Ultrasonographic evaluation of the gallbladder motor function in the diagnosis and prognosis of intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is characterized by skin pruritus, elevated liver enzymes, and increased serum total bile acids. Several previous studies have revealed that the fasting and ejection volumes of the gallbladder in cholestasis of pregnancy are greater than those in normal pregnancy. The goal of this study was to explore the gallbladder volume and evaluate the diagnostic and prognostic value of ultrasound in ICP. METHODS: We prospectively recruited a cohort of 60 ICP patients at the First Affiliated Hospital of Xi'an Jiaotong University, Shaanxi, China from January 2020 to December 2021 and compared their data with those from healthy pregnant women (n = 60). The gallbladder volume was evaluated by real-time ultrasound examination after overnight fasting and at 30, 60, 120, and 180 min after a liquid test meal of 200 mL, and the ejection fraction was calculated. Continuous data between two groups were compared by Student's t test. Differences were considered significant for p < 0.05. The diagnostic and prognostic value of the volume and ejection function of the gallbladder was analyzed by the receiver operating characteristic (ROC) curve. RESULTS: The ICP group had significantly higher gallbladder basal volume (43.49 ± 1.34 cm3 vs. 26.66 ± 0.83 cm3, p < 0.01) and higher ejection fraction compared with the healthy group. The ejection fraction higher than 54.55% at 120 min might predict ICP diagnosis with 96.67% sensitivity and 88.33% specificity, and an AUC of 0.9739 (95% CI 0.9521-0.9956), while the gallbladder volume higher than 12.52 cm3 at 60 min might predict ICP severity with 59.18% sensitivity and 72.73% specificity, and an AUC of 0.7319 (95% CI 0.5787-0.8852). CONCLUSION: Our results indicate abnormal volume and ejection function of the gallbladder in patients with ICP. The ejection fraction at 120 min can assist in the diagnosis if ICP exists, and the gallbladder volume at 60 min may assess the degree of severity of ICP.

Lipopolysaccharide Inhibits Autophagy and Promotes Inflammatory Responses via p38 MAPK-Induced Proteasomal Degradation of Atg13 in Hepatic Stellate Cells.

Background: Inflammation plays a critical role in the progression of acute-on-chronic liver failure (ACLF). Atg13 is a vital regulatory component of the ULK1 complex, which plays an essential role in the initiation of autophagy. Previously, hepatic stellate cells (HSCs) were considered to be noninflammatory cells that contribute only to hepatic fibrosis. Recently, it has been found that HSCs can secrete inflammatory cytokines and participate in hepatic inflammation. Autophagy and proteasome-mediated degradation constitute two major means of protein turnover in cells. Autophagy has been shown to regulate inflammation, but it is unclear whether ubiquitin (Ub)-proteasome system (UPS) is involved in inflammatory responses in HSCs during ACLF. Methods: Clinical data were collected from ACLF patients, and surgically resected paraffin-embedded human ACLF liver tissue specimens were collected. The expression of Atg13 was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Secretion of IL-1ß was assessed by ELISA. Atg13 was knocked down by siRNA in LX2 cells. Coimmunoprecipitation assay was used to detect protein binding and polyubiquitination of Atg13. In vitro tests with LX2 cells were performed to explore the effects and regulation of p38 MAPK, Atg13, UPS, autophagy, and inflammation. Results: Serum lipopolysaccharide (LPS) was positively associated with disease severity in ACLF patients, and p38 MAPK was overexpressed in ACLF liver tissue. We evaluated the role of Atg13 in HSC inflammation and explored the possible underlying mechanisms. Inflammatory factors were upregulated via activation of p38 MAPK and inhibition of autophagy in LX-2 cells. Expression of Atg13 was decreased in LPS-incubated LX2 cells. Atg13 knockdown markedly inhibited autophagy and promoted LPS-induced inflammation in LX2 cells. Our in vitro experiments also showed that LPS induced depletion of Atg13 via UPS, and this process was dependent on p38 MAPK. Conclusions: LPS induces proteasomal degradation of Atg13 via p38 MAPK, thereby participating in the aggravation of LPS-induced autophagy inhibition and inflammatory responses in LX2 cells. Atg13 serves as a mediator between autophagy and proteasome. Modulation of Atg13 or proteasome activity might be a novel strategy for treating HSC inflammation.

Serum Nitric Oxide Level Serves as a Potential Prognostic Biomarker in ACLF Patients.

Background and Aim: Fewer than 50% of patients with acute-on-chronic liver failure (ACLF) recover spontaneously, and without liver transplantation, ACLF is associated with high death rates. Nitric oxide (NO) has a role in the pathogenesis of various liver disorders. We investigated if serum NO level could be used as a biomarker to predict the severity and prognosis of patients with ACLF. Methods: Between January 2018 and September 2020, a retrospective cohort of 120 ACLF patients, as well as healthy and cirrhotic controls, was investigated. The serum NO levels were measured using a commercial ELISA kit, and Kaplan-Meier survival analysis was conducted. Results: ACLF patients had significantly higher serum NO levels than healthy and cirrhotic controls. Multivariate analysis indicated that the serum NO level (HR=1.078, 95% CI 1.031-1.126, P<0.01), as well as the Model for End-stage Liver Disease (MELD) score, may be an affordable, easily available, and significant independent predictive marker for mortality. In ACLF patients, a serum NO level of > 53.5 µmol/L was associated with a significant increase in the risk of mortality or liver transplantation. A combination of serum NO level and MELD score to assess the severity and prognosis of ACLF patients showed enhanced performance. Conclusion: Based on serum NO levels at the time of hospital admission, ACLF patients may be divided into high-risk and low-risk groups. The combination of serum NO level and MELD score is more closely linked to the patient's outcome than either value alone. This method might be used to evaluate patient prognoses and select candidates for liver transplantation.

High levels of serum superoxide dismutase as a biomarker of intrahepatic cholestasis of pregnancy in patients with viral hepatitis B.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is characterized by skin pruritus and impaired liver function. Hepatitis B virus (HBV) infection increases the risk of developing ICP. HBV infection is associated with oxidative stress, which has been proven to participate in the development of ICP. The goal of this study was to explore the relationship among HBV, oxidative stress, and ICP, and investigate whether a biomarker of oxidative stress may predict the diagnosis and severity of ICP. METHODS: We induced a retrospective cohort of 70 ICP patients from January 2019 to December 2020, and compared their data with those from healthy pregnant women (n = 70). Serum levels of an oxidative stress marker superoxide dismutase (SOD) were examined using an enzyme-linked immunosorbent assay (ELISA). Diagnostic and prognostic values of serum SOD were analyzed by receiver operating characteristic (ROC) curve. RESULTS: Pregnant women in the ICP group had significantly higher level of serum SOD (243.24 ± 12.57 U/L vs 98.70 ± 2.95 U/L, p < 0.01) and a higher rate of HBV infection (51.53% vs 25.71%, p < 0.05) compared with the control group. HBsAg-positive ICP patients had a higher levels of serum SOD (287.24 ± 19.21 U/L vs 196.65 ± 11.75 U/L, p < 0.01) compared with HBsAg-negative ICP patients. A serum SOD level > 121.4 U/mL might be used to predict ICP, while a serum SOD level > 274.6 U/mL might predict ICP severity. CONCLUSION: HBV infection promotes oxidative stress during the pathogenesis of ICP. Serum levels of SOD could be used to predict ICP diagnosis and severity. Modification of oxidative stress might be a treatment target for ICP.

Prognostic value of plasma level of superoxide dismutase in HBV-related acute-on-chronic liver failure.

BACKGROUND: Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is the most prevalent type of ACLF in China. The mortality rate of HBV-ACLF has decreased in recent years due to advances in treatment therapies; however, it is still above 50%. Many cases of HBV-ACLF are caused by HBV reactivation due to discontinuation of nucleoside analog treatment. The present study focused on plasma levels of superoxide dismutase (SOD) in HBV-ACLF patients and investigated whether the plasma level of SOD is a useful biomarker in assessing disease severity and predicting outcomes of HBV-ACLF patients, including patients treated with Entecavir (ETV) and patients who were withdrawn from ETV treatment. METHODS: Plasma samples and clinical data from 200 HBV-ACLF patients and from age- and sex-matched cirrhotic and healthy controls were collected and analyzed. Plasma levels of SOD were measured using an ELISA commercial kit. RESULTS: Among the HBV-ACLF patients, in the ETV withdrawal group, the mortality rate was higher than in the ETV group (69.95% vs 46.71%, P < 0.05). Moreover, HBV-DNA and SOD plasma levels were higher in the ETV withdrawal group than in the ETV group (Log10(HBV-DNA): 6.49 ± 0.24 vs 4.79 ± 0.14, P < 0.01; SOD: 463.1 ± 27.61 U/mL vs 397.2 ± 10.97 U/mL, P < 0.05). The mortality and liver transplantation rates were significantly higher in HBV-ACLF patients with plasma levels of SOD > 428 U/mL than in patients with plasma SOD levels ≤ 428 U/mL. CONCLUSIONS: Reactivation of HBV and elevated oxidative stress caused by discontinuation of ETV treatment are crucial factors in the pathogenesis of HBV-ACLF. Plasma level of SOD may serve as a useful biomarker in estimating disease severity and predicting outcomes of HBV-ACLF patients who stop ETV treatment.

Thymosin ß4 Protects against Cardiac Damage and Subsequent Cardiac Fibrosis in Mice with Myocardial Infarction.

Background: Inflammation is a critical factor in the development and progression of myocardial infarction and cardiac fibrosis. Thymosin ß4 (Tß4) alleviates the disease process via protective antioxidant and anti-inflammatory mechanisms. Although Tß4 has been shown to have a protective effect in myocardial infarction, its impact on cardiac fibrosis has not been well reported. In this study, we evaluated the influence of exogenous Tß4 on myocardial infarction and cardiac fibrosis and explored the possible underlying mechanism. Methods: Real-time quantitative reverse-transcription PCR (qRT-PCR), immunohistochemistry (IHC), and Western blot were used to analyze Tß4 expression in acute myocardial infarction (AMI) cardiac tissues. The effects of intraperitoneal adeno-associated virus-Tß4 (AAV-Tß4) on ligation-induced AMI in mice were studied using cardiac function parameters, and RT-PCR, Western blot, HE staining, Masson staining, and IHC were used to assess the degree of myocardial fibrosis. The effects of Tß4 were confirmed in vitro using mouse cardiac myocytes and myofibroblasts. Results: Tß4 was shown to be significantly elevated in mice AMI cardiac tissues. In mice, AAV-Tß4 induced exogenous expression of Tß4 significantly reduced oxidative damage, inflammation, cardiac dysfunction, and fibrosis. H2O2 inhibited mitophagy and increased inflammation in mouse cardiac myocytes via oxidative stress, and Tß4 substantially reduced mitophagy inhibition and inflammasome activation in myocytes caused by H2O2. Furthermore, Tß4 decreased cardiac myofibroblast growth and reduced TGF-ß1-induced activation. Conclusions: AAV-Tß4 induced expression of Tß4 reduced inflammation, heart damage, and eventual fibrosis in vivo. Tß4 helped to reduce oxidative stress, promote mitophagy, and alleviate inflammation and fibrosis. Exogenous supplementation of Tß4 might be a promising therapeutic agent for treating myocardial infarction as well as cardiac fibrosis.

Serum superoxide dismutase level is a potential biomarker of disease prognosis in patients with HEV-induced liver failure.

BACKGROUND: Viral hepatitis E clinically ranges from self-limiting hepatitis to lethal liver failure. Oxidative stress has been shown to mediate hepatic inflammation during HBV-induced liver failure. We investigated whether a biomarker of oxidative stress may be helpful in assessing severity and disease outcomes of patients with HEV-induced liver failure. METHODS: Clinical data were obtained from patients with HEV-induced acute viral hepatitis (AVH, n = 30), acute liver failure (ALF, n = 17), and acute-on-chronic liver failure (ACLF, n = 36), as well as from healthy controls (HC, n = 30). The SOD and HMGB1 levels were measured in serum by ELISA. HL-7702 cells were cultured and stimulated by serum from HEV-infected patients or by HMGB1; oxidative status was investigated by CellROX and apoptosis was investigated by flow cytometry. RESULTS: Patients with HEV-induced liver failure (including ALF and ACLF) showed increased SOD levels compared with HEV-AVH patients and healthy controls. SOD levels > 400 U/mL were associated with a significantly higher risk of mortality in HEV-ALF and HEV-ACLF patients. Serum from HEV-infected patients led to ROS accumulation, HMGB1 secretion, and apoptosis in HL-7702 cells. Antioxidant treatment successfully inhibited HEV-induced HMGB1 secretion, and HMGB1 promoted apoptosis in HL-7702 cells. CONCLUSION: HEV increased oxidative stress in the pathogenesis of HEV-induced hepatic diseases. Early testing of serum SOD may serve as a predictor of both HEV-ALF and HEV-ACLF outcomes. Moreover, development of strategies for modulating oxidative stress might be a potential target for treating HEV-induced liver failure patients.

Purity determination of pyributicarb by internal standard correction-high-performance liquid chromatography-quantitative nuclear magnetic resonance.

An internal standard correction-high-performance liquid chromatography-quantitative nuclear magnetic resonance (ISC-HPLC-qNMR) procedure was established as a reliable quantitative method for complex organic compounds with low purity in order to solve the risk of qNMR inaccuracy because of insufficient resolution of impurity peaks from the selected quantitative peak. This method collects a small quantity of target analyte from low-purity organics by LC. After drying and re-dissolving in deuterated solvent containing internal standard, the solution was analyzed by 1H NMR and HPLC. Another solution prepared by accurately weighing unpurified low-purity substance and internal standard was analyzed by HPLC. Based on the theoretical derivation derived from the Beer-Lambert law, using the ratio of the HPLC peak areas of two solutions as correction, the purity was then calculated without the same reference as target analyte. Compared to previous methods with similar selectivity and accuracy, it has advantages such as a less purified sample is required, time for lyophilization is reduced by half, and sample preparation is more controllable. The proposed method was verified by analysis of a suite of six commercially available, high-purity compounds, and the difference of results between it and direct qNMR was within 0.1%. The result of pyributicarb using ISC-HPLC-qNMR was 97.6% (U = 0.5%; k = 2), and the reference value was 97.61% (U = 0.22%; k = 2). The results demonstrate that the proposed method provides a new way for reference material producers to calibrate lower-purity organics and has the potential advantage of accurate quantification of lower-purity organics.

Simultaneous determination of polybrominated diphenyl ethers and hydroxylated analogues in human serum using high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

Polybrominated diphenyl ethers (PBDEs) and their hydroxylated (OH-BDE) analogues are known as contaminants with potential risks to human health. In this work, a HPLC-ICP-MS method was developed for simultaneous determination of four polybrominated diphenyl ethers (BDE-47, -99, -153 and -209) and four hydroxylated analogues (3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, 5'-OH-BDE-99) in human serum. Solid-phase extraction (SPE) was employed as the primary extraction and purification procedure. By using mixtures of acetone and hexane with different rations, OH-PBDEs and PBDEs were efficiently recovered during elution procedure. With additional ultrasonic assisted extraction of PBDEs from solid residue after protein precipitation, the recoveries of investigated analytes ranged from 79.1 %to 89.9%. Due to the characteristics of inductively coupled plasma (ICP), organic matrix effects were effectively eliminated. Good sensitivity and precision were also obtained. The limits of quantification ranged from 0.060 to 0.081 ng mL-1 and inter-/intra-day relative standard deviations were all below 4%. In addition, compound-independent calibration (CIC) was investigated and the spray chamber efficiency might be a key factor for CIC application among different kinds of brominated chemicals. Finally, the developed method was successfully applied for analysis of 20 human serum samples collected from the Tianjin city. This work provided an alternative approach for simultaneous determination of halogenated chemicals by quantification of halogen element with one-time pretreatment, especially for human serum samples with limited sample volume.

Determination of bisphenol analogues in food-contact plastics using diode array detector, charged aerosol detector and evaporative light-scattering detector.

Freshness protection packages and preservative films are widely used food-contact plastic made of polyethylene. Diode array detector (DAD), charged aerosol detector (CAD) and evaporative light-scattering detector (ELSD) were evaluated for determination of 6 bisphenols (bisphenol A, bisphenol S, bisphenol F, bisphenol B, bisphenol AF and tetrabromobisphenol A.) in polyethylene. DAD presented better parameters including limit of quantification (LOQs) ranging from 0.05 to 0.5 µg/g with relative standard deviations (RSDs, n = 5) lower than 1% at two concentration levels. CAD and ELSD are universal detectors with relative consistent response parameters for different analogues which have potential application by using single calibrant for quantification of multiple analytes. Matrix effects were barely observed on three detectors. Samples of freshness protection packages and preservative films were further analyzed and preliminary profiles of bisphenols in products from Beijing market was obtained. Bisphenol S have become most abundant analogue instead of bisphenol A in investigated products.

Development and evaluation of microwave-assisted and ultrasound-assisted methods based on a quick, easy, cheap, effective, rugged, and safe sample preparation approach for the determination of bisphenol analogues in serum and sediments.

Microwave- and ultrasound-assisted methods based on a quick, easy, cheap, effective, rugged, and safe sample preparation approach followed by high-performance liquid chromatography with tandem mass spectrometry were developed for the simultaneous determination of eight bisphenol analogues in serum and sediment. The developed methods provided satisfactory extraction efficiency for the energy provided by microwaves and ultrasound. Compositions of commercial sorbents (primary secondary amine, MgSO4 , octadecyl-modified silica, and graphitized carbon black) were evaluated. The ultrasound-assisted method was suited for serum using primary secondary amine, MgSO4 , and octadecyl-modified silica as sorbents and a mixture of hexane and ethyl acetate as extraction solvent. The microwave-assisted method worked better for sediment with tetrahydrofuran and methanol as solvents and primary secondary amine, MgSO4 , octadecyl-modified silica, and graphitized carbon black as sorbents. Other experimental parameters, such as extraction temperature and time, were also optimized. The inter- and intraday relative standard deviations ranged from 2.7 to 5.5%. The limits of detection were between 0.1 and 1.0 ng/mL for serum and between 0.1 and 0.5 ng/g dry weight for sediment. The proposed methods were successfully applied to seven sediment and 20 human serum samples. The results showed that the developed methods were practical for the analysis and biomonitoring of bisphenols in sera and sediment.

Determination of avermectins by the internal standard recovery correction - high performance liquid chromatography - quantitative Nuclear Magnetic Resonance method.

Quantitative Nuclear Magnetic Resonance (qNMR) is widely used to determine the purity of organic compounds. For the compounds with lower purity especially molecular weight more than 500, qNMR is at risk of error for the purity, because the impurity peaks are likely to be incompletely separated from the peak of major component. In this study, an offline ISRC-HPLC-qNMR (internal standard recovery correction - high performance liquid chromatography - qNMR) was developed to overcome this problem. It is accurate by excluding the influence of impurity; it is low-cost by using common mobile phase; and it extends the applicable scope of qNMR. In this method, a mix solution of the sample and an internal standard was separated by HPLC with common mobile phases, and only the eluents of the analyte and the internal standard were collected in the same tube. After evaporation and re-dissolution, it was determined by qNMR. A recovery correction factor was determined by comparison of the solutions before and after these procedures. After correction, the mass fraction of analyte was constant and it was accurate and precise, even though the sample loss varied during these procedures, or even in bad resolution of HPLC. Avermectin B1a with the purity of ~93% and the molecular weight of 873 was analyzed. Moreover, the homologues of avermectin B1a were determined based on the identification and quantitative analysis by tandem mass spectrometry and HPLC, and the results were consistent with the results of traditional mass balance method. The result showed that the method could be widely used for the organic compounds, and could further promote qNMR to become a primary method in the international metrological systems.

Development, comparison and application of sorbent-assisted accelerated solvent extraction, microwave-assisted extraction and ultrasonic-assisted extraction for the determination of polybrominated diphenyl ethers in sediments.

Fast and selective analytical methods were developed based on sorbent-assisted mechanism and microwave-assisted extraction (SA-MAE), accelerate solvent extraction (SA-ASE) and ultrasonic-assisted extraction (SA-UAE) for the determination of polybrominated diphenyl ethers (PBDEs) in sediments. The experimental parameters, such as extraction conditions and sorbent amounts, were optimized according to Taguchi Orthogonal Arrays. The accuracy of developed SA-methods was a satisfactory ranging from 71% to 118%. The inter/intra-day RSDs were <10% indicating a good method precision. The limits of quantification (LOQ) for target BDEs were ≤1.0ng/g dry weight (dw) with an exception of BDE 209 which was 10.0ng/g dw. The proposed methods were validated by the analysis of PBDEs in standard reference materials (SRM 1944) and the method performances were compared with each other. The results approved the feasibility of SA-methods for PBDEs analysis in sediments. Meanwhile, the optimization processes indicated the mixed sorbents mainly worked on matrix effects elimination. The compositions of sorbents deserved careful optimization because different characteristics of the matrix and extraction intensity may produce various matrix effects. In addition, the developed SA-ASE method was successfully applied on real environmental samples collected from a typical polluted area. The data and calculation suggested local environmental contamination pattern and potential pollution source.

Certified reference materials (GBW09170 and 09171) of creatinine in human serum.

Creatinine is the most widely used clinical marker for assessing renal function. Concentrations of creatinine in human serum need to be carefully checked in order to ensure accurate diagnosis of renal function. Therefore, development of certified reference materials (CRMs) of creatinine in serum is of increasing importance. In this study, two new CRMs (Nos. GBW09170 and 09171) for creatinine in human serum have been developed. They were prepared with mixtures of several dozens of healthy people's and kidney disease patient's serum, respectively. The certified values of 8.10, 34.1 mg/kg for these two CRMs have been assigned by liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) method which was validated by using standard reference material (SRM) of SRM909b (a reference material obtained from National Institute of Standards and Technology, NIST). The expanded uncertainties of certified values for low and high concentrations were estimated to be 1.2 and 1.1%, respectively. The certified values were further confirmed by an international intercomparison for the determination of creatinine in human serum (Consultative Committee for Amount of Substance, CCQM) of K80 (CCQM-K80). These new CRMs of creatinine in human serum pool are totally native without additional creatinine spiked for enrichment. These new CRMs are capable of validating routine clinical methods for ensuring accuracy, reliability and comparability of analytical results from different clinical laboratories. They can also be used for instrument validation, development of secondary reference materials, and evaluating the accuracy of high order clinical methods for the determination of creatinine in human serum.