Brivanib in combination with Notch3 silencing shows potent activity in tumour models.

BACKGROUND: Sorafenib is the first targeted agent proven to improve survival of patients with advanced hepatocellular carcinoma (HCC) and it has been used in first line treatments with heterogeneous response across patients. Most of the promising agents evaluated in first-line or second-line phase III trials for HCC failed to improve patient survival. The absence of molecular characterisation, including the identification of pathways driving resistance might be responsible for these disappointing results. METHODS: 2D DIGE and MS analyses were used to reveal proteomic signatures resulting from Notch3 inhibition in HepG2 cells, combined with brivanib treatment. The therapeutic potential of Notch3 inhibition combined with brivanib treatment was also demonstrated in a rat model of HCC and in cell lines derived from different human cancers. RESULTS: Using a proteomic approach, we have shown that Notch3 is strongly involved in brivanib resistance through a p53-dependent regulation of enzymes of the tricarboxylic acid (TCA), both in vitro and in vivo. CONCLUSION: We have demonstrated that regulation of the TCA cycle is a common mechanism in different human cancers, suggesting that Notch3 inhibitors combined with brivanib treatment may represent a strong formulation for the treatment of HCC as well as Notch3-driven cancers.

Molecular and functional characterization of CD133+ stem/progenitor cells infused in patients with end-stage liver disease reveals their interplay with stromal liver cells.

BACKGROUND AIMS: Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133+ stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. METHODS: Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133+ cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133+and LX-2 hepatic stellate cells. RESULTS: We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133+ SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133+ SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. DISCUSSION: We demonstrated that the interaction between CD133+ SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy.

Posttranscriptional changes of serum albumin: clinical and prognostic significance in hospitalized patients with cirrhosis.

UNLABELLED: Beside the regulation of fluid distribution, human serum albumin (HSA) carries other activities, such as binding, transport, and detoxification of many molecules. In patients with cirrhosis, HSA exhibits posttranscriptional alterations that likely affect its functions. This study aimed at identifying the structural HSA alterations occurring in cirrhosis and determining their relationship with specific clinical complications and patient survival. One hundred sixty-eight patients with cirrhosis, 35 with stable conditions and 133 hospitalized for acute clinical complications, and 94 healthy controls were enrolled. Posttranscriptional HSA molecular changes were identified and quantified by using a high-performance liquid chromatography/electrospray ionization mass spectrometry technique. Clinical and biochemical parameters were also recorded and hospitalized patients were followed for up to 1 year. Seven HSA isoforms carrying one or more posttranscriptional changes were identified. Altered HSA isoforms were significantly more represented in patients than in healthy controls. Conversely, the native, unchanged HSA isoform was significantly reduced in cirrhosis. Native HSA and most altered isoforms correlated with both Child-Pugh and Model for End-Stage Liver Disease scores. In hospitalized patients, oxidized and N-terminal truncated isoforms were independently associated with ascites, renal impairment, and bacterial infection. Finally, the native HSA and cysteinylated/N-terminal truncated isoforms were predictors of 1-year survival, with greater prognostic accuracy than total serum albumin concentration. CONCLUSIONS: Extensive posttranscriptional changes of HSA, involving several molecular sites and increasing in parallel with disease severity, occur in patients with cirrhosis. Altered isoforms are independently associated with specific clinical complications, whereas the residual, native HSA isoform independently predicts patient survival. These findings support the concept of the "effective albumin concentration," which implies that the global HSA function is related not only to its serum concentration, but also to the preservation of its structural integrity.

Ischaemia-modified albumin: a marker of bacterial infection in hospitalized patients with cirrhosis.

BACKGROUND & AIMS: Patients with cirrhosis present structural changes of human serum albumin (HSA) affecting non-oncotic functions. Ischaemia-modified albumin (IMA), which reflects the capacity to bind cobalt, has been associated to patient mortality during acute-on-chronic liver failure. This study aimed to assess whether circulating IMA is elevated in advanced cirrhosis and its relationship with severity of cirrhosis and specific complications. METHODS: A total of 127 cirrhotic patients hospitalized for an acute complication of the disease and 44 healthy controls were enrolled. Plasma IMA and IMA to albumin ratio (IMAr) were measured with a cobalt-binding assay. HSA isoforms carrying post-transcriptional molecular changes were assessed with HPLC-ESI-MS. The effect of endotoxemia on IMA was evaluated in rats with CCl4 -cirrhosis. RESULTS: IMA/IMAr is significantly higher in cirrhotic patients than in controls, but no correlations were found with prognostic scores. IMA did not correlate with the altered HSA isoforms. Ascites, renal impairment and hepatic encephalopathy did not influence IMA/IMAr levels. In contrast, IMA/IMAr is significantly higher in infected than non-infected patients. ROC curves showed that IMA/IMAr had similar discriminating performances for bacterial infection as C-reactive protein (CRP). Moreover, CRP and IMA were independently associated with bacterial infection. Consistently, endotoxin injection significantly increased IMA in cirrhotic, but not in healthy rats. CONCLUSIONS: IMA is elevated in patients with advanced cirrhosis. The IMA level does not correlate with disease severity scores, but it is specifically associated to bacterial infection, showing a discriminating performance similar to CRP. Further investigations to assess IMA as a novel diagnostic test for bacterial infection are advocated.

Liver tumorigenicity promoted by microRNA-221 in a mouse transgenic model.

UNLABELLED: MicroRNA-221 (miR-221) is one of the most frequently and consistently up-regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR-221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR-221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down-regulation of miR-221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2-modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules. CONCLUSIONS: This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy.

The endocannabinoid system in advanced liver cirrhosis: pathophysiological implication and future perspectives.

Endogenous cannabinoids (EC) are ubiquitous lipid signalling molecules providing different central and peripheral effects that are mediated mostly by the specific receptors CB1 and CB2. The EC system is highly upregulated during chronic liver disease and consistent experimental and clinical findings indicate that it plays a role in the pathogenesis of liver fibrosis and fatty liver disease associated with obesity, alcohol abuse and hepatitis C. Furthermore, a considerable number of studies have shown that EC and their receptors contribute to the pathogenesis of the cardio-circulatory disturbances occurring in advanced cirrhosis, such as portal hypertension, hyperdynamic circulatory syndrome and cirrhotic cardiomyopathy. More recently, the EC system has been implicated in the development of ascites, hepatic encephalopathy and the inflammatory response related to bacterial infection. Rimonabant, a selective CB1 antagonist, was the first drug acting on the EC system approved for the treatment of obesity. Unfortunately, it has been withdrawn from the market because of its neuropsychiatric side effects. Compounds able to target selectively the peripheral CB1 receptors are under evaluation. In addition, molecules stimulating CB2 receptor or modulating the activity of enzymes implicated in EC metabolism are promising areas of pharmacological research. Liver cirrhosis and the related complications represent an important target for the clinical application of these compounds.

Reversal of liver fibrosis by the antagonism of endocannabinoid CB1 receptor in a rat model of CCl(4)-induced advanced cirrhosis.

The endocannabinoid system is involved in the pathogenesis of liver fibrosis. Although many substances have been proved to reduce fibrosis in experimental models of chronic liver injury, most of them appear to be effective only if given as a prophylactic or early treatment. This study aimed to explore the effect of pharmacological antagonism of the endocannabinoid cannabinoid type 1 (CB1) receptor started after the stage of full-blown cirrhosis had been reached. Wistar-Han rats with carbon tetrachloride (CCl(4))-induced cirrhosis were randomized to receive the CB1 receptor antagonist Rimonabant (10 mg/kg/day) or the vehicle for 2 weeks. Age-matched healthy rats served as controls. Liver fibrosis was assessed using Sirius red staining, hydroxyproline concentration and α-smooth muscle actin expression. Hepatic gene expression of mediators of fibrogenesis and inflammation were evaluated by real-time PCR. We also assessed the hepatic expression of CB1 and CB2 receptors and that of the enzymes implicated in the endocannabinoid metabolism. Fibrosis was significantly reduced in rats treated with Rimonabant compared with rats receiving the vehicle. CB1 receptor antagonism limited the gene upregulation of fibrogenic and inflammatory mediators occurring in untreated cirrhotic rats. CB1 and CB2 receptor expression was increased in cirrhotic animals. Interestingly, pharmacological CB1 receptor antagonism was associated with a further induction of the CB2 receptor expression. Regression of fibrosis can be achieved by pharmacological blockade of the CB1 receptor even when started in an advanced stage of the disease. This effect is associated with the suppression of pro-fibrogenic and inflammatory mediators and may have been indirectly favoured by the induction of CB2 receptor expression.

An innovative hyperbaric hypothermic machine perfusion protects the liver from experimental preservation injury.

PURPOSE: Hypothermic machine perfusion systems seem more effective than the current static storage to prevent cold ischemic liver injury. Thus, we test an innovative hyperbaric hypothermic machine perfusion (HHMP), which combines hyperbaric oxygenation of the preservation solution and continuous perfusion of the graft. METHODS: Rat livers were preserved with Celsior solution according to 4 different modalities: normobaric static preservation; hyperbaric static preservation at 2 atmosphere absolute (ATA); normobaric dynamic preservation, with continuous perfusion; hyperbaric dynamic preservation, with continuous perfusion at 2 ATA. After 24 h cold preservation, we assessed different parameters. RESULTS: Compared to baseline, livers preserved with the current static storage showed severe ultrastructural damage, glycogen depletion and an increased oxidative stress. Normobaric perfused livers showed improved hepatocyte ultrastructure and ameliorated glycogen stores, but they still suffered a significant oxidative damage. The addition of hyperbaric oxygen produces an extra benefit by improving oxidative injury and by inducing endothelial NO synthase (eNOS) gene expression. CONCLUSIONS: Preservation by means of the present innovative HHMP reduced the liver injury occurring after the current static cold storage by lowering glycogen depletion and oxidative damage. Interestingly, only the use of hyperbaric oxygen was associated to a blunted oxidative stress and an increased eNOS gene expression.

[Effect of the pharmacological antagonism of the endocannabinoid receptors on the sodium balance in liver cirrhosis]. / Effetto dell'antagonismo farmacologico dei recettori per gli endocannabinoidi sull'equilibrio idrosodico nella cirrosi epatica. Prospettive terapeutiche.

The endocannabinoids are endogenous mediators implicated in many physiologic and pathophysiologic processes. It has been recently shown that they contribute to the pathogenesis of liver fibrosis and hemodynamic alterations of cirrhosis. The pharmacological modulation of the endocannnabinoid system represents a potential target for the treatment of liver diseases.

Cannabinoid type 1 receptor antagonism delays ascites formation in rats with cirrhosis.

BACKGROUND & AIMS: Endocannabinoids contribute to hemodynamic abnormalities of cirrhosis. Whether this favors renal sodium retention and ascites formation is unknown. We determined whether cannabinoid type 1 receptor antagonism prevents sodium retention and ascites formation in preascitic cirrhotic rats. METHODS: Once renal sodium handling was impaired, rats with carbon tetrachloride-induced cirrhosis were randomized to receive either vehicle or rimonabant (3 [group 1] or 10 [group 2] mg x kg(-1) x day(-1)) for 2 weeks. Natriuresis, sodium intake, and sodium balance were measured daily. At the end of the protocol, systemic hemodynamics, renal blood flow, ascites volume, and liver fibrosis were assessed. RESULTS: A significant reduction in ascites formation (group 1: 54%; group 2: 10%; vehicle: 90%) and volume (group 1: 1.6 +/- 0.3 mL; group 2: 0.5 mL; vehicle: 5.5 +/- 0.8 mL) occurred in treated rats. Rimonabant significantly improved sodium balance during week 2 (group 1: 0.98 +/- 0.08 mmol; group 2: 0.7 +/- 0.08 mmol; vehicle: 3.05 +/- 0.11 mmol). Both treated groups showed lower cardiac output and higher mean arterial pressure, peripheral vascular resistance, and renal blood flow (P < .05). Liver fibrosis was reduced in group 2 by 30% (P < .05 vs vehicle). Mean arterial pressure inversely correlated with sodium balance (R = -0.61; P = .003), but not with fibrosis score. CONCLUSIONS: Rimonabant improves sodium balance and delays decompensation in preascitic cirrhosis. This is achieved though an improvement in systemic and renal hemodynamics, although it cannot be excluded that the antifibrotic effect of the drug may play a role.

Circulating and hepatic endocannabinoids and endocannabinoid-related molecules in patients with cirrhosis.

BACKGROUND/AIMS: Endocannabinoids include anandamide (AEA) and 2-arachidonoylglycerol (2-AG). Endocannabinoid-related molecules like oleoyl-ethanolamine (OEA) and palmitoyl-ethanolamine (PEA) have also been identified. AEA contributes to the pathogenesis of cardiovascular alterations in experimental cirrhosis, but data on the endocannabinoid system in human cirrhosis are lacking. Thus, we aimed to assess whether circulating and hepatic endocannabinoids are upregulated in cirrhotic patients and whether their levels correlate with systemic haemodynamics and liver function. METHODS: The endocannabinoid levels were measured in peripheral and hepatic veins and liver tissue by isotope-dilution liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Systemic haemodynamics were assessed by the transthoracic electrical bioimpedance technique. Portal pressure was evaluated by hepatic venous pressure gradient. RESULTS: Circulating AEA and, to a greater extent, PEA and OEA were significantly higher in cirrhotic patients than in controls. PEA and OEA were also increased in the cirrhotic liver tissue. AEA, OEA and PEA levels were significantly higher in peripheral than in the hepatic veins of cirrhotic patients, while the opposite occurred for 2-AG. Finally, circulating AEA, OEA and PEA correlated with parameters of liver function, such as serum bilirubin and international normalized ratio. No correlations were found with systemic haemodynamics. CONCLUSIONS: The endocannabinoid system is upregulated in human cirrhosis. Peripheral AEA is increased in patients with a high model of end-stage liver disease score and may reflect the extent of liver dysfunction. In contrast, the 2-AG levels, the other major endocannabinoid, are not affected by cirrhosis. The upregulation of the endocannabinoid-related molecules, OEA and PEA, is even greater than that of AEA, prompting pharmacological studies on these compounds.

A novel model of CCl4-induced cirrhosis with ascites in the mouse.

BACKGROUND/AIMS: The current approaches to study the molecular mechanisms involved in the pathophysiology of liver diseases often rely on the use of transgenic mice. However, experimental models of decompensated cirrhosis have not been clearly established in mice. Thus, we aimed to set an efficient and well-tolerated protocol to induce cirrhosis in mice able to progress up to the ascitic stage. METHODS: C57BL/6N mice received CCl(4) subcutaneously, intraperitoneally or by inhalation. In the latter group, gaseous CCl(4) was administered according to three different schedules: increasing exposure times, twice weekly (traditional protocol; TP), short inhalation cycles, twice or three times weekly. RESULTS: Portal hypertension, sodium retention, and ascites developed in all groups between 11 and 15 weeks. Mortality reached 70% in the TP group, but it was only 0-10% with all other protocols. All the inhalation groups had significantly more ascites at sacrifice than those receiving CCl(4) subcutaneously and intraperitoneally. Extensive abdominal adhesions and evidence of enhanced hepatic inflammation, as suggested by the increased gene expression of pro-inflammatory cytokines in liver tissue, were found in the intraperitoneal group, while large granulomas at the injection site and marked neutrophil infiltration of lungs developed in the subcutaneous group. No extra-hepatic damage could be detected in mice inhaling CCl(4). CONCLUSIONS: The use of short cycles of CCl(4) inhalation represents a novel, safe, and effective method to induce decompensated cirrhosis in mice. Intraperitoneal CCl(4) leads instead to abdominal adhesions precluding a correct evaluation of ascites, while subcutaneous CCl(4) causes an unwanted systemic inflammatory response.

The Analysis of Food Intake in Patients with Cirrhosis Waiting for Liver Transplantation: A Neglected Step in the Nutritional Assessment.

Patients with cirrhosis waiting for liver transplantation (LT) frequently present a nutritional disorder, which represents an independent predictor of morbidity and mortality before and after transplantation. Thus, a proper assessment of the food intake by using different methods, such as food records, food frequency questionnaires, and 24 h recall, should be deemed an important step of the nutritional management of these patients. The available published studies indicate that the daily food intake is inadequate in the majority of waitlisted patients. These findings were confirmed by our experience, showing that the daily intake of total calories, proteins and carbohydrates was inadequate in approximately 85-95% of patients, while that of lipids and simple carbohydrates was inadequate in almost 50% of them. These data highlight the need to implement an effective educational program provided by certified nutritionists or dieticians, who should work in close collaboration with the hepatologist to provide a nutritional intervention tailored to the individual patient requirements.

MicroRNA-Based Prophylaxis in a Mouse Model of Cirrhosis and Liver Cancer.

Most hepatocellular carcinomas (HCCs) arise in the context of chronic liver disease and/or cirrhosis. Thus, chemoprevention in individuals at risk represents an important but yet unproven approach. In this study, we investigated the ability of microRNA (miRNA)-based molecules to prevent liver cancer development in a cirrhotic model. To this end, we developed a mouse model able to recapitulate the natural progression from fibrosis to HCC, and then we tested the prophylactic activity of an miRNA-based approach in the model. The experiments were carried out in the TG221 transgenic mouse, characterized by the overexpression of miR-221 in the liver and predisposed to the development of liver tumors. TG221 as well as wild-type mice were exposed to the hepatotoxin carbon tetrachloride (CCl4) to induce chronic liver damage. All mice developed liver cirrhosis, but only TG221 mice developed nodular lesions in 100% of cases within 6 months of age. The spectrum of lesions ranged from dysplastic foci to carcinomas. To investigate miRNA-based prophylactic approaches, anti-miR-221 oligonucleotides or miR-199a-3p mimics were administered to TG221 CCl4-treated mice. Compared to control animals, a significant reduction in number, size, and, most significantly, malignant phenotype of liver nodules was observed, thus demonstrating an important prophylactic action of miRNA-based molecules. In summary, in this article, we not only report a simple model of liver cancer in a cirrhotic background but also provide evidence for a potential miRNA-based approach to reduce the risk of HCC development.

The epigenetically regulated miR-494 associates with stem-cell phenotype and induces sorafenib resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, especially in patients not amenable for curative treatments. The multi-kinase inhibitor sorafenib represents the first-line treatment option for advanced HCC; nevertheless, its effectiveness is limited due to tumor heterogeneity as well as innate or acquired drug resistance, raising the need for new therapeutic strategies. MicroRNAs (miRNAs) involvement in treatment response as well as their safety and efficacy in preclinical models and clinical trials have been widely documented in the oncologic field, including HCC. Here, we identified miR-494 upregulation in a subgroup of human and rat HCCs with stem cell-like characteristics, as well as multiple epigenetic mechanisms involved in its aberrant expression in HCC cell lines and patients. Moreover, we identified p27, puma and pten among miR-494 targets, contributing to speed up cell cycle progression, enhance survival potential in stressful conditions and increase invasive and clonogenic capabilities. MiR-494 overexpression increased sorafenib resistance via mTOR pathway activation in HCC cell lines and, in line, high miR-494 levels associated with decreased sorafenib response in two HCC animal models. A sorafenib-combined anti-miR-494-based strategy revealed an enhanced anti-tumor potential with respect to sorafenib-only treatment in our HCC rat model. In conclusion, our findings suggested miR-494 as a possible therapeutic target as well as a candidate biomarker for patient stratification in advanced HCC.

Therapeutic dosages of aspirin counteract the IL-6 induced pro-tumorigenic effects by slowing down the ribosome biogenesis rate.

Chronic inflammation is a risk factor for the onset of cancer and the regular use of aspirin reduces the risk of cancer development. Here we showed that therapeutic dosages of aspirin counteract the pro-tumorigenic effects of the inflammatory cytokine interleukin(IL)-6 in cancer and non-cancer cell lines, and in mouse liver in vivo. We found that therapeutic dosages of aspirin prevented IL-6 from inducing the down-regulation of p53 expression and the acquisition of the epithelial mesenchymal transition (EMT) phenotypic changes in the cell lines. This was the result of a reduction in c-Myc mRNA transcription which was responsible for a down-regulation of the ribosomal protein S6 expression which, in turn, slowed down the rRNA maturation process, thus reducing the ribosome biogenesis rate. The perturbation of ribosome biogenesis hindered the Mdm2-mediated proteasomal degradation of p53, throughout the ribosomal protein-Mdm2-p53 pathway. P53 stabilization hindered the IL-6 induction of the EMT changes. The same effects were observed in livers from mice stimulated with IL-6 and treated with aspirin. It is worth noting that aspirin down-regulated ribosome biogenesis, stabilized p53 and up-regulated E-cadherin expression in unstimulated control cells also. In conclusion, these data showed that therapeutic dosages of aspirin increase the p53-mediated tumor-suppressor activity of the cells thus being in this way able to reduce the risk of cancer onset, either or not linked to chronic inflammatory processes.

Albumin Homodimers in Patients with Cirrhosis: Clinical and Prognostic Relevance of a Novel Identified Structural Alteration of the Molecule.

Decompensated cirrhosis is associated to extensive post-transcriptional changes of human albumin (HA). This study aims to characterize the occurrence of HA homodimerization in a large cohort of patients with decompensated cirrhosis and to evaluate its association with clinical features and prognosis. HA monomeric and dimeric isoforms were identified in peripheral blood by using a HPLC-ESI-MS technique in 123 cirrhotic patients hospitalized for acute decompensation and 50 age- and sex-comparable healthy controls. Clinical and biochemical parameters were recorded and patients followed up to one year. Among the monomeric isoforms identified, the N- and C-terminal truncated and the native HA underwent homodimerization. All three homodimers were significantly more abundant in patients with cirrhosis, acute-on-chronic liver failure and correlate with the prognostic scores. The homodimeric N-terminal truncated isoform was independently associated to disease complications and was able to stratify 1-year survival. As a result of all these changes, the monomeric native HA was significantly decreased in patients with cirrhosis, being also associated with a poorer prognosis. In conclusion homodimerization is a novel described structural alteration of the HA molecule in decompensated cirrhosis and contributes to the progressive reduction of the monomeric native HA, the only isoform provided of structural and functional integrity.

Mass spectrometry characterization of circulating human serum albumin microheterogeneity in patients with alcoholic hepatitis.

Human serum albumin (HSA) is the most abundant plasma protein, endowed with several biological properties unrelated to its oncotic power, such as antioxidant and free-radicals scavenging activities, binding and transport of many endogenous and exogenous substances, and regulation of endothelial function and inflammatory response. These non-oncotic activities are closely connected to the peculiarly dynamic structure of the albumin molecule. HSA undergoes spontaneous structural modifications, mainly by reaction with oxidants and saccharides; however, patients with cirrhosis show extensive post-transcriptional changes at several molecular sites of HSA, the degree of which parallels the severity of the disease. The present work reports the development and application of an innovative LC-MS analytical method for a rapid and reproducible determination of the relative abundance of HSA isoforms in plasma samples from alcoholic hepatitis (AH) patients. A condition of severe oxidative stress, similar to that observed in AH patients, is associated with profound changes in circulating HSA microheterogeneity. More interestingly, the high resolution provided by the analytical platform allowed the monitoring of novel oxidative products of HSA never reported before.

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.