Growing Human Hepatocellular Tumors Undergo a Global Metabolic Reprogramming.

Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis, high morbidity and mortality concerning with lack of effective diagnosis and high postoperative recurrence. Similar with other cancers, HCC cancer cells have to alter their metabolism to adapt to the changing requirements imposed by the environment of the growing tumor. In less vascularized regions of tumor, cancer cells experience hypoxia and nutrient starvation. Here, we show that HCC undergoes a global metabolic reprogramming during tumor growth. A combined proteomics and metabolomics analysis of paired peritumoral and tumor tissues from 200 HCC patients revealed liver-specific metabolic reprogramming and metabolic alterations with increasing tumor sizes. Several proteins and metabolites associated with glycolysis, the tricarboxylic acid cycle and pyrimidine synthesis were found to be differentially regulated in serum, tumor and peritumoral tissue with increased tumor sizes. Several prognostic metabolite biomarkers involved in HCC metabolic reprogramming were identified and integrated with clinical and pathological data. We built and validated this combined model to discriminate against patients with different recurrence risks. An integrated and comprehensive metabolomic analysis of HCC is provided by our present work. Metabolomic alterations associated with the advanced stage of the disease and poor clinical outcomes, were revealed. Targeting cancer metabolism may deliver effective therapies for HCC.

Effects of Vitamin D Supplementation on Serum 25-Hydroxyvitamin D Concentrations in Cirrhotic Patients: A Randomized Controlled Trial.

BACKGROUND: The liver is crucial for 25-hydroxyvitamin D (25(OH)D) metabolism, and vitamin D deficiency is highly prevalent in patients with cirrhosis and predicts adverse outcomes. We aimed to evaluate whether vitamin D supplementation in patients with cirrhosis is effective in increasing 25(OH)D serum concentrations. Secondary outcome measures included liver function tests (aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), and alkaline phosphatase (AP)), albumin, International Normalized Ratio (INR), bilirubin, the liver fibrosis marker hyaluronic acid, and parameters of mineral metabolism including parathyroid hormone (PTH). METHODS: This is a double-center, double-blind, placebo-controlled study conducted from December 2013 to May 2014 at the Medical University of Graz, and the hospital Hoergas-Enzenbach, Austria. We enrolled 36 consecutive patients with cirrhosis and 25(OH)D concentrations below 30 ng/mL. Study participants were randomly allocated to receive either 2800 International Units of vitamin D3 per day as oily drops (n = 18) or placebo (n = 18) for 8 weeks. RESULTS: Thirty-three study participants (mean (SD) age: 60 (9) years; 21% females; 25(OH)D: 15.6 (7.4) ng/mL) completed the trial. The mean treatment effect (95% CI) for 25(OH)D was 15.2 (8.0 to 22.4) ng/mL (p < 0.001). There was no significant effect on any secondary outcome. CONCLUSIONS: In this randomized controlled trial, vitamin D supplementation increases 25(OH)D serum concentrations, even in cirrhotic patients.

Association of homoarginine and methylarginines with liver dysfunction and mortality in chronic liver disease.

Previous studies on arginine metabolites reported an association of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) with liver dysfunction and an inverse relation of homoarginine (hArg) with cardiovascular risk. The aim of the present study was to investigate the relationships between hArg, ADMA, SDMA, and the dimethylarginine score (DAS, i.e., ADMA + SDMA) and liver dysfunction and survival in chronic liver disease. In 94 consecutive cirrhotic patients admitted to our outpatient liver clinic, serum levels of hArg, ADMA, and SDMA were measured by HPLC at baseline. Patients were followed with respect to mortality. In the entire study cohort (age 58.5 ± 11.2 years; 31 % females), the serum concentrations were 1.94 ± 0.90 µM for homoarginine, 0.90 ± 0.22 µM for ADMA, and 0.70 (0.60-0.93) µM for SDMA. ADMA correlated with both Child-Pugh and MELD scores, while SDMA, DAS, and hArg correlated with MELD score only. Thirty patients (32 %) died during a median follow-up of 3.5 years. Age- and sex-adjusted Cox proportional hazard ratios (HR) per µM (with 95 % confidence intervals) showed that hArg was associated with decreased mortality [HR 0.59 (0.37-0.96)], whereas mortality was increased in patients with higher ADMA [HR 3.78 (0.98-14.60)], SDMA [HR 6.54 (3.15-13.59)] and DAS [HR 4.13 (2.26-7.56)]. Only SDMA and DAS remained significantly associated with mortality after additional adjustments for either Child-Pugh stage or MELD score. In conclusion, in cirrhotic patients seen in an outpatient liver clinic, hArg as well as the dimethylarginines ADMA and SDMA was related to long-term mortality. In particular, SDMA predicts mortality independently of both Child-Pugh stage and MELD score.

Asymmetric dimethylarginine responses during interferon-α-induced depression in patients with chronic hepatitis C infection.

OBJECTIVE: The aim of this study was to examine the association of depressive symptoms with asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Patients with chronic hepatitis C infection were examined during interferon-α (IFN-α) treatment, which is often associated with treatment-induced depression. The associations between IFN-α-induced depressive symptoms with ADMA and SDMA levels were prospectively investigated until 3 months after treatment. METHODS: Psychiatric and biological assessments were obtained at six different time points: before, during (at 1, 3, 6, and 9 months), and after the end of IFN-α treatment. RESULTS: During IFN-α treatment, 22 (53.7%) patients fulfilled the criteria for a treatment-related depressive disorder at least once during treatment. The increase in ADMA levels from baseline (depression group: 0.63 [0.08] µM, no depression group: 0.69 [0.08] µM) in response to IFN-α treatment was considerably higher in patients with IFN-α treatment-induced depressive episodes compared with patients without treatment-induced depressive episodes (3 months after the start of treatment: depression group: 0.72 [0.08] µM, no depression group: 0.72 [0.11] µM; ADMA: repeated-measure design analysis of variance [time × depression]: F(5,151) = 2.446, p = .036). The increase in SDMA was not associated with treatment-induced depression. CONCLUSIONS: Depression in response to IFN-α treatment is associated with elevated ADMA levels. These findings are relevant to nitric oxide-related biological pathways linking depression to increased cardiovascular disease risk. Future studies are needed to clarify the role of serotonin in these pathways and may lead to preventative treatment strategies.

Human nonmercaptalbumin-2: a novel prognostic marker in chronic liver failure.

Albumin is the major plasma protein with several important biological functions. Various disturbances of albumin function have been demonstrated in end-stage liver disease. These functional disturbances may be related to oxidative modifications of albumin at cysteine-34, including the irreversibly oxidized human nonmercaptalbumin-2 (HNA2). The aim of the present study was to relate oxidative modification of albumin to short-term prognosis in chronic liver failure. Patients with advanced cirrhosis (N = 39), acute-on-chronic liver failure (N = 15), and healthy controls (N = 15) were investigated. Three fractions of albumin were separated by high performance liquid chromatography according to the redox state of cysteine-34. The HNA2 fraction was markedly increased in cirrhotic patients vs. controls and correlated with the degree of chronic liver failure as well as laboratory parameters of liver dysfunction. The HNA2 level tended to be a better predictor of short-term mortality than the model for end stage liver disease with respect to both 30-day mortality (area under the receiver operating characteristic curve [AUROC] 0.87 vs. 0.81, NS) and 90-day mortality (AUROC 0.87 vs. 0.82, NS). In multivariate analysis of prognostic variables, HNA2 was the only remaining predictor of 90-day mortality. Our results suggest that HNA2, a marker of chronic oxidative stress, is related to liver dysfunction and mortality in cirrhosis and may represent a novel biomarker of chronic liver failure.

A biopsychosocial model of interferon-alpha-induced depression in patients with chronic hepatitis C infection.

BACKGROUND: The aim of this prospective study was to gain a more comprehensive picture of the biopsychosocial effects of interferon-α (IFN-α) treatment of patients with chronic hepatitis C (HCV). The predictors of depressive development and changes in health-related quality of life, life satisfaction and cognitive ability were measured with the inclusion of the social context. Furthermore, the effects of IFN-α treatment on indoleamine 2,3-dioxygenase, the level of tryptophan supply in the brain, the development of neurotoxic kynurenine metabolites and the thyroid glands were investigated. Therefore, for the first time the conditions for the development of depressive episodes in HCV patients treated with IFN-α were examined over the entire period of treatment as well as 3 months later, applying a holistic biopsychosocial model. METHOD: Psychiatric and biological assessments were carried out at 6 different times: before, during (at 1, 3, 6 and 9 months) and after the end of IFN-α treatment. RESULTS: During IFN-α treatment 22 (53.7%) of 41 patients fulfilled the criteria for a treatment-related depressive disorder at least once during treatment. Contributing factors are tryptophan depletion (tryptophan to competing amino acids quotient), increased neurotoxic challenge (kynurenine to kynurenic acid quotient), less social support, female gender, preexisting psychiatric vulnerability, means of transmission, low financial security, impaired sexual satisfaction, small circle of friends, impaired physical role, strong body pain, low general health and vitality, reduced social functioning, impaired mental health and impaired emotional role. CONCLUSIONS: The awareness of relevant risk factors of IFN-α treatment-induced depression is essential to develop preventative treatment strategies.

Oxidative albumin damage in chronic liver failure: relation to albumin binding capacity, liver dysfunction and survival.

BACKGROUND & AIMS: Impaired binding function of albumin has been demonstrated in end-stage liver disease. This and other functional disturbances of albumin may be related to oxidative stress which is believed to play an important role in the pathogenesis of liver failure as well as sepsis. The aim of the present study was to relate oxidative modification of albumin to loss of albumin binding function in advanced chronic liver failure and in sepsis. METHODS: Patients with decompensated cirrhosis or sepsis and healthy controls were investigated. Three fractions of albumin were separated by chromatography according to the redox state of cysteine-34: non-oxidized human mercaptalbumin, reversibly oxidized human non-mercaptalbumin-1, and irreversibly oxidized human non-mercaptalbumin-2 (HNA2). Binding properties of albumin site II were measured using dansylsarcosine as a ligand. RESULTS: Both in cirrhotic and septic patients, fractions of oxidized albumin were increased and binding capacity for dansylsarcosine was decreased. Mass spectroscopy confirmed specific oxidation of cysteine-34. In cirrhotic patients, dansylsarcosine binding correlated strongly with liver function parameters and moderately with HNA2. Baseline levels of HNA2 accurately predicted 30-day and 90-day survival in cirrhotic patients and this was confirmed in an external validation cohort. CONCLUSIONS: Our results suggest that oxidative damage impairs binding properties of albumin. In advanced liver disease, reduced binding capacity of albumin site II is mainly related to impaired liver function. The plasma level of HNA2 is closely related to survival and may represent a novel biomarker for liver failure.

Pleuro-Pulmonary Nocardiosis as Opportunistic Infection in a Patient with Chronic Hepatitis C under Combination Treatment with Pegylated Interferon, Ribavirin, and Boceprevir.

Nocardiosis is an infrequent but serious pulmonary infection caused by Gram-positive aerobic actinomycetes. In this paper, we report on a 48-year-old patient with pleuropulmonary nocardiosis and cirrhosis due to chronic hepatitis C virus infection treated with triple antiviral treatment complicated by prolonged neutropenia.

Comparison of two molecular assays with conventional blood culture for diagnosis of sepsis.

In this small study, the LightCycler® SeptiFast, and the SepsiTest™ were compared with blood culture. The SeptiFast showed a higher sensitivity (42.9%) and specificity (88.2%) when compared to blood culture than the SepsiTest™ (28.6 and 85.3%). The SeptiFast provides more species specific results, although the identification panel is smaller.

Increase of HCV RNA concentration during hemodialysis treatment in patients with chronic hepatitis C.

BACKGROUND: In hemodialysis (HD) patients, a decrease of serum HCV RNA concentration during HD has been reported. OBJECTIVES: To evaluate the effect of two different extracorporeal blood treatments, HD and hemodiafiltration (HDF), on HCV RNA concentration under standardized conditions. STUDY DESIGN: Eleven chronic HD patients with chronic hepatitis C (CHC) and thirty-three non-uremic patients with CHC as controls were studied. Blood samples were collected at baseline (t=0 min), 30 min (t=30), and 180 min (t=180) after start of HD or HDF. HCV RNA concentrations were determined by a real-time PCR assay. Values obtained 30 min and 180 min after start of HD or HDF were adjusted according to the ultrafiltration-induced hemoconcentration. RESULTS: Baseline HCV RNA concentrations were found to be similar in dialysis patients and controls (2.9E+06 vs. 5.8E+06 IU/ml). After adjustment for hemoconcentration, no significant differences of HCV RNA concentrations were observed when HD versus HDF treatments and blood samples collected pre versus those collected post membrane were compared. Adjusted HCV RNA concentrations increased by 13% (not significant) at 30 min and by 56% (p<0.001) at 180 min after start of HD or HDF. Inhibitory effects on PCR through heparin and uremic toxins could be excluded. CONCLUSIONS: In contrast to recent publications, a significant increase of serum HCV RNA within 180 min after start of HD or HDF was observed. Changes in serum HCV RNA concentration are independent from HD and HDF procedures, dialysis membrane, heparin concentration, and uremic toxins.

Correction of plasma concentrations for effects of hemoconcentration or hemodilution.

The removal of plasma water during hemodialysis and ultrafiltration usually leads to a decrease in plasma volume and to a concomitant increase in the concentration of components not removed by that process. At a baseline hematocrit of 35% the relative change of a component measured per unit plasma volume is almost twice as large as the concomitant change in hematocrit or hemoglobin concentration measured per unit blood volume. Thus, to asses whether the change of a plasma component results from the volume change or from other aspects of the intervention, the plasma concentration measured per unit plasma volume has to be divided by the hemoconcentration for the plasma compartment h(p) = H(1) (100 - H(0))/(H(0)[100 - H(1)]), where H is the hematocrit in percent and where indices 0 and 1 refer to the condition before and after intervention, and not by the hemoconcentration for the blood compartment h(H) = H(1)/H(0), as it is frequently done.

Association of 25-hydroxyvitamin D levels with liver dysfunction and mortality in chronic liver disease.

BACKGROUND: Previous studies suggest that chronic liver disease may be related to vitamin D deficiency. It is, however, not known whether 25(OH)D levels are associated with incident hepatic decompensation and mortality in chronic liver failure. AIMS: We aimed to evaluate whether 25(OH)D serum levels are associated with Child-Pugh (CP) score, model for end-stage liver disease (MELD) score, occurrence of hepatic decompensation, and survival in patients with cirrhosis. METHODS: We enrolled 75 consecutive cirrhotic patients admitted to our outpatient liver clinic (32% females; age: 58 ± 11 years; aetiology alcohol in 61%). At baseline, 25(OH)D was determined and the degree of liver dysfunction was estimated by CP and MELD score. Thereafter patients were followed-up with respect to hepatic decompensation and mortality. RESULTS: 25(OH)D levels averaged 16.0 ± 9.2 ng/ml and were inversely correlated with MELD score (r = -0.34, P = 0.003) and CP score (r = -0.21, P = 0.080). Thirty-seven patients developed hepatic decompensation and 24 patients died during a median follow-up of 3.6 years. Age- and gender-adjusted relative risk (with 95% confidence interval) was 6.37 (1.75-23.2; P = 0.005) for hepatic decompensation and 4.31 (1.38-13.5; P = 0.012) for mortality within the first vs the third 25(OH)D tertile but these associations were largely attenuated towards non-significant trends after additional adjustments for CP or MELD score. CONCLUSIONS: Our findings show a significant association of 25(OH)D with the degree of liver dysfunction and suggest that low 25(OH)D levels may predict hepatic decompensation and mortality in patients with chronic liver failure.

Low-dose atorvastatin improves dyslipidemia and vascular function in patients with primary biliary cirrhosis after one year of treatment.

OBJECTIVE: Primary biliary cirrhosis (PBC) is frequently associated with hypercholesterolemia and with an increased cardiovascular morbidity and mortality. Statins lower serum cholesterol levels and may thus improve the cardiovascular risk in PBC patients. The aim of our study was to prospectively examine the efficacy of low-dose atorvastatin on cholestasis as well as cardiovascular risk markers such as dyslipidemia and vascular function in patients with PBC. METHODS: Nineteen patients with early-stage (biopsy proven and AMA positive) PBC and low-density lipoprotein cholesterol (LDL-C) above 130mg/dL were included in this single-center study and treated with atorvastatin 10mg per day for one year. RESULTS: Concentrations of total cholesterol, LDL-C, LDL triglycerides, oxLDL, IgG and sVCAM-1 decreased significantly after 48 weeks of atorvastatin treatment. Flow-mediated dilation (FMD) of the brachial artery as an indicator of vascular function significantly increased, while carotid artery intima-media thickness and vascular wall stiffness did not progress under treatment. No statistical differences in liver enzymes were observed except a transient increase of alkaline phosphatase. CONCLUSION: Treatment with low-dose atorvastatin is safe in early-stage PBC, effectively reduces total cholesterol, LDL-C, LDL triglycerides, oxLDL and sVCAM-1 and improves vascular function as reflected by FMD, without affecting cholestasis progression. Therefore, statin therapy should be considered in PBC patients with additional risk factors for cardiovascular disease.

Reliable detection and quantitation of viral nucleic acids in oral fluid: Liquid phase-based sample collection in conjunction with automated and standardized molecular assays.

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.

Oxidative damage of albumin in advanced liver disease.

Albumin has a number of biological functions and the serum albumin level is related to prognosis in advanced liver disease. Oxidative stress is believed to play an important role in the pathogenesis of liver failure. The aim of the present study was to characterize oxidative modification of albumin in patients with various degrees of liver failure and to investigate implications for its binding function. Patients with liver cirrhosis (n=10), acute-on-chronic liver failure (n=8) and healthy controls (n=15) were included in the study. Three fractions of albumin were separated by HPLC according to the redox state of cysteine-34 and detected by fluorescence as well as UV absorption. Carbonyl groups were measured as a marker of oxidative modification in plasma proteins and, by western blotting, on albumin. Progressive oxidative modification of albumin was found with increasing severity of liver failure indicated by an increased content of carbonyl groups and oxidation of cysteine-34. Fluorescence properties of albumin were altered by oxidation and, in patients with acute-on-chronic liver failure, by high plasma levels of bilirubin. This alteration of albumin fluorescence by bilirubin provides evidence for a preferred binding of bilirubin to the fully reduced form of albumin.

Atorvastatin in patients with primary biliary cirrhosis and incomplete biochemical response to ursodeoxycholic acid.

UNLABELLED: Statin therapy may target both hypercholesterolemia and cholestasis in primary biliary cirrhosis (PBC). However, little is known about the efficacy and safety of statins in PBC. The aim of this single-center study was therefore to prospectively examine the effects of atorvastatin on serum markers of cholestasis, aminotransferases, and lipid and bile acid metabolism as well as inflammatory and immunological markers in patients with PBC. Fifteen patients with early-stage PBC and an incomplete biochemical response to ursodeoxycholic acid (UDCA) therapy (defined as alkaline phosphatase 1.5-fold above the upper limit of normal after 1 year) were treated with atorvastatin 10 mg/day, 20 mg/day, and 40 mg/day for 4 weeks, respectively. Serum levels of alkaline phosphatase increased during atorvastatin 20 mg and 40 mg (P < 0.05), whereas leucine aminopeptidase and gamma-glutamyltransferase remained unchanged. No statistical differences in overall serum ALT, AST, bilirubin, and IgM levels were observed. However, atorvastatin was discontinued in 1 out of 15 patients because of ALT 2-fold above baseline, and 2 patients showed ALT elevations 3-fold above the upper limit of normal at the end of the atorvastatin treatment period. Serum total cholesterol and low-density lipoprotein cholesterol levels decreased by 35% and 49%, respectively (P < 0.001). Precursors of cholesterol biosynthesis (lanosterol, desmosterol, lathosterol) showed a similar pattern. No changes in serum bile acid levels and composition were observed during treatment. CONCLUSION: Atorvastatin does not improve cholestasis in PBC patients with an incomplete biochemical response to UDCA but effectively reduces serum cholesterol levels.

Removal of bile acids by two different extracorporeal liver support systems in acute-on-chronic liver failure.

Acute-on-chronic liver failure (ACLF) is accompanied by marked intrahepatic cholestasis leading to accumulation of cytotoxic bile acids. Extracorporeal liver support systems efficiently remove bile acids, but their effect on bile acid composition in ACLF is unknown. The aim of the present study was to compare elimination of individual plasma bile acids by albumin dialysis (Molecular Adsorbents Recirculating System, MARS) and fractionated plasma separation (Prometheus). Eight consecutive patients with ACLF underwent alternating 6-hour sessions with MARS or Prometheus in a randomized, cross-over design. Serum samples were obtained before, during, and after each treatment, and individual bile acids including cholic acid and chenodeoxycholic acid (CDCA) were measured by gas chromatography. MARS and Prometheus removed total bile acids to a similar extent (reduction ratio, 45% and 46%, respectively). Both devices cleared cholic acid more efficiently than did CDCA. The molar fraction of CDCA (fCDCA) was elevated at baseline and correlated with the degree of liver dysfunction. Prometheus but not MARS treatments further increased fCDCA. Although both devices eliminate total bile acids to a similar extent, clearance of individual bile acids is different, leading to a slight change of the bile acid profile toward hydrophobic bile acids during Prometheus treatments.