Citrate Dialysate with and without Magnesium Supplementation in Hemodiafiltration: A Comparative Study Versus Acetate.

The choice of dialysate buffer in hemodialysis is crucial, with acetate being widely used despite complications. Citrate has emerged as an alternative because of its favorable effects, yet concerns persist about its impact on calcium and magnesium levels. This study investigates the influence of citrate dialysates (CDs) with and without additional magnesium supplementation on CKD-MBD biomarkers and assesses their ability to chelate divalent metals compared to acetate dialysates (ADs). A prospective crossover study was conducted in a single center, involving patients on thrice-weekly online hemodiafiltration (HDF). The following four dialysates were compared: two acetate-based and two citrate-based. Calcium, magnesium, iPTH, iron, selenium, cadmium, copper, zinc, BUN, albumin, creatinine, bicarbonate, and pH were monitored before and after each dialysis session. Seventy-two HDF sessions were performed on eighteen patients. The CDs showed stability in iPTH levels and reduced post-dialysis total calcium, with no significant increase in adverse events. Magnesium supplementation with CDs prevented hypomagnesemia. However, no significant differences among dialysates were observed in the chelation of other divalent metals. CDs, particularly with higher magnesium concentrations, offer promising benefits, including prevention of hypomagnesemia and stabilization of CKD-MBD parameters, suggesting citrate as a viable alternative to acetate. Further studies are warranted to elucidate long-term outcomes and optimize dialysate formulations. Until then, given our results, we recommend that when a CD is used, it should be used with a 0.75 mmol/L Mg concentration rather than a 0.5 mmol/L one.

Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice.

RNA helicase DHX15 plays a significant role in vasculature development and lung metastasis in vertebrates. In addition, several studies have demonstrated the overexpression of DHX15 in the context of hepatocellular carcinoma. Therefore, we hypothesized that this helicase may play a significant role in liver regeneration, physiology, and pathology. Dhx15 gene deficiency was generated by CRISPR/Cas9 in zebrafish and by TALEN-RNA in mice. AUM Antisense-Oligonucleotides were used to silence Dhx15 in wild-type mice. The hepatocellular carcinoma tumor induction model was generated by subcutaneous injection of Hepa 1-6 cells. Homozygous Dhx15 gene deficiency was lethal in zebrafish and mouse embryos. Dhx15 gene deficiency impaired liver organogenesis in zebrafish embryos and liver regeneration after partial hepatectomy in mice. Also, heterozygous mice presented decreased number and size of liver metastasis after Hepa 1-6 cells injection compared to wild-type mice. Dhx15 gene silencing with AUM Antisense-Oligonucleotides in wild-type mice resulted in 80% reduced expression in the liver and a significant reduction in other major organs. In addition, Dhx15 gene silencing significantly hindered primary tumor growth in the hepatocellular carcinoma experimental model. Regarding the potential use of DHX15 as a diagnostic marker for liver disease, patients with hepatocellular carcinoma showed increased levels of DHX15 in blood samples compared with subjects without hepatic affectation. In conclusion, Dhx15 is a key regulator of liver physiology and organogenesis, is increased in the blood of cirrhotic and hepatocellular carcinoma patients, and plays a key role in controlling hepatocellular carcinoma tumor growth and expansion in experimental models.

Characterisation of the urinary steroid profile of patients with nonfunctioning adrenal incidentalomas: A matched controlled cross-sectional study.

AIM: To identify alterations in steroid metabolism in patients with nonfunctioning adrenal incidentalomas (NFAIs) through the analysis of their urinary steroid profile (USP). METHODS: Cross-sectional study with one study group (NFAIs, cortisol post dexamethasone suppression test [DST] ≤ 1.8 µg/dl [49.7 nmol/L]) and 2 control groups: patients with autonomous cortisol secretion (ACS group, cortisol post-DST > 1.8 µg/dl (49.7 nmol/L) and patients without adrenal tumours (healthy-adrenal group). Twenty-four-hour urine collections for USP measurement (total and free fraction of 51 24 h-urine specimens) were obtained from 73 participants (24 with NFAIs, 24 without AIs, and 25 with ACS). USP was determined by gas chromatography coupled to mass spectrometry. Patients of the three groups were matched according to sex, age (±5 years-old) and body mass index (±5 kg/m2 ). RESULTS: Compared to healthy-adrenal controls, patients with NFAIs had a lower excretion of androgen metabolites (230.5 ± 190.12 vs. 388.7 ± 328.58 µg/24 h, p = .046) and a higher excretion of urinary free cortisol (UFC) (54.3 ± 66.07 vs. 25.4 ± 11.16 µg/24 h, p = .038). UFC was above the reference range in 20.8% of patients in the NFAI, compared to 0% in the healthy-adrenal group (p = .018). Patients with ACS had a higher prevalence of hypertension, dyslipidemia, and diabetes than patients with NFAIs or the control group. A lower excretion of androgen metabolites (218.4 ± 204.24 vs. 231 ± 190 µg/24 h, p = .041) and a nonsignificant higher excretion of glucocorticoid metabolites (2129.6 ± 1195.96 vs. 1550.8 ± 810.03 µg/24 h, p = .180) was found in patients with ACS compared to patients with NFAIs. CONCLUSION: NFAIs seem to secrete a subtle, yet clinically relevant, excess of glucocorticoids. Future studies are needed to confirm our findings; and to identify metabolic alterations associated with an increased cardiometabolic risk.

The Integration of Nanomedicine with Traditional Chinese Medicine: Drug Delivery of Natural Products and Other Opportunities.

The integration of progressive technologies such as nanomedicine with the use of natural products from traditional medicine (TM) provides a unique opportunity for the longed-for harmonization between traditional and modern medicine. Although several actions have been initiated decades ago, a disparity of reasons including some misunderstandings between each other limits the possibilities of a truly complementation. Herein, we analyze some common challenges between nanomedicine and traditional Chinese medicine (TCM). These challenges, if solved in a consensual way, can give a boost to such harmonization. Nanomedicine is a recently born technology, while TCM has been used by the Chinese people for thousands of years. However, for these disciplines, the regulation and standardization of many of the protocols, especially related to the toxicity and safety, regulatory aspects, and manufacturing procedures, are under discussion. Besides, both TCM and nanomedicine still need to achieve a wider social acceptance. Herein, we first briefly discuss the strengths and weaknesses of TCM. This analysis serves to focus afterward on the aspects where TCM and nanomedicine can mutually help to bridge the existing gaps between TCM and Western modern medicine. As discussed, many of these challenges can be applied to TM in general. Finally, recent successful cases in scientific literature that merge TCM and nanomedicine are reviewed as examples of the benefits of this harmonization.

Tcf20 deficiency is associated with increased liver fibrogenesis and alterations in mitochondrial metabolism in mice and humans.

BACKGROUND & AIMS: Transcription co-activator factor 20 (TCF20) is a regulator of transcription factors involved in extracellular matrix remodelling. In addition, TCF20 genomic variants in humans have been associated with impaired intellectual disability. Therefore, we hypothesized that TCF20 has several functions beyond those described in neurogenesis, including the regulation of fibrogenesis. METHODS: Tcf20 knock-out (Tcf20-/- ) and Tcf20 heterozygous mice were generated by homologous recombination. TCF20 gene genotyping and expression was assessed in patients with pathogenic variants in the TCF20 gene. Neural development was investigated by immufluorescense. Mitochondrial metabolic activity was evaluated with the Seahorse analyser. The proteome analysis was carried out by gas chromatography mass-spectrometry. RESULTS: Characterization of Tcf20-/- newborn mice showed impaired neural development and death after birth. In contrast, heterozygous mice were viable but showed higher CCl4 -induced liver fibrosis and a differential expression of genes involved in extracellular matrix homeostasis compared to wild-type mice, along with abnormal behavioural patterns compatible with autism-like phenotypes. Tcf20-/- embryonic livers and mouse embryonic fibroblast (MEF) cells revealed differential expression of structural proteins involved in the mitochondrial oxidative phosphorylation chain, increased rates of mitochondrial metabolic activity and alterations in metabolites of the citric acid cycle. These results parallel to those found in patients with TCF20 pathogenic variants, including alterations of the fibrosis scores (ELF and APRI) and the elevation of succinate concentration in plasma. CONCLUSIONS: We demonstrated a new role of Tcf20 in fibrogenesis and mitochondria metabolism in mice and showed the association of TCF20 deficiency with fibrosis and metabolic biomarkers in humans.

Evaluation of Body Composition in Patients With and Without Adrenal Tumors and Without Overt Hypersecretory Syndromes.

OBJECTIVE: To compare body composition between patients with autonomous cortisol secretion (ACS), those with nonfunctioning adrenal incidentalomas (NFAIs), and control subjects without adrenal tumors. METHODS: A cross-sectional study was performed, incluidng the following 3 groups: patients with ACS (cortisol post-dexamethasone suppression test [DST] >1.8 µg/dL), NFAIs (cortisol post-DST ≤ 1.8 µg/dL), and patients without adrenal tumors (control group). Patients of the 3 groups were matched according to age (±5 years), sex, and body mass index (±5 kg/m2). Body composition was evaluated by bioelectrical impedance and abdominal computed tomography (CT) and urinary steroid profile by gas chromatography mass spectrometry. RESULTS: This study enrolled 25 patients with ACS, 24 with NFAIs, and 24 control subjects. Based on CT images, a weak positive correlation between the serum cortisol level post-DST and subcutaneous fat area (r = 0.3, P =.048) was found. As assessed by bioelectrical impedance, lean mass and bone mass were positively correlated with the excretion of total androgens (r = 0.56, P <.001; and r = 0.58, P <.001, respectively); visceral mass was positively correlated with the excretion of glucocorticoid metabolites and total glucocorticoids (r = 0.28, P =.031; and r = 0.42, P =.001, respectively). Based on CT imaging evaluation, a positive correlation was observed between lean mass and androgen metabolites (r = 0.30, P =.036) and between visceral fat area, total fat area, and visceral/total fat area ratio and the excretion of glucocorticoid metabolites (r = 0.34, P =.014; r = 0.29, P =.042; and r = 0.31, P =.170, respectively). CONCLUSION: The urinary steroid profile observed in adrenal tumors, comprising a low excretion of androgen metabolites and high excretion of glucocorticoid metabolites, is associated with a lower lean mass and bone mass and higher level of visceral mass in patients with adrenal tumors.

Evaluation of Metabolic Changes in Acute Intermittent Porphyria Patients by Targeted Metabolomics.

Acute intermittent porphyria (AIP) is an inherited rare hepatic disorder due to mutations within the hydroxymethylbilane gene. AIP patients with active disease overproduce aminolevulinic acid (ALA) and porphobilinogen (PBG) in the liver which are exported inducing severe neurological attacks. Different hepatic metabolic abnormalities have been described to be associated with this condition. The goal of this research was to explore the metabolome of symptomatic AIP patients by state-of-the art liquid chromatography-tandem mass spectrometry (LC-MS/MS). A case versus control study including 18 symptomatic AIP patients and 33 healthy controls was performed. Plasmatic levels of 51 metabolites and 16 ratios belonging to four metabolic pathways were determined. The results showed that the AIP patients presented significant changes in the two main areas of the metabolome under study: (a) the tryptophan/kynurenine pathway with an increase of tryptophan in plasma together with increase of the kynurenine/tryptophan ratio; and (b) changes in the tricarboxylic acid cycle (TCA) including increase of succinic acid and decrease of the fumaric acid/succinic acid ratio. We performed a complementary in vitro study adding ALA to hepatocytes media that showed some of the effects on the TCA cycle were parallel to those observed in vivo. Our study confirms in plasma previous results obtained in urine showing that AIP patients present a moderate increase of the kynurenine/tryptophan ratio possibly associated with inflammation. In addition, it also reports changes in the mitochondrial TCA cycle that, despite requiring further research, could be associated with an energy misbalance due to sustained overproduction of heme-precursors in the liver.

Validation of a Microwave-Assisted Derivatization Gas Chromatography-Mass Spectrometry Method for the Quantification of 2-Hydroxybutyrate in Human Serum as an Early Marker of Diabetes Mellitus.

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.

Bespoken Nanoceria: An Effective Treatment in Experimental Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Despite the availability of new-generation drugs, hepatocellular carcinoma (HCC) is still the third most frequent cause of cancer-related deaths worldwide. Cerium oxide nanoparticles (CeO2 NPs) have emerged as an antioxidant agent in experimental liver disease because of their antioxidant, anti-inflammatory, and antisteatotic properties. In the present study, we aimed to elucidate the potential of CeO2 NPs as therapeutic agents in HCC. APPROACH AND RESULTS: HCC was induced in 110 Wistar rats by intraperitoneal administration of diethylnitrosamine for 16 weeks. Animals were treated with vehicle or CeO2 NPs at weeks 16 and 17. At the eighteenth week, nanoceria biodistribution was assessed by mass spectrometry (MS). The effect of CeO2 NPs on tumor progression and animal survival was investigated. Hepatic tissue MS-based phosphoproteomics as well as analysis of principal lipid components were performed. The intracellular uptake of CeO2 NPs by human ex vivo perfused livers and human hepatocytes was analyzed. Nanoceria was mainly accumulated in the liver, where it reduced macrophage infiltration and inflammatory gene expression. Nanoceria treatment increased liver apoptotic activity, while proliferation was attenuated. Phosphoproteomic analysis revealed that CeO2 NPs affected the phosphorylation of proteins mainly related to cell adhesion and RNA splicing. CeO2 NPs decreased phosphatidylcholine-derived arachidonic acid and reverted the HCC-induced increase of linoleic acid in several lipid components. Furthermore, CeO2 NPs reduced serum alpha-protein levels and improved the survival of HCC rats. Nanoceria uptake by ex vivo perfused human livers and in vitro human hepatocytes was also demonstrated. CONCLUSIONS: These data indicate that CeO2 NPs partially revert the cellular mechanisms involved in tumor progression and significantly increase survival in HCC rats, suggesting that they could be effective in patients with HCC.

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and ß-Hydroxybutyrate/Acetoacetate in Biological Samples.

The metabolic ratios lactate/pyruvate and ß-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, ß-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and ß-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

Cerium Oxide Nanoparticles: Advances in Biodistribution, Toxicity, and Preclinical Exploration.

Antioxidant nanoparticles have recently gained tremendous attention for their enormous potential in biomedicine. However, discrepant reports of either medical benefits or toxicity, and lack of reproducibility of many studies, generate uncertainties delaying their effective implementation. Herein, the case of cerium oxide is considered, a well-known catalyst in the petrochemistry industry and one of the first antioxidant nanoparticles proposed for medicine. Like other nanoparticles, it is now described as a promising therapeutic alternative, now as threatening to health. Sources of these discrepancies and how this analysis helps to overcome contradictions found for other nanoparticles are summarized and discussed. For the context of this analysis, what has been reported in the liver is reviewed, where many diseases are related to oxidative stress. Since well-dispersed nanoparticles passively accumulate in liver, it represents a major testing field for the study of new nanomedicines and their clinical translation. Even more, many contradictory works have reported in liver either cerium-oxide-associated toxicity or protection against oxidative stress and inflammation. Based on this, finally, the intention is to propose solutions to design improved nanoparticles that will work more precisely in medicine and safely in society.

Potential value of urinary amatoxin quantification in patients with hepatotoxic mushroom poisoning.

BACKGORUND & AIMS: Mushroom poisoning with Amanita phalloides or similar species can lead to liver failure with 10-30% mortality rates. We aimed at defining the prognostic value of urinary amatoxin quantification in patients with hepatotoxic mushroom poisoning. METHODS: Data from 32 patients with hepatotoxic mushroom poisoning (Hospital Clínic Barcelona, 2002-16) in whom urinary amatoxins were determined (ELISA) were retrospectively reviewed. Correlations between urinary amatoxin and collected baseline variables with outcomes including hepatotoxicity (ALT>1000 U/L), severe acute liver injury (ALI, prothrombin <50%), acute liver failure (ALF, ALI and encephalopathy), transplantation/death and hospital length-of-stay, were evaluated. RESULTS: 12/32 patients developed increased aminotransferase activity. Among the 13/32 amatoxin negative patients, 1 developed ALI and 12/13 no hepatotoxicity. Among the 19/32 amatoxin positive patients, 8/19 (42%) developed hepatotoxicity, including 5 who progressed to severe ALI, of whom 3 developed ALF (2 deaths, 1 transplantation). Urinary amatoxin and prothrombin were independent predictors of hepatotoxicity, ALT peak values (along with age) and hospital length-of-stay. In positive amatoxins patients, urinary concentrations > 55 ng/ml (or a baseline prothrombin ≤ 83%), were associated to hepatotoxicity (presented by 8/9 patients with ALT>1000 U/L). Among 5 patients with urinary amatoxin ≥ 70 ng/ml, 4 developed severe ALI. CONCLUSIONS: In patients with hepatotoxic mushroom poisoning, a negative urinary amatoxin quantification within 72h of intake ruled out the risk of hepatotoxicity in 92% of patients, whereas positive urinary amatoxins were associated with hepatotoxicity and severe ALI. Concentrations >55 ng/ml and ≥ 70 ng/ml were predictive of hepatotoxicity and severe ALI, respectively.

Cerium oxide nanoparticles improve liver regeneration after acetaminophen-induced liver injury and partial hepatectomy in rats.

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.

Cerium Oxide Nanoparticles Protect against Oxidant Injury and Interfere with Oxidative Mediated Kinase Signaling in Human-Derived Hepatocytes.

Cerium oxide nanoparticles (CeO2NPs) possess powerful antioxidant properties, thus emerging as a potential therapeutic tool in non-alcoholic fatty liver disease (NAFLD) progression, which is characterized by a high presence of reactive oxygen species (ROS). The aim of this study was to elucidate whether CeO2NPs can prevent or attenuate oxidant injury in the hepatic human cell line HepG2 and to investigate the mechanisms involved in this phenomenon. The effect of CeO2NPs on cell viability and ROS scavenging was determined, the differential expression of pro-inflammatory and oxidative stress-related genes was analyzed, and a proteomic analysis was performed to assess the impact of CeO2NPs on cell phosphorylation in human hepatic cells under oxidative stress conditions. CeO2NPs did not modify HepG2 cell viability in basal conditions but reduced H2O2- and lipopolysaccharide (LPS)-induced cell death and prevented H2O2-induced overexpression of MPO, PTGS1 and iNOS. Phosphoproteomic analysis showed that CeO2NPs reverted the H2O2-mediated increase in the phosphorylation of peptides related to cellular proliferation, stress response, and gene transcription regulation, and interfered with H2O2 effects on mTOR, MAPK/ERK, CK2A1 and PKACA signaling pathways. In conclusion, CeO2NPs protect HepG2 cells from cell-induced oxidative damage, reducing ROS generation and inflammatory gene expression as well as regulation of kinase-driven cell survival pathways.

Translational evidence of prothrombotic and inflammatory endothelial damage in Cushing syndrome after remission.

OBJECTIVE: Sustained evidence from observational studies indicates that after remission of Cushing syndrome (CS) a cardiovascular risk phenotype persists. Here, we performed a translational study in active CS and CS in remission (RCS) to evaluate the subclinical cardiometabolic burden and to explore the direct pro-inflammatory and prothrombotic potential of their sera on the endothelium in an in vitro translational atherothrombotic cell model. PATIENTS: Cross sectional study. The groups were (n = 9/group): I. RCS; II. Active CS (ACS) and III. Controls (CTR), all matched for age, body mass index, sex, without other hormonal deficits. DESIGN: We evaluated in vivo: cardiometabolic profile; endothelial markers (sVCAM-1, NO); endothelial dysfunction (FMD); intima-media thickness and body composition (DEXA). In vitro endothelial cells (EC) were exposed to sera taken from the different subjects to evaluate inflammatory EC response (tisVCAM) and thrombogenicity of the generated extracellular matrix (ECM): von Willebrand factor (VWF) and platelet reactivity. RESULTS: Three of the 9 RCS subjects were on glucocorticoid replacement therapy (GC-RT). Patients on GC-RT had a shorter period of time in stable remission. In vivo analysis ACS showed typically metabolic features, while cardiometabolic markers reached statistical significance for RCS only for Hs-CRP (P < .01). In vitro:EC exposed to ACS and RCS sera displayed increased tisVCAM-1 (P < .01 for ACS and P < .05 for RCS vs CTR), VWF (P < .01 for ACS and P < .05 for RCS vs CTR) and platelet adhesion on ECM (P < .01 for ACC and P < .05 for RCS vs CTR). No statistically significant differences were observed between GC-RT RSC and RCS without GC-RT. CONCLUSIONS: The sera of premenopausal women with CS in remission, without atherothrombotic disease, contain circulatory endothelial deleterious factors with a direct thrombogenic and pro-inflammatory endothelial effect that could increase cardiovascular risk.

The GALA study: relationship between galectin-3 serum levels and short- and long-term outcomes of patients with acute heart failure.

OBJECTIVE: We tested the hypothesis that early measurement of galectin-3 at the emergency department (ED) during an episode of acute heart failure (AHF) allows predicting short- and long-term outcomes. METHODS: We performed an exploratory study including 115 patients consecutively diagnosed with AHF in a single ED. Clinical and analytical variables were recorded. The primary endpoint was 30-day all-cause mortality, and secondary endpoints were 30-day composite outcome (death, rehospitalization or ED reconsultation, whichever first) and 1-year mortality. RESULTS: Seven patients (6.1%) died within 30 days and 43 (37.4%) within 1 year. The 30-day composite endpoint was observed in 21.1% of patients. Galectin-3 was correlated with NT-proBNP and the glomerular filtration rate but not with age and s-cTnI. Measured at time of ED arrival, galectin-3 showed good discriminatory capacity for 30-day mortality (AUC ROC: 0.732; 95% CI 0.512-0.953; p = 0.041) but not for 1-year mortality (0.521; 0.408-0.633; p = 0.722). Patients with galectin-3 concentrations >42 µg/L had an OR = 7.67(95%CI = 1.57-37.53; p = 0.012) for 30-day mortality. Conversely, NT-proBNP only showed predictive capacity for 1-year mortality (0.642; 0.537-0.748; p = 0.014). Patients with NT-proBNP concentrations >5400 ng/L had an OR = 4.34 (95%CI = 1.93-9.77; p < 0.001) for 1-year mortality. These increased short- (galectin-3) and long-term (NT-proBNP) risks remained significant after adjustment for age or renal function. s-cTnI failed in both short- and long term death prediction. No biomarker predicted the short-term composite endpoint. CONCLUSION: These results suggest that galectin-3 could help to monitor the risk of short-term mortality in unselected patients with AHF attended in the ED.

Cerium oxide nanoparticles reduce steatosis, portal hypertension and display anti-inflammatory properties in rats with liver fibrosis.

BACKGROUND & AIMS: Cerium oxide nanoparticles (CeO2NPs) have proven to behave as free radical scavengers and/or anti-inflammatory agents. The aim of the study was to determine whether CeO2NPs display hepatoprotective properties in experimental chronic liver disease. METHODS: Systemic and hepatic effects of nanoparticles were assessed in CCl4-treated rats receiving CeO2NPs or vehicle twice weekly for two weeks and CCl4 treatment was continued for 8 additional weeks. Thereafter, mean arterial pressure and portal pressure (PP) were assessed and serum samples obtained to measure standard hepatic and renal function tests. Organ and subcellular distribution of NPs were assessed using mass spectrometry (ICP-MS) and transmission electron microscopy. Liver samples were obtained to evaluate steatosis, α-SMA expression, macrophage infiltration, apoptosis and mRNA expression of oxidative stress, inflammatory or vasoactive related genes. RESULTS: Most CeO2NPs were located in the liver and it reduced hepatic steatosis, ameliorated systemic inflammatory biomarkers and improved PP without affecting mean arterial pressure. In addition, a marked reduction in mRNA expression of inflammatory cytokines (TNFα, IL1ß, COX-2, iNOS), ET-1 and messengers related to oxidative (Epx, Ncf1, Ncf2) or endoplasmic reticulum (Atf3, Hspa5) stress signaling pathways was observed in the liver of rats receiving CeO2NPs. This was associated with reduced macrophage infiltration and reduced abundance of caspase-3, α-SMA and inflammatory cytokines. CONCLUSIONS: CeO2NPs administration to CCl4-treated rats protects against chronic liver injury by reducing liver steatosis and portal hypertension and markedly attenuating the intensity of the inflammatory response, thereby suggesting that CeO2NPs may be of therapeutic value in chronic liver disease.

Overexpression of angiopoietin-2 in rats and patients with liver fibrosis. Therapeutic consequences of its inhibition.

BACKGROUND & AIMS: Studies in experimental models of cirrhosis showed that anti-angiogenic treatments may be effective for the treatment of liver fibrosis. In this context, angiopoietins are potential therapeutic targets as they are involved in the maintenance and stabilization of newly formed blood vessels. In addition, angiopoietin-2 is expressed in fibrotic livers and its inhibition in tumours results in vessel stability. Therefore, our study was aimed to assess the therapeutic utility of inhibiting angiopoietin-2. METHODS: Circulating levels of angiopoietin-1 and angiopoietin-2 were quantified by ELISA in CCl4 -treated rats and in patients with cirrhosis. In vivo blockade of angiopoietin-2 in rats with liver fibrosis was performed with a chemically programmed antibody, CVX-060. RESULTS: High levels of angiopoietin-2 were found in the systemic and suprahepatic circulation of cirrhotic patients and the ratio angiopoietin-1/angiopoietin-2 inversely correlated with prognostic models for alcoholic liver disease. Chronic treatment of CCl4 -treated rats with CVX-060 was associated with a significant decrease in inflammatory infiltrate, normalization of the hepatic microvasculature and reduction in VCAM-1 vascular expression. The anti-angiopoietin-2 treatment was also associated with less liver fibrosis and with lower levels of circulating transaminases. CVX-060 treatment was not associated with either vascular pruning in healthy tissue or compensatory overexpression of VEGF. CONCLUSIONS: Inhibition of angiopoietin-2 is an effective and safe treatment for liver fibrosis in CCl4 -treated rats, acting mainly through the induction of vessel normalization and the attenuation of hepatic inflammatory infiltrate. Therefore, inhibition of angiopoietin-2 offers a therapeutic alternative for liver fibrosis.

LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

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, 6ß-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 6ß-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.

The use of saliva and blood progesterone to profile the menstrual cycles of youth professional football players.

Background: Understanding individual ovarian hormone cycles and their relationship with health, performance and injuries is highly important to practitioners supporting female athletes. Venous blood sampling is the current gold standard for measuring the ovarian hormones, but the invasive nature of this method presents a major barrier in sport environments. Saliva analysis may offer an alternative method as it is non-invasive, allowing the sample to be collected "in situ", with relative ease, necessary in applied sport environments. Objective: The aims of this study were: (i) To compare the concentration of progesterone between capillary blood and saliva, (ii) To assess the efficacy of weekly measurements of progesterone for determining if ovulation has occurred in elite eumenorrheic football players, and (iii) To establish a saliva criteria cut-off for establishing ovulation and assessing the sensitivity, specificity and accuracy values of the method. Methodology: Twenty-one professional and semi-professional, Spanish league female football players (18.6 ± 1.5 years, 58.1 ± 6.0 kg, 164.0 ± 4.8 cm) with natural menstrual cycles, completed the study. Capillary blood and saliva samples were collected from each participant on twelve occasions each separated by at least 7 days. All samples were collected in the morning, following an overnight fast. Results: According to luteal phase serum progesterone concentrations, 11 out of 21 (52%) players presented with menstrual irregularities (oligomenorrheic n = 6, anovulatory n = 4, amenorrhoeic n = 1). A significant correlation was observed between plasma and saliva progesterone in the estimated eumenorrheic group (r = 0.80, p = <0.001, 95% CI 0.72-0.86). The association between serum and saliva progesterone was weaker in the oligomenorrheic group (r = 0.47, p = <0.001, 95% CI 0.27-0.64) and was not present in the anovulatory or amenorrhoeic groups. Conclusions: Salivary measurements of progesterone are well correlated with capillary blood when taken during eumenorrheic menstrual cycles and presents a viable, non-invasive method of establishing characteristic progesterone fluctuations in applied sport settings. The strength of the association appears to be concentration dependent. A luteal phase saliva progesterone (P4) >50 pg/ml and >1.5× follicular baseline has good sensitivity, specificity, and accuracy to indicate ovulation compared to established criteria for serum progesterone.