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Introduction: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography–high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. Methods: The present study aimed to validate a simple routine LC–HRMS method to quantify cortisol, cortisone, 6β-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. Results: The LC-HRMS method involved liquid–liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6β-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). Conclusion: A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Nanoparticle (NP) pharmacokinetics significantly differ from traditional small molecule principles. From this emerges the need to create new tools and concepts to harness their full potential and avoid unnecessary risks. Nanoparticle pharmacokinetics strongly depend on size, shape, surface functionalisation, and aggregation state, influencing their biodistribution, accumulation, transformations, and excretion profile, and hence their efficacy and safety. Today, while NP biodistribution and nanoceria biodistribution have been studied often at short times, their long-term accumulation and excretion have rarely been studied. In this work, 3 nm nanoceria at 5.7 mg/kg of body weight was intravenously administrated in a single dose to healthy mice. Biodistribution was measured in the liver, spleen, kidney, lung, brain, lymph nodes, ovary, bone marrow, urine, and faeces at different time points (1, 9, 30, and 100 days). Biodistribution and urinary and faecal excretion were also studied in rats placed in metabolic cages at shorter times. The similarity of results of different NPs in different models is shown as the heterogeneous nanoceria distribution in organs. After the expectable accumulation in the liver and spleen, the concentration of cerium decays exponentially, accounting for about a 50% excretion of cerium from the body in 100 days. Cerium ions, coming from NP dissolution, are most likely excreted via the urinary tract, and ceria nanoparticles accumulated in the liver are most likely excreted via the hepatobiliary route. In addition, nanoceria looks safe and does not damage the target organs. No weight loss or apathy was observed during the course of the experiments.

Abstract Background and aims Cerium oxide nanoparticles are effective scavengers of reactive oxygen species and have been proposed as a treatment for oxidative stress-related diseases. Consequently, we aimed to investigate the effect of these nanoparticles on hepatic regeneration after liver injury by partial hepatectomy and acetaminophen overdose. Methods All the in vitro experiments were performed in HepG2 cells. For the acetaminophen and partial hepatectomy experimental models, male Wistar rats were divided into three groups: (1) nanoparticles group, which received 0.1 mg/kg cerium nanoparticles i.v. twice a week for 2 weeks before 1 g/kg acetaminophen treatment, (2) N-acetyl-cysteine group, which received 300 mg/kg of N-acetyl-cysteine i.p. 1 h after APAP treatment and (3) partial hepatectomy group, which received the same nanoparticles treatment before partial hepatectomy. Each group was matched with vehicle-controlled rats. Results In the partial hepatectomy model, rats treated with cerium oxide nanoparticles showed a significant increase in liver regeneration, compared with control rats. In the acetaminophen experimental model, nanoparticles and N-acetyl-cysteine treatments decreased early liver damage in hepatic tissue. However, only the effect of cerium oxide nanoparticles was associated with a significant increment in hepatocellular proliferation. This treatment also reduced stress markers and increased cell cycle progression in hepatocytes and the activation of the transcription factor NF-κB in vitro and in vivo. Conclusions Our results demonstrate that the nanomaterial cerium oxide, besides their known antioxidant capacities, can enhance hepatocellular proliferation in experimental models of liver regeneration and drug-induced hepatotoxicity.

Circulating levels of 2-hydroxybutyrate (2HB) are highly related to glycemic status in different metabolomic studies. According to recent evidence, 2HB is an early biomarker of the future development of dysglycemia and type 2 diabetes mellitus and may be causally related to the progression of normal subjects to impaired fasting glucose or insulin resistance. In the present study, we developed and validated a simple, specific and sensitive gas chromatography-mass spectrometry (GC-MS) method specifically intended to quantify serum levels of 2HB. Liquid–liquid extraction with ethyl acetate was followed by 2 min of microwave-assisted derivatization. The method presented acceptable accuracy, precision and recovery, and the limit of quantification was 5 µM. Levels of 2HB were found to be stable in serum after three freeze-thaw cycles, and at ambient temperature and at a temperature of 4 °C for up to 24 h. Extracts derivatized under microwave irradiation were stable for up to 96 h. No differences were found in 2HB concentrations measured in serum or plasma EDTA samples. In summary, the method is useful for a rapid, precise and accurate quantification of 2HB in serum samples assessed for the evaluation of dysglycemia and diabetes mellitus.

Oxidative stress induced by the overproduction of free radicals or reactive oxygen species (ROS) has been considered as a key pathogenic mechanism contributing to the initiation and progression of injury in liver diseases. Consequently, during the last few years antioxidant substances, such as superoxide dismutase (SOD), resveratrol, colchicine, eugenol, and vitamins E and C have received increasing interest as potential therapeutic agents in chronic liver diseases. These substances have demonstrated their efficacy in equilibrating hepatic ROS metabolism and thereby improving liver functionality. However, many of these agents have not successfully passed the scrutiny of clinical trials for the prevention and treatment of various diseases, mainly due to their unspecificity and consequent uncontrolled side effects, since a minimal level of ROS is needed for normal functioning. Recently, cerium oxide nanoparticles (CeO2NPs) have emerged as a new powerful antioxidant agent with therapeutic properties in experimental liver disease. CeO2NPs have been reported to act as a ROS and reactive nitrogen species (RNS) scavenger and to have multi-enzyme mimetic activity, including SOD activity (deprotionation of superoxide anion into oxygen and hydrogen peroxide), catalase activity (conversion of hydrogen peroxide into oxygen and water), and peroxidase activity (reducing hydrogen peroxide into hydroxyl radicals). Consequently, the beneficial effects of CeO2NPs treatment have been reported in many different medical fields other than hepatology, including neurology, ophthalmology, cardiology, and oncology. Unlike other antioxidants, CeO2NPs are only active at pathogenic levels of ROS, being inert and innocuous in healthy cells. In the current article, we review the potential of CeO2NPs in several experimental models of liver disease and their safety as a therapeutic agent in humans as well.

Colorectal cancer (CRC) is one of the most common cancers in the developed countries, and nearly 70% of patients with CRC develop colorectal liver metastases (CRLMs). During the last decades, several scores have been proposed to predict recurrence after CRLM resection. However, these risk scoring systems do not accurately reflect the prognosis of these patients. Therefore, this investigation was designed to identify a proteomic profile in human hepatic tumor samples to classify patients with CRLM as “mild” or “severe” based on the 5-year survival. The study was performed on 85 CRLM tumor samples. Firstly, to evaluate any distinct tumor proteomic signatures between mild and severe CRLM patients, a training group of 57 CRLM tumor samples was characterized by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, and a classification and regression tree (CART) analysis was subsequently performed. Finally, 28 CRLM tumor samples were used to confirm and validate the results obtained. Based on all the protein peaks detected in the training group, the CART analysis was generated, and four peaks were considered to be the most relevant to construct a diagnostic algorithm. Indeed, the multivariate model yielded a sensitivity of 85.7% and a specificity of 86.1%, respectively. In addition, the receiver operating characteristic (ROC) curve showed an excellent diagnostic accuracy to discriminate mild from severe CRLM patients (area under the ROC: 0.903). Finally, the validation process yielded a sensitivity and specificity of 68.8% and 83.3%, respectively. We identified a proteomic profile potentially useful to determine the prognosis of CRLM patients based on the 5-year survival.

At present, several procedures are used for staging liver fibrosis. However, these methods may involve clinical complications and/or present diagnostic uncertainty mainly in the early stages of the disease. Thus, this study was designed to unveil new non-invasive biomarkers of liver fibrosis in an in vivo model of fibrosis/cirrhosis induction by CCl4 inhalation by using a label-free quantitative LC-MS/MS approach. We analyzed 94 serum samples from adult Wistar rats with different degrees of liver fibrosis and 36 control rats. Firstly, serum samples from 18 CCl4-treated rats were clustered into three different groups according to the severity of hepatic and the serum proteome was characterized by label-free LC-MS/MS. Furthermore, three different pooled serum samples obtained from 16 control Wistar rats were also analyzed. Based on the proteomic data obtained, we performed a multivariate analysis which displayed three main cell signaling pathways altered in fibrosis. In cirrhosis, more biological imbalances were detected as well as multi-organ alterations. In addition, hemopexin and signal-induced proliferation-associated 1 like 1 (SIPA1L1) were selected as potential serum markers of liver fibrogenesis among all the analyzed proteins. The results were validated by ELISA in an independent group of 76 fibrotic/cirrhotic rats and 20 controls which confirmed SIPA1L1 as a potential non-invasive biomarker of liver fibrosis. In particular, SIPA1L1 showed a clear diminution in serum samples from fibrotic/cirrhotic rats and a great accuracy at identifying early fibrotic stages. In conclusion, the proteomic analysis of serum samples from CCl4-treated rats has enabled the identification of SIPA1L1 as a non-invasive marker of early liver fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic accumulation of lipids. Antisteatotic effects of cerium oxide nanoparticles (CeO2NPs) have recently been shown in animal models of liver disease. However, it is unclear whether the activity of CeO2NPs is related solely to the decrease in oxidative stress or, in addition, they directly decrease liver fatty acid accumulation. To address this question, in this work, we used an in vitro model of hepatocellular steatosis, exposing HepG2 cells to oleic and palmitic acid. Cell uptake of CeO2NPs and their effect on oxidative stress and viability of hepatic cells cultured with H2O2 were also evaluated. Results show that CeO2NPs were uptaken by HepG2 cells and reduced oxidative stress and improved cell viability. Treatment with oleic and palmitic acid increased lipogenesis and the content of different fatty acids. CeO2NPs reduced palmitic and stearic acid and most fatty acids consisting of more than 18 carbon atoms. These effects were associated with significant changes in elongase and desaturase activity. In conclusion, CeO2NPs directly protected HepG2 cells from cell injury in oxidative stress conditions and reduced fatty acid content in steatotic conditions by inducing specific changes in fatty acid metabolism, thus showing potential in the treatment of NAFLD.

Background: The molecular mechanisms by which myocardial ischemia translates into ventricular remodeling remain unclear. Methods: We investigated whether hypoxia and proinflammatory cytokines are specific inducers of remodeling signals in an in vitro model of cultured adult human ventricular myocytes (AC16 cells). Results:Hypoxia modified the ratio of matrix remodeling factors by increasing the aminoterminal propeptide of type III procollagen (PIIINP) and reducing tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1) secretion in AC16 cells. These effects, however, were not associated with either modifications in expression of matrix metalloproteinase type 2, collagen-I or metalloproteinase activity. Hypoxia does, actually increase the production of the cardiac antifibrogenic growth factors, Apelin and VEGF, through an Hypoxia Inducible Factor type 1-dependent mechanism. Concerning proinflammatory signaling pathways, IL1β emerged as a powerful inducer of matrix turnover, since it significantly enhanced PIIINP, TIMP-1 and hyaluronic acid production and increased metalloproteinase activity. In contrast, TNFα did not modify matrix turnover but markedly induced the production of Apelin and VEGF. Conclusion: Hypoxia and increased TNFα activity likely exert cardioprotective actions by activating the cardiac antifibrogenic factors Apelin and VEGF. In contrast, IL1β is a strong promoter of interstitial collagen remodeling that may contribute to ventricular dilation and heart failure in the ischemic myocardium.