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OBJECTIVE: The objective of this study was to establish a methodology for determining carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) concentrations in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test results were also used for clinical aging research. METHODS: Human plasma samples were incubated with aqueous perfluorovaleric acid (NFPA), succeeded by precipitation utilizing trichloroacetic acid, hydrolysis facilitated by hydrochloric acid, nitrogen drying, and ultimate re-dissolution utilizing NFPA, followed by filtration. Cotinine-D3 was added as an internal standard. The separation was performed on an Agela Venusil ASB C18 column (50 mm × 4.6 mm, 5 µm) with a 5 mmol/L NFPA and acetonitrile/water of 60:40 (v/v) containing 0.15% formic acid. The multiple reaction monitoring mode was used for detecting CML, CEL, and cotinine-D3, with ion pairs m/z 205.2 > 84.1 (for quantitative) and m/z 205.2 > m/z 130.0 for CML, m/z 219.1 > 84.1 (for quantitative) and m/z 219.1 > m/z 130.1 for CEL, and m/z 180.1 > 80.1 for cotinine-D3, respectively. RESULTS: The separation of CML and CEL was accomplished within a total analysis time of 6 minutes. The retention times of CML, CEL, and cotinine-D3 were 3.43 minutes, 3.46 minutes, and 4.50 minutes, respectively. The assay exhibited linearity in the concentration range of 0.025-1.500 µmol/L, with a lower limit of quantification of 0.025 µmol/L for both compounds. The relative standard deviations of intra-day and inter-day were both below 9%, and the relative errors were both within the range of ±4%. The average recoveries were 94.24% for CML and 97.89% for CEL. CONCLUSION: The results indicate that the developed methodology is fast, highly sensitive, highly specific, reproducible, and suitable for the rapid detection of CML and CEL in clinical human plasma samples. The outcomes of the clinical research project on aging underscored the important indicative significance of these two indicators for research on human aging.
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Lisina , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Lisina/análise , Lisina/química , Cotinina , Gerociência , Produtos Finais de Glicação Avançada/análise , Produtos Finais de Glicação Avançada/química , Cromatografia Líquida de Alta PressãoRESUMO
OBJECTIVE: To study the digestive stability of 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) protein and phosphinothricina cetyltransferase(PAT) protein in simulated gastric fluid. METHODS: The component of simulated gastric fluid was based on the method of target protein digestive stability in simulative gastric and intestinal in national standard of the People's Republic of China(Published by the Ministry of Agriculture No. 869-2-2007). The test model of stability of different protein to digestion in Simulated Gastric Fluid was established by dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis(SDS-PAGE)and western blot. The degradation of EPSPS protein and PAT protein in simulated gastric fluid at different digestion time points were analyzed. RESULTS: The experiment showed that EPSPS protein and PAT protein were completely digested within 15 s in simulated gastric fluid, no any remain of protein was detected by SDS-PAGE and Western blot, indicating that EPSPS protein and PAT protein were easily digested in the simulated gastric. CONCLUSION: EPSPS protein and PAT protein do not have immunogenicity after digestion with simulated gastric fluid.
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Digestão , Proteínas , Western Blotting , China , Eletroforese em Gel de PoliacrilamidaRESUMO
OBJECTIVE: To evaluate the ability for the detection of 5 metal elements in serum in the laboratories of disease prevention and control system. METHODS: The samples for calcium, magnesium, iron, copper and zinc detection were distributed to 48 laboratories of disease prevention and control system. Inductively coupled plasma mass spectrometry( ICP-MS) analysis or self-selected determination method were allowed to use during detection for each laboratory. The results were analyzed by robust statistical analysis and Z value was used to evaluate the detection ability. RESULTS: Of the laboratories involved in the study, 40 reported results of metal elements detection. Among them, 29 laboratories had satisfactory results, and 11 laboratories had unsatisfactory or suspicious results. The laboratory pass rate of this inter-laboratory comparison was60. 4%. CONCLUSION: The detection level of calcium, magnesium, iron, copper and zinc in serum in disease prevention and control system is generally satisfactory, but the detection ability of some laboratories needs to be further improved.
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Monitoramento Ambiental , Poluentes Ambientais/sangue , Metais/sangue , Cálcio , Cobre , Ferro , Magnésio , ZincoRESUMO
Thymic stromal lymphopoietin (TSLP) is a member of the IL-2 cytokine family and has been widely recognized as a master regulator of type 2 inflammatory responses at barrier surfaces. Recent studies found dysregulation of the TSLP-TSLP receptor (TSLPR) pathway is associated with the pathogenesis of not only allergic diseases but also a wide variety of cancers including both solid tumors and hematological tumors. Thus, the blockade of TSLP represents an attractive therapeutic strategy for allergic diseases and cancer. In this study, we report the development of a novel humanized anti-TSLP monoclonal antibody (mAb) HZ-1127. Binding affinity, specificity, and ability of HZ-1127 in inhibiting TSLP were tested. HZ-1127 selectively binds to the TSLP cytokine with high affinity and specificity. Furthermore, HZ-1127 dramatically inhibits TSLP-dependent STAT5 activation and is more potent than Tezepelumab, which is an FDA-approved humanized mAb against TSLP for severe asthma treatment in inhibiting TSLP-induced CCL17 and CCL22 chemokines secretion in human peripheral blood mononuclear cells. Our pre-clinical study demonstrates that HZ-1127 may serve as a potential therapeutic agent for allergic diseases and cancer.
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In this study, we developed a method for determining cotinine and 3-hydroxycotinine in human serum and established a methodology for an in-depth study of tobacco exposure and health. After the proteins in the human serum samples were precipitated with acetonitrile, they were separated on a ZORBAX SB-Phenyl column with a mobile phase of methanol encompassing 0.3% formic acid-water encompassing 0.15% formic acid. The measurement was performed on an API5500 triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Cotinine, 3-hydroxycotinine, and cotinine-d3 isotope internal standards were held for 2.56 minutes, 1.58 minutes, and 2.56 minutes, respectively. In serum, the linear range was 0.05 to 500 ng·mL-1 for cotinine and 0.50 to 1250 ng·mL-1 for 3-hydroxycotinine. The lower limit of quantification (LLOQ) was 0.05 ng·mL-1 and 0.5 ng·mL-1 for cotinine and 3-hydroxycotinine, respectively. The intra-day and inter-day relative standard deviations were <11%, and the relative errors were withinâ ±â 7%. Moreover, the mean extraction recoveries of cotinine and 3-hydroxycotinine were 98.54% and 100.24%, respectively. This method is suitable for the rapid determination of cotinine and 3-hydroxycotinine in human serum because of its rapidity, sensitivity, strong specificity, and high reproducibility. The detection of cotinine levels in human serum allows for the identification of the cutoff value, providing a basis for differentiation between smoking and nonsmoking populations.
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Cotinina , Espectrometria de Massas em Tandem , Humanos , Cotinina/sangue , Cotinina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Reprodutibilidade dos Testes , Limite de DetecçãoRESUMO
Dysregulated elevation of interleukin-6 (IL-6) signaling is implicated in the pathogenesis of multiple pathophysiological states, and the functional neutralization of the IL-6 pathway with monoclonal antibodies has been proven an effective therapeutic method in treating various diseases with abnormally enhanced IL-6 signaling, and its clinical indications are expanding. Here, we report that by using the conventional hybridoma technology and humanization mutation method, we develop a novel humanized anti-IL-6 receptor (IL-6R) antibody-namely, HZ0412a. In our study, we found that HZ0412a exhibits higher binding affinity to soluble recombinant human IL-6R than tocilizumab. Importantly, in contrast to tocilizumab-a humanized anti-IL-6R antibody approved by the US Food and Drug Administration for the treatment of rheumatoid arthritis, juvenile idiopathic arthritis, giant cell arteritis and Castleman's disease-HZ0412a does not significantly affect the binding of IL-6 to IL-6R. Further analysis revealed that HZ0412a prevents IL-6R from binding to gp130 in vitro, while tocilizumab has a minimal effect under the same condition. Using various cell-based assays, we demonstrate that HZ0412a is noninferior to tocilizumab in inhibiting IL-6 signaling. Finally, we showed that HZ0412a is well tolerated in cynomolgus monkeys after a single subcutaneous injection at a dose of 1 or 5 mg/kg. Taken together, our results indicated that HZ0412a targets an epitope on human IL-6R that is different from that of tocilizumab, and the epitope region is essential for the interaction between IL-6R and gp130. This distinctive mode of action plus its high affinity to IL-6R led to the high potency of HZ0412a in suppressing in vitro IL-6 signaling.
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Aim: Liu-Wei-Luo-Bi (LWLB) granules was a Chinese compound prescription for treating diabetic peripheral neuropathy (DPN). The aim of this study was to investigate the effect of LWLB granules on diabetic mice with peripheral neuropathy and to elucidate the potential mechanism based on an untargeted metabolomics approach. Methods: One hundred forty db/db mice were randomly divided into seven groups: the Control group, DPN group, Mudan (MD) granules group, Epalrestat (Epa) group, and the LWLB low, medium, or high dose (LW-l, LW-m, or LW-h) group. After 12 weeks of treatment, body weight, blood glucose, mechanical pain threshold, motor conduction velocity (MCV), sensory conduction velocity (SCV), and Pathological Organization of the Sciatic and Caudal Nerves in mice were measured. Serum samples were collected for untargeted metabolomics analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and multivariate statistics. Disease-related pathways were screened out with function enrichment analyses of candidate biomarkers. Results: LWLB granules can improve the peripheral neuropathy of type 2 diabetic mice with peripheral nerve conduction disorders, mainly through significantly improving the nerve conduction velocity (P < 0.05) and lowering the mechanical pain threshold (P < 0.05). A total of 43 metabolites were identified as potential biomarkers related to the therapeutic effect of LWLB granules. Fifty, 4, and 26; 23, 4, and 22; and 24, 1, and 16 biomarkers were discovered in the LW-l, LW-m, and LW-h groups at the 4th, 6th, and 12th weeks, respectively. Five, three, seven, five, and four metabolic pathways were found in MD, Epa, LW-l, LW-m, and LW-h groups, respectively. The arginine biosynthesis pathway is the overlapping pathway in LW-l, LW-m, and LW-h groups. Conclusion: LWLB granules have an obvious neuroprotective effect on diabetic peripheral neuropathy, and the metabolism mechanism of LWLB is mainly related to the arginine biosynthesis pathway on diabetic db/db mice with peripheral neuropathy.
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PURPOSE: Paclitaxel liposome (Lipusu) is the first commercialized liposomal formulation of paclitaxel. There has been little data collected on the pharmacokinetics (PK) of paclitaxel liposome, especially in relation to patient use. This study aimed to build a population pharmacokinetic (PopPK) model and further explore the exposure-safety relationship for paclitaxel liposome in patients with non-small cell lung cancer (NSCLC). METHODS: Data from 45 patients with a total of 349 plasma concentrations were analyzed. The PopPK model was built using the non-linear mixed effect modeling technique. RESULTS: The PK of paclitaxel liposome were well described by a three-compartment model with first-order elimination. For a dose of 175 mg m-2, the estimated clearance of total plasma paclitaxel was 21.55 L h-1. Age, sex, body weight, total bilirubin, albumin, serum creatinine, and creatinine clearance did not influence the paclitaxel PK. Exposure to paclitaxel had no significant change in the presence of the traditional Chinese medicine, aidi injection. The exploratory exposure-safety relationship was well described by a generalized linear regression model. Higher probabilities of grade >1 neutropenia were observed in patients with higher exposure to paclitaxel. CONCLUSION: This PopPK model adequately described the PK of paclitaxel liposome in patients with NSCLC. Predicted exposure of paclitaxel did not change in the presence of the traditional Chinese medicine, aidi injection. The exposure-safety analysis suggested that a higher risk of neutropenia was correlated with higher exposure to paclitaxel.
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Xiao-xu-ming decoction (XXMD) prescription is a traditional Chinese prescription that has been widely used to treat theoplegia and the sequela of theoplegia. Modern pharmacological research has also indicated that the active fraction from XXMD is able to treat cardiovascular diseases and Alzheimer's disease. In the study reported here, high-performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS) was developed to identify active compounds and their metabolites after oral administration of active fraction from Xiao-xu-ming decoction to rats, using parent mass list triggered data-dependent multiple-stage mass analysis at a resolving power of 100,000 in the external calibration mode. The mass accuracies obtained for full-scan MS were within 2 ppm in most cases. Fifteen constituents were identified in the active fraction from XXMD and the biological samples of rats. The fragmentation behaviors of these constituents were summarized which would be helpful for structural characterization. The profiles of the constituents in the active fraction and biological samples of rats were obtained which provided us with much information for a better understanding of the chemical basis of the pharmacologic actions of XXMD.