatitis (SAH) is associated with iron accumulation in hepatocytes/macrophages. This possibly correlates with inflammation and stress but the exact mechanism still remains obscure. To understand the role of iron and the mechanisms of systemic iron-overload, a transcriptomic study of liver and Peripheral Blood -Mononuclear-Cells (PBMCs) was undertaken in SAH patients, with and without hepatic iron-overload. Our results show that iron-overload in hepatocytes/macrophages is due to an increased expression of iron-loading receptors and CD163 signaling cascade. Increase in labile iron pool induces expression of iron-loading, oxidative-stress and inflammatory genes along with expression of CD163 and ADAM17. Increased liver iron correlated with circulatory iron, TNF-α, macrophage activation (sCD163) and peroxide-stress in CD163+macrophages in patients who were iron-overloaded and died. Circulatory TNF-α and sCD163 levels were associated with poor outcome. Temporal iron/Fenton stress induced in healthy monocyte-derived-macrophage (MDM)/Tohoku-Hospital-Pediatrics-1(THP1) cells showed higher expression of iron-regulatory, inflammatory and oxidative-stress genes. These genes could be suppressed by iron-chelation. These results suggest that iron mediates inflammation through ADAM17 induction, resulting in macrophage activation and increased shedding of TNF-α and sCD163. These events could be inhibited with iron chelation or with ADAM17-blockade, postulating a therapeutic strategy for SAH patients with iron overload.

Baseline urine metabolic phenotype in patients with severe alcoholic hepatitis and its association with outcome.

Severe alcoholic hepatitis (SAH) has a high mortality rate, and corticosteroid therapy is effective in 60% patients. This study aimed to investigate a baseline metabolic phenotype that could help stratify patients not likely to respond to steroid therapy and to have an unfavorable outcome. Baseline urine metabolome was studied in patients with SAH using ultra-high performance liquid chromatography and high-resolution mass spectrometry. Patients were categorized as responders (Rs, n = 52) and nonresponders (NRs, n = 8) at day 7 according to the Lille score. Multivariate projection analysis identified metabolites in the discovery cohort (n = 60) and assessed these in a validation cohort of 80 patients (60 Rs, 20 NRs). A total of 212 features were annotated by using metabolomic/biochemical/spectral databases for metabolite identification. After a stringent selection procedure, a total of nine urinary metabolites linked to mitochondrial functions significantly discriminated nonresponders, most importantly by increased acetyl-L-carnitine (12-fold), octanoylcarnitine (4-fold), decanoylcarnitine (4-fold), and alpha-ketoglutaric acid (2-fold) levels. Additionally, urinary acetyl-L-carnitine and 3-hydroxysebasic acid discriminated nonsurvivors (P < 0.01). These urinary metabolites significantly correlated to severity indices and mortality (r > 0.3; P < 0.01) and were associated with nonresponse (odds ratio >3.0; P < 0.001). In the validation cohort, baseline urinary acetyl-L-carnitine documented an area under the receiver operating curve of 0.96 (0.85-0.99) for nonresponse prediction and a hazard ratio of 3.5 (1.5-8.3) for the prediction of mortality in patients with SAH. Acetyl-L-carnitine at a level of >2,500 ng/mL reliably segregated survivors from nonsurvivors (P < 0.01, log-rank test) in our study cohort. Conclusion: Urinary metabolome signatures related to mitochondrial functions can predict pretherapy steroid response and disease outcome in patients with SAH. (Hepatology Communications 2018;2:628-643).

Molecular Ellipticity of Circulating Albumin-Bilirubin Complex Associates With Mortality in Patients With Severe Alcoholic Hepatitis.

BACKGROUND & AIMS: Hyperbilirubinemia and hypoalbuminemia are features of hepatic dysfunction that associate with disease severity. This is because hepatic insufficiency causes hypoalbuminemia, which indirectly increases the circulating levels of free bilirubin. Circular dichroism (CD) spectroscopy can be used to quantify the molecular ellipticity (ME) of the albumin-bilirubin complex, and might associate with the severity or outcome of severe alcoholic hepatitis (SAH). METHODS: We performed a cross-sectional study of 265 patients with SAH admitted in the Department of Hepatology, Institute of Liver and Biliary Sciences in New Delhi, India from January 2014 through January 2016. Blood samples were collected and patients were followed for 12 months or death. The molar ratios of bilirubin: albumin and albumin-bilirubin complexes were determined for a discovery cohort (30 patients who survived the study period and 60 patients who did not survive) and compared with those of 60 patients with alcoholic cirrhosis and 30 healthy individuals (controls). Optical activities of albumin-bilirubin complexes in blood samples were determined by CD spectroscopy and compared among groups. Findings were validated in a separate cohort of 150 patients with SAH from the same institute. We studied the correlation between ME and albumin binding capacity (ABiC). RESULTS: The molar ratio of bilirubin: albumin was higher in patients with SAH than with alcoholic cirrhosis or controls (P < .05). Patients with SAH had different CD spectra and higher ME than the other groups (P < .01); ME correlated with model for end-stage liver disease score (with and without Na) and discriminant function (r2 > .3; P < .01). ME values above a cut off of 1.84 mdeg predicted 3-month mortality in patients with SAH with an area under receiver operating characteristic curve of 0.87 (95% CI, 0.79-0.95), a 77% positive predictive value, and a 90% negative predictive value. The hazard ratio and concordance index of ME values for 3-month mortality in patients with SAH was 10% higher than the hazard ratio and concordance index of model for end-stage liver disease score. In patients with SAH, there was an inverse correlation between ME and ABiC (r2 > 0.7; P < .01). We observed a significant reduction in ABiC with increasing levels of bilirubin in vitro prepared albumin-bilirubin complex. CONCLUSION: In a cross-sectional study of patients with SAH, we associated ME of the albumin-bilirubin complex, measured by CD spectroscopy, with outcomes of patients with SAH. Increased loading of bilirubin on albumin could explain reduced albumin function. Bilirubin removal by albumin dialysis might benefit patients with SAH.

Hepatitis B Core Antibody Negativity in a Chronic Hepatitis B Infected Patient: Report of an Unusual Serological Pattern.

Diagnosis of Hepatitis B Virus (HBV) infection is established by the presence of various hepatitis B serological and molecular markers. Expression of these serological markers may vary in natural course of HBV infection. We report a case of an unusual HBV serological pattern in a Chronic Hepatitis B (CHB) infected patient demonstrating absence of Hepatitis B core Antibody (Anti-HBc) in spite of presence of Hepatitis B surface Antigen (HBsAg) and HBV DNA. Since, anti-HBc represents a reliable serological marker for past exposure of HBV infection, therefore we emphasize on the presence of such unusual serological pattern which could lead to doubts in the interpretation of results.

Pre-therapy liver transcriptome landscape in Indian and French patients with severe alcoholic hepatitis and steroid responsiveness.

Patients with severe alcoholic hepatitis (SAH) not responding to glucocorticoid therapy have higher mortality, though they do not differ in their baseline clinical characteristics and prognostic scores from those who respond to therapy. We hypothesized that the baseline hepatic gene expression differs between responders (R) and non-responders (NR). Baseline liver transcriptome was compared between R and NR in Indian (16 each) and French (5 NR, 3 R) patients with SAH. There were differentially expressed genes (DEGs) between NR and R, in Indian (1106 over-expressed, 96 under-expressed genes) and French patients (65 over-expressed, 142 under-expressed genes). Indian NR had features of hepatocyte senescence and French NR exhibited under-expression of genes involved in cell division, indicating a central defect in the capacity of hepatocytes for self-renewal in both populations. Markers of hepatic progenitor cell proliferation were either very few (Indian patients) or absent (French patients). No DEGs were enriched in inflammatory pathways and there were no differences in nuclear receptor subfamily 3 group C member 1 (NR3C1) transcript expression and splicing between NR and R. Our results reveal that baseline hepatic transcriptome is reflective of subsequent glucocorticoid non-response and indicate impaired regenerative potential of the liver as an underlying phenomenon in NR.