Biomarkers For Liver Disease Research Articles

GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases and hepatocellular carcinoma (HCC) and Golgi protein 73 (GP73) is a serum biomarker for liver diseases and HCC. However, the mechanism underlying GP73 regulates HCV infection is largely unknown. Here, we revealed that GP73 acts as a novel negative regulator of host innate immunity to facilitate HCV infection. GP73 expression is activated and correlated with interferon-beta (IFN-β) production during HCV infection in patients’ serum, primary human hepatocytes (PHHs) and human hepatoma cells through mitochondrial antiviral signaling protein (MAVS), TNF receptor-associated factor 6 (TRAF6) and mitogen-activated protein kinase kinase/extracellular regulated protein kinase (MEK/ERK) pathway. Detailed studies revealed that HCV infection activates MAVS that in turn recruits TRAF6 via TRAF-interacting-motifs (TIMs), and TRAF6 subsequently directly recruits GP73 to MAVS via coiled-coil domain. After binding with MAVS and TRAF6, GP73 promotes MAVS and TRAF6 degradation through proteasome-dependent pathway. Moreover, GP73 attenuates IFN-β promoter, IFN-stimulated response element (ISRE) and nuclear factor κB (NF-κB) promoter and down-regulates IFN-β, IFN-λ1, interleukin-6 (IL-6) and IFN-stimulated gene 56 (ISG56), leading to the repression of host innate immunity. Finally, knock-down of GP73 down-regulates HCV infection and replication in Huh7-MAVSR cells and primary human hepatocytes (PHHs), but such repression is rescued by GP73m4 (a mutant GP73 resists to GP73-shRNA#4) in Huh7-MAVSR cells, suggesting that GP73 facilitates HCV infection. Taken together, we demonstrated that GP73 acts as a negative regulator of innate immunity to facilitate HCV infection by interacting with MAVS/TRAF6 and promoting MAVS/TRAF6 degradation. This study provides new insights into the mechanism of HCV infection and pathogenesis, and suggests that GP73 is a new potential antiviral target in the prevention and treatment of HCV associated diseases.

Oxidized LDL, Gamma-Glutamyltransferase and Adverse Outcomes in Older Adults.

Gamma-glutamyltransferase (GGT) is a biomarker of liver disease and oxidative stress which was associated with all-cause and cardiovascular (CV) mortality in the general population and in patients with high risk conditions. This study aims at assessing whether oxLDL modifies the relationship between GGT, all-cause, and CV mortality in elderly individuals from the general population. Observational longitudinal study. Population-based cohort of older individuals (>65years) free of liver disease. One thousand and thirty-eight individuals from the Invecchiare in Chianti (InCHIANTI) study. Serum GGT level, oxidized low-density lipoprotein (oxLDL), CV comorbidities, all-cause and CV mortality. The median age of the study population (n=1,038) was 74years (inter-quartile range: 69-79), 152 individuals (15%) had past CV events. During a median follow-up of 9years, 401 individuals died, 168 of them (42%) for CV causes. In adjusted analyses, GGT predicted all-cause mortality (HR for 20U/L increase in serum GGT: 1.11, 95% CI: 1.02-1.21, P=.02) and CV mortality (HR: 1.17, 95% CI: 1.03-1.33; P=.02). Furthermore, in an analysis for interaction circulating oxLDL amplified the effect of GGT on all-cause mortality (P=.003). Circulating oxLDL amplifies the effect of GGT on mortality in the elderly. The mechanism for this association remains unknown and requires further research, including studying the potential role of GGT in oxidative stress. These results are consistent with the hypothesis of a causal role of GGT in the CV morbidity and mortality in older individuals and indicate that oxLDL plays a crucial role in the interpretation of the link between GGT and the risk of adverse clinical events in this population.

Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease.

Basement membranes provide structural support to epithelium, endothelium, muscles, fat cells, Schwann cells, and axons. Basement membranes are multifunctional: they modulate cellular behavior, regulate organogenesis, promote tissue repair, form a barrier to filtration and tumor metastasis, bind growth factors, and mediate angiogenesis. All basement membranes contain type IV collagen (Col IV), laminin, nidogen, and perlecan. Col IV and laminin self-assemble into two independent supramolecular networks that are linked to nidogen and perlecan to form a morphological discernable basement membrane/basal lamina. The triple helical region, 7S domain and NCI domain of Col IV, laminin and laminin fragment P1 have been evaluated as noninvasive fibrosis biomarkers of alcoholic liver disease, viral hepatitis, and nonalcoholic fatty liver disease. Elevated serum Col IV and laminin are related to degrees of fibrosis and severity of hepatitis, and may reflect hepatic basement membrane metabolism. But the serum assays have not been linked to disclosing the anatomical sites and lobular distribution of perisinusoidal basement membrane formation in the liver. Hepatic sinusoids normally lack a basement membrane, although Col IV is a normal matrix component of the space of Disse. In liver disease, laminin deposits in the space of Disse and codistributes with Col IV, forming a perisinusoidal basement membrane. Concomitantly, the sinusoidal endothelium loses its fenestrae and is transformed into vascular type endothelium. These changes lead to capillarization of hepatic sinusoids, a significant pathology that impairs hepatic function. Accordingly, codistribution of Col IV and laminin serves as histochemical marker of perisinusoidal basement membrane formation in liver disease. Anat Rec, 300:1371-1390, 2017. © 2017 Wiley Periodicals, Inc.

Phosphodiester content measured in human liver by in vivo 31P MR spectroscopy at 7 tesla

PurposePhosphorus (31P) metabolites are emerging liver disease biomarkers. Of particular interest are phosphomonoester and phosphodiester (PDE) “peaks” that comprise multiple overlapping resonances in 31P spectra. This study investigates the effect of improved spectral resolution at 7 Tesla (T) on quantifying hepatic metabolites in cirrhosis.MethodsFive volunteers were scanned to determine metabolite T1s. Ten volunteers and 11 patients with liver cirrhosis were scanned at 7T. Liver spectra were acquired in 28 min using a 16‐channel 31P array and 3D chemical shift imaging. Concentrations were calculated using γ‐adenosine‐triphosphate (γ‐ATP) = 2.65 mmol/L wet tissue.ResultsT1 means ± standard deviations: phosphatidylcholine 1.05 ± 0.28 s, nicotinamide‐adenine‐dinucleotide (NAD+) 2.0 ± 1.0 s, uridine‐diphosphoglucose (UDPG) 3.3 ± 1.4 s. Concentrations in healthy volunteers: α‐ATP 2.74 ± 0.11 mmol/L wet tissue, inorganic phosphate 2.23 ± 0.20 mmol/L wet tissue, glycerophosphocholine 2.34 ± 0.46 mmol/L wet tissue, glycerophosphoethanolamine 1.50 ± 0.28 mmol/L wet tissue, phosphocholine 1.06 ± 0.16 mmol/L wet tissue, phosphoethanolamine 0.77 ± 0.14 mmol/L wet tissue, NAD+ 2.37 ± 0.14 mmol/L wet tissue, UDPG 2.00 ± 0.22 mmol/L wet tissue, phosphatidylcholine 1.38 ± 0.31 mmol/L wet tissue. Inorganic phosphate and phosphatidylcholine concentrations were significantly lower in patients; glycerophosphoethanolamine concentrations were significantly higher (P < 0.05).ConclusionWe report human in vivo hepatic T1s for phosphatidylcholine, NAD+, and UDPG for the first time at 7T. Our protocol allows high signal‐to‐noise, repeatable measurement of metabolite concentrations in human liver. The splitting of PDE into its constituent peaks at 7T may allow more insight into changes in metabolism. Magn Reson Med 78:2095–2105, 2017. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Evaluation of a combinational use of serum microRNAs as biomarkers for liver diseases

Circulating microRNAs (miRNA) have been widely recognized as a novel noninvasive biomarker in a variety of physiological and pathological conditions. In order to assess the sensitivity and reliability of potential miRNAs as diagnostic markers for liver disease related to viral infection, alcohol abuse, or chemical exposure, we collected serum samples from 326 participants and evaluated the single and combination diagnostic values of three serum miRNAs (miR-122, miR-125b, miR-192) compared with a conventional marker ALT. We found that serum miR-122 is significantly elevated in patients with active HBV. MiR-125b increased in HCV positive patients, whereas miR-192 and miR-122 were associated with chemical-induced liver injury. None of the aforementioned miRNAs were shown to increase significantly in alcohol-related liver injuries. Furthermore, we analyzed different combinations and found that a set of miR-122 and miR-125b enhanced the sensitivity of detecting liver injury. Among the 58 ALI/ALF patients, miR-122 responded more rapidly than ALT in successful treatments. Patients with spontaneous recovery from ALI/ALF showed significantly higher serum levels of miR-122 and miR-125b compared to non-recovered patients. In conclusion, our results suggest that the combination of miR-122 and miR-125b may serve as efficient biomarkers for liver injury and may be of a prognostic value in predicting ALI/ALF outcome.

Metabonomics Research Progress on Liver Diseases.

Metabolomics as the new omics technique develops after genomics, transcriptomics, and proteomics and has rapid development at present. Liver diseases are worldwide public health problems. In China, chronic hepatitis B and its secondary diseases are the common liver diseases. They can be diagnosed by the combination of history, virology, liver function, and medical imaging. However, some patients seldom have relevant physical examination, so the diagnosis may be delayed. Many other liver diseases, such as drug-induced liver injury (DILI), alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD), and autoimmune liver diseases, still do not have definite diagnostic markers; the diagnosis consists of history, medical imaging, and the relevant score. As a result, the clinical work becomes very complex. So it has broad prospects to explore the specific and sensitive biomarkers of liver diseases with metabolomics. In this paper, there are several summaries which are related to the current research progress and application of metabolomics on biomarkers of liver diseases.

Circulating microRNAs: Possible role as non-invasive diagnostic biomarkers in liver disease

Liver is the central organ for metabolism and the hepatocytes metabolize several drugs, hepatotoxins, alcohol, etc. Continuous exposure of the hepatocytes to these toxins result in various chronic diseases, such as alcoholic liver disease, non-alcoholic fatty liver disease, viral hepatitis and hepatocellular carcinoma. Although several diagnostic methods, such as serum markers, liver biopsy or imaging studies are currently available, most of these are either invasive or detect the disease at advanced stages. Hence, there is a need for new molecular markers that can be used for early detection of the disease. MicroRNAs (miRNAs) are naturally occurring, 20-22 nucleotide long, non-coding RNA molecules that regulate the gene expression at post-transcriptional levels, thereby modulating various biological functions. Their expression is deregulated under pathological conditions, and recent studies showed that they are secreted and can be detected in various body fluids. Since the cellular changes occur at earlier stages of the disease, detecting miRNAs in the body fluids could make them as potential novel biomarkers. Albeit, the difficulties in standardization procedures, cost and availability should be addressed before using them in the clinical arena. This review highlights the possible role of secreted miRNAs to use as early non-invasive diagnostic markers for liver disease.

Quantitative analysis of total serum glycome in human and mouse.

Model mice are frequently used in drug discovery research. Knowledge of similarities and differences between the mouse and human glycomes is critical when model mice are used for the discovery of glycan-related biomarkers and targets for therapeutic intervention. Since few comparative glycomic studies between human and mouse have been conducted, we performed a comprehensive comparison of the major classes of glycans in human and mouse sera using mass spectrometric and liquid chromatographic analyses. Up to 131 serum glycans, including N-glycans, free oligosaccharides (fOSs), glycosaminoglycans, O-glycans, and glycosphingolipid (GSL)-glycans, were quantified. In both serum samples, N-glycans were the most abundant in the total serum glycome, while fOSs were the least abundant. As expected, the diversity of sialic acid (i.e. Neu5Ac vs. Neu5Gc) was the major species difference between human and mouse in terms of N- and O-glycosylation, while GSL-glycomic profiles were completely different, even when the sialic acid diversity was taken into consideration. Furthermore, total serum glycomics of STAM mouse were unveiled as an initial step to identify novel biomarkers of liver diseases, with which we could identify several glycans with expression significantly increased or decreased expression.

Emerging role of exosomes in liver physiology and pathology.

Exosomes can mediate intercellular communication by conveying various bioactive molecules. Plentiful evidence suggests that exosomes are involved in many liver diseases including hepatitis C virus infection, hepatitis B virus infection, hepatocellular carcinoma, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease, and alcoholic liver disease. Moreover, exosomes are present in nearly all human body fluids. Therefore, exosomal miRNA or proteins have the potential to be novel biomarkers of liver diseases. In the treatment of liver diseases, exosomes could participate in adaptive immune response and mesenchymal stem cell-based therapy. Exosomes can also be used as vehicles for genetic materials and drug delivery.

Differential effects of hyaluronan synthase 3 deficiency after acute vs chronic liver injury in mice.

BackgroundHyaluronan (HA) is a ubiquitous extracellular matrix (ECM) glycosaminoglycan synthesized by three different enzymes, hyaluronan synthase (HAS)1, 2, and 3. HA synthesis mediated by HAS3 promotes inflammation and is pathogenic in animal models of human lung and intestinal disease. Liver fibrosis is a common endpoint to chronic liver injury and inflammation for which there is no cure. Although plasma HA is a commonly used biomarker for liver disease, if and how HA contributes to disease pathogenesis remains unclear. Here, we tested the hypothesis that HA synthesized by HAS3 enhances inflammation and fibrosis. To test this hypothesis, we exposed wild-type or Has3−/− mice to carbon tetrachloride (CCl4) once (acute) or ten (chronic) times.ResultsHAS3-deficient mice exhibited increased hepatic injury and inflammatory chemokine production 48 h after acute CCl4; this was associated with a threefold reduction in plasma HA levels and alterations in the proportions of specific molecular weight HA polymer pools. Hepatic accumulation of fibrosis-associated transcripts was also greater in livers from HAS3-deficient mice compared to controls after acute CCl4 exposure. Surprisingly, fibrosis was not different between genotypes. Hepatic matrix metalloproteinase (MMP)13 mRNA and MMP13 activity was greater in livers from Has3-null mice after chronic CCl4; this was prevented by a MMP13-specific inhibitor. Collectively, these data suggest that Has3, or more likely HA produced by HAS3, limits hepatic inflammation after acute injury and attenuates MMP13-mediated matrix metabolism after chronic injury.ConclusionsThese data suggest that HA should be investigated further as a novel therapeutic target for acute and chronic liver disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-016-0041-5) contains supplementary material, which is available to authorized users.

MicroRNAs as Biomarkers for Liver Disease and Hepatocellular Carcinoma.

Serum levels of liver enzymes, such as alanine transaminase, aspartate transaminase, and α-fetoprotein, provide insight into liver function and are used during treatment of liver disease, but such information is limited. In the case of hepatocellular carcinoma (HCC), which is often not detected until an advanced stage, more sensitive biomarkers may help to achieve earlier detection. Serum also contains microRNAs, a class of small non-coding RNAs that play an important role in regulating gene expression. miR-122 is specific to the liver and correlates strongly with liver enzyme levels and necroinflammatory activity, and other microRNAs are correlated with the degree of fibrosis. miR-122 has also been found to be required for hepatitis C virus (HCV) infection, whereas other microRNAs have been shown to play antiviral roles. miR-125a-5p and miR-1231 have been shown to directly target hepatitis B virus (HBV) transcripts, and others are up- or down-regulated in infected individuals. MicroRNA profiles also differ in the case of HBV and HCV infection as well as between HBeAg-positive and negative patients, and in patients with occult versus active HBV infection. In such patients, monitoring of changes in microRNA profiles might provide earlier warning of neoplastic changes preceding HCC.

Acute and Chronic Hepatitis E Virus Infection in Human Immunodeficiency Virus-Infected U.S. Women.

Exposure to hepatitis E virus (HEV) is common in the United States, but there are few data on prevalence of HEV/human immunodeficiency virus (HIV) coinfection in U.S. We tested 2,919 plasma samples collected from HIV-infected (HIV(+)) women and men enrolled in U.S. cohort studies for HEV viremia using a high-throughput nucleic acid testing (NAT) platform. NAT(+) samples were confirmed by real-time polymerase chain reaction. Samples were selected for testing primarily on the basis of biomarkers of liver disease and immune suppression. Prevalence of HEV viremia was 3 of 2,606 and 0 of 313 in tested plasma samples collected from HIV(+) women and men, respectively. All HEV isolates were genotype 3a. Based on follow-up testing of stored samples, 1 woman had chronic HEV infection for >4 years whereas 2 women had acute HEV detectable at only a single study visit. To our knowledge, this is the first reported case of chronic HEV infection in an HIV(+) U.S. individual. We also confirm that chronic HEV infection can persist despite a CD4(+) count >200 cells/mm(3). Overall, though, these data suggest that HEV infection is rare in the HIV(+) U.S. population.

Acute Injury in Natural Diet-Induced Fatty Livers - A Model for Therapy Development

Given the diabesity and metabolic syndrome epidemics, fatty liver disease is reaching epidemic proportions. Relatively indolent, this disease is often asymptomatic and the patient is often made aware of its presence only during a routine physical exam. Nevertheless, fatty livers are more susceptible to insult compared to their non-fatty counterparts and persons with fatty livers are at increased risk for morbidity and mortality following consumption of commonly used substances such as alcohol (EtOH) and acetaminophen (APAP). We have developed a rat model of natural diet-induced fatty liver disease and evaluated the effects of two commonly used substances viz. EtOH and APAP in this phenotype. High fat diet (HFD) fed animals exhibited steatosis, liver inflammation and liver fibrosis with an increase in serum aspartate aminotransferase. Bolus administration of EtOH, which was without effect on the livers from standard diet fed animals, had a profound and adverse impact on the HFD fatty liver. Similarly, APAP administration which was without effect on liver function tests in control animals, also provoked an increase in liver enzymes in HFD animals. Treatment with the poly(ADP-ribose) polymerase-1 inhibitor (PARP-1), veliparib, reduced the increase in liver function tests secondary to EtOH and APAP. This model forms the framework for identification of fatty liver disease biomarkers given that this disease is relatively asymptomatic but fraught with risk for acute injury. This model also forms the framework for evaluation of novel drugs for acute injury in fatty livers especially given that current strategies for management of acute liver failure in non-fatty livers are inadequate. Also, relevant patents related to the use of liver biomarkers as diagnostic are discussed. Keywords: Acetaminophen, acute, alcohol, fibrosis, injury, liver, steatosis.

Biomarkers in Pediatric Liver Disease.

The chronic nature of liver diseases in children and adults merits close follow-up for disease progression and/or treatment evaluation. Disease progression involves injury to liver cells resulting in cell death, varying degrees of inflammation, steatosis depending on the insult, oxidative stress, and eventually fibrosis and cirrhosis unless the process is modified with treatment or spontaneous recovery. Inflammation, cell death, and fibrosis are the three major processes that determine the outcome of liver disease irrespective of the etiology. Markers to measure the activity or status of these parameters in a dynamic way, particularly via noninvasive methods, are urgently required. In this chapter, we summarize recent advances in the identification of biomarkers of liver diseases: biomarkers corresponding to inflammation, cell death, fibrosis, and the development of malignancy.

Oxidative Stress as Estimated by Gamma-Glutamyl Transferase Levels Amplifies the Alkaline Phosphatase-Dependent Risk for Mortality in ESKD Patients on Dialysis.

Alkaline phosphatase (Alk-Phos) is a powerful predictor of death in patients with end-stage kidney disease (ESKD) and oxidative stress is a strong inducer of Alk-Phos in various tissues. We tested the hypothesis that oxidative stress, as estimated by a robust marker of systemic oxidative stress like γ-Glutamyl-Transpeptidase (GGT) levels, may interact with Alk-Phos in the high risk of death in a cohort of 993 ESKD patients maintained on chronic dialysis. In fully adjusted analyses the HR for mortality associated with Alk-Phos (50 IU/L increase) was progressively higher across GGT quintiles, being minimal in patients in the first quintile (HR: 0.89, 95% CI: 0.77–1.03) and highest in the GGT fifth quintile (HR: 1.13, 95% CI: 1.03–1.2) (P for the effect modification = 0.02). These findings were fully confirmed in sensitivity analyses excluding patients with preexisting liver disease, excessive alcohol intake, or altered liver disease biomarkers. GGT amplifies the risk of death associated with high Alk-Phos levels in ESKD patients. This observation is compatible with the hypothesis that oxidative stress is a strong modifier of the adverse biological effects of high Alk-Phos in this population.

Prevalence and incidence of liver dysfunction and assessment of biomarkers of liver disease in HIV-infected Asian children.

We determined the prevalence and incidence of liver dysfunction before and after initiation of combination antiretroviral therapy (cART) in the TREAT Asia Pediatric HIV Observational Database. Data from children initiated on cART between 2 and 18 years of age with baseline alanine aminotransferase (ALT) available before and at least once after cART initiation in TREAT Asia Pediatric HIV Observational Database between 2008 and 2012 were analyzed. Prevalence and incidence of liver dysfunction and biomarkers including the aspartate aminotransferase to platelet ratio index and FIB4 index (a noninvasive panel to stage liver disease) were assessed. Data from 1930 children were included. Their median age was 6.9 years; 49% were male; 98% were perinatally infected and 94% were initiated on non-nucleoside reverse transcriptase-based cART regimens. Before cART, the prevalence of ALT ≥3 times the upper limit of normal (×ULN) was 5.8%. There were 8.5% of children with aspartate aminotransferase to platelet ratio index >1.5 (suggestive of liver fibrosis) and 2.7% with FIB4 index >1.3 (predictive of possible cirrhosis). Among the 1143 cases with normal baseline ALT (≤1×ULN), the incidence of ALT 3×ULN after cART was 1.19 of 1000 person-months (95% confidence interval: 0.93-1.51). Two of 350 with available tests (0.6%) met Hy's law (ALT >3×ULN and total bilirubin >2×ULN). By multivariate analysis, baseline hemoglobin <7.5 g/dL was a predictor of ALT >3×ULN, whereas age 5-9 years at cART initiation was protective for liver dysfunction. We demonstrated a low prevalence and incidence of liver dysfunction before and after cART initiation in children with normal baseline chemistries. In this population facing life-long cART, prospective surveillance for emergence of liver disease is warranted.

Circulating microRNAs as noninvasive diagnostic biomarkers of liver disease in children with cystic fibrosis.

Cystic fibrosis liver disease (CFLD), resulting from progressive hepatobiliary fibrosis, causes significant morbidity and mortality in up to 20% of children with cystic fibrosis (CF). Both pathogenesis and early detection of CFLD are elusive. Current diagnostic procedures to detect early CFLD and stage fibrosis severity are inadequate. Recent studies highlight a role for microRNAs (miRNAs) in the pathogenesis of many diseases and have suggested that serum miRNAs could be used as diagnostic biomarkers. We profiled circulating serum miRNA levels in patients with CFLD (n = 52), patients with CF without liver disease (CFnoLD, n = 30), and non-CF pediatric controls (n = 20). Extracted RNA was subjected to polymerase chain reaction (PCR) array of 84 miRNAs detectable in human serum. Seven candidate miRNAs identified were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), normalizing data to geNorm-determined stable reference genes, miR-19b and miR-93. miR-122 was significantly elevated in patients with CFLD versus patients with CFnoLD and controls (P < 0.0001). miR-25 (P = 0.0011) and miR-21 (P = 0.0133) were elevated in patients with CFnoLD versus patients with CFLD and controls. CFLD was discriminated by both miR-122 (area under the curve [AUC] 0.71, P = 0.002) and miR-25 (AUC 0.65, P = 0.026). Logistic regression combining 3 miRNAs (-122, -25, -21) was greatly predictive of detecting CFLD (AUC 0.78, P < 0.0001). A combination of 6 miRNAs (-122, -21, -25, -210, -148a, -19a) distinguished F0 from F3-F4 fibrosis (AUC 0.73, P = 0.04), and miR-210 combined with miR-22 distinguished F0 fibrosis from any fibrosis, that is, F1-F4 (AUC 0.72, P = 0.02). These data provide the first evidence of changes to circulating miRNA levels in CF, suggesting that serum-based miRNA analysis may complement and extend current CFLD screening strategies with potential to predict early hepatic fibrosis.

Citrus limonin glucoside supplementation decreased biomarkers of liver disease and inflammation in overweight human adults

Mixtures of limonoid glucosides demonstrated health benefits in human and animal studies; however, the specific metabolic effects of purified citrus limonin glucoside (LG) in humans are unknown. We determined effects of LG on circulating biomarkers of chronic inflammatory diseases such as nonalcoholic fatty liver disease (NAFLD), diabetes, CVD, and cancer in a cross-over, placebo controlled, double-blind study in overweight/obese individuals. LG had no specific adverse effects. It did not alter circulating concentrations of blood lipids, lipoproteins or their particle sizes, glucose, insulin, hematological parameters, and markers of inflammation except MMP-9 and TNF-α which were decreased by 38.7% and 10.7%, respectively. LG significantly decreased concentrations of liver proteins: gamma-glutamyl transferase (33.8%), alanine aminotransferase (13.1%), alkaline phosphatase (10.1%), and complement C3 (6.4%). Since liver enzymes are elevated in metabolic syndrome, NAFLD, diabetes, CVD, and chronic kidney disease and liver cancer, LG may be useful in the prevention and/or treatment of those diseases.

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings.Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC–MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity.

Global MicroRNA Analysis in Primary Hepatocyte Cultures.

MicroRNAs are small non-coding molecules that regulate gene expression and in return affect diverse biological functions, including those involved in toxicity and development of disease. Recent evidence suggests that microRNAs play an important role in liver pathologies, like viral hepatitis, alcoholic liver, hepatocellular carcinoma, or drug-induced liver injury. Furthermore, numerous studies demonstrated the high potential of microRNAs as promising non-invasive biomarkers of liver disease or as relevant targets for therapeutic treatment. This chapter describes a method for global microRNA analysis of primary hepatocytes by high-throughput sequencing. The method comprises the isolation of high-quality total RNA, analysis of microRNA sequencing data, and the validation of the findings by reverse transcriptase quantitative polymerase chain reaction analysis.