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
1
H 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.
Abstract
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and predicting outcomes of HBV-ACLF patients, including patients treated with Entecavir (ETV) and patients who were withdrawn from ETV treatment.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 (
Log
10
(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.
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.
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.
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.
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.
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.
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.
The ICP group had significantly higher gallbladder basal volume (43.49 ± 1.34 cm
vs. 26.66 ± 0.83 cm
, 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 cm
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).
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.
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.
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.
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.
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.
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-1β 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.
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.
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•Low cost HPLC-qNMR by common mobile phase; replace to deuterated solvent after HPLC.•Accurate assessment of complex compound with low purity by excluding all impurities.•Internal standard recovery correction method was used to correct the sample loss.•Assessment for highly overlapped peaks in NMR and incompletely resolved HPLC peaks.•The result is accurate and precise, though the sample loss varied during replacement.
Background. Inflammation is a critical factor in the development and progression of myocardial infarction and cardiac fibrosis. Thymosin β4 (Tβ4) alleviates the disease process via protective ...antioxidant and anti-inflammatory mechanisms. Although Tβ4 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 Tβ4 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 Tβ4 expression in acute myocardial infarction (AMI) cardiac tissues. The effects of intraperitoneal adeno-associated virus-Tβ4 (AAV-Tβ4) 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 Tβ4 were confirmed in vitro using mouse cardiac myocytes and myofibroblasts. Results. Tβ4 was shown to be significantly elevated in mice AMI cardiac tissues. In mice, AAV-Tβ4 induced exogenous expression of Tβ4 significantly reduced oxidative damage, inflammation, cardiac dysfunction, and fibrosis. H2O2 inhibited mitophagy and increased inflammation in mouse cardiac myocytes via oxidative stress, and Tβ4 substantially reduced mitophagy inhibition and inflammasome activation in myocytes caused by H2O2. Furthermore, Tβ4 decreased cardiac myofibroblast growth and reduced TGF-β1-induced activation. Conclusions. AAV-Tβ4 induced expression of Tβ4 reduced inflammation, heart damage, and eventual fibrosis in vivo. Tβ4 helped to reduce oxidative stress, promote mitophagy, and alleviate inflammation and fibrosis. Exogenous supplementation of Tβ4 might be a promising therapeutic agent for treating myocardial infarction as well as cardiac fibrosis.
In this paper, in order to clarify the influence of structural parameters of laminates on the modal characteristics of high-aspect-ratio wings under prestress, the CFD/CSD coupling method was used to ...study the modal characteristics of nonlinear structures under the influence of layering angle, layering unbalanced coefficient, and layering reference direction. The results show that the first six order modal frequencies of wing structure increase with the increase of layering angle, and the increment change of frequency increases with the increase of layering angle. The frequency results of positive and negative layering angles are basically the same, and there is no great difference. The modal frequency of airfoil structure is not very sensitive to the change of unbalanced coefficient. The modal frequencies obtained by the mixed angle layering scheme are obviously larger than those obtained by the layering scheme composed of only two angles. With the change of the reference direction of laying, the lowest frequency is generally present in the 0° reference direction and has one or two minima in each order of modal frequency. The layer reference direction angle mainly in the second quadrant is beneficial to the enhancement of modal frequency. After determining the related layer parameters, the appropriate adjustment of the layer reference direction will be beneficial to change the vibration characteristics of the wing structure.
The use of redundant binary (RB) arithmetic in the design of high-speed digital multipliers is beneficial due to its high modularity and carry-free addition. To reduce the number of partial products, ...a high-radix-modified Booth encoding algorithm is desired. However, its use is hampered by the complexity of generating the hard multiples and the overheads resulting from negative multiples and normal binary (NB) to RB number conversion. This paper proposes a new RB Booth encoding scheme to circumvent these problems. The idea is to polarize two adjacent Booth encoded digits to directly form an RB partial product to avoid the hard multiple of high-radix Booth encoding without incurring any correction vector. The proposed method leads to lower encoding and decoding complexity than the recently proposed RB Booth encoder. Synthesis results using Artisan TSMC 0.18-mum standard-cell library show that the RB multipliers designed with our proposed Booth encoding algorithm exhibit on average 14% higher speed and 17% less energy-delay product than the existing multiplication algorithms for a gamut of power-of-two word lengths from 8 to 64 b.
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