ABSTRACT
The treatment and prevention of pathogenic diseases by lactic acid bacteria (LAB) has attracted more and more attention. As a special LAB, Levilactobacillus brevis (L. brevis) has relatively less research on its antibacterial infection in vivo, and its protective effect and mechanism still need to be fully studied. In this study, we selected L. brevis 23017, which can regulate the intestinal immunity of the host animal and resist pathogen infection, to evaluate its protective role and potential molecular mechanisms in the mouse model of S. typhimurium C7731 infection. As expected, we confirmed that L. brevis 23017 reduced the diarrhea rate and increased the daily weight gain and survival rate of the mouse model, and inhibited S. typhimurium colonization in the jejunum and liver. It also reduced the level of oxidative damage and protected the integrity of intestinal tissue by increasing the activity of intestinal antioxidant enzymes (SOD, GSH-Px and T-AOC). From the perspective of intestinal mucosal barrier injury and repair, it was confirmed that L. brevis 23017 could increase the expression levels of intestinal tight junction proteins (ZO-1 and OCLN). Our research results also show that L. brevis 23017 inhibits the inflammatory response and promotes the occurrence of cellular immunity in the body by promoting the increase in IL-10 and inhibiting IL-13 in serum and intestinal tissue. Notably, L. brevis 23017 increased total secretory immunoglobulin A (SIgA) levels in the intestine, which were closely associated with elevated levels of IL-5, IL-13, pIgR, j-chain, and IgAα-chain. In addition, L. brevis 23017 increased the expression of antioxidant proteins Nrf2, NQO1, and HO-1 associated with Nrf2 signaling to inhibit intestinal oxidative damage. This mechanism may be responsible for its protective effect against S. typhimurium-infected intestine. Our study provides new evidence and theoretical support for the analysis of the anti-bacterial infection effect and mechanism of L. brevis, which will contribute to the development of L. brevis and the treatment of pathogenic bacteria intestinal infection.
ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (Bu Gu Zhi) is the fruit of Psoralea corylifolia L. (PCL) and has been used for centuries in traditional Chinese medicine formulas to treat osteoporosis (OP). A new drug called "BX" has been developed from PCL, but its mechanism for treating OP is not yet fully understood. AIM OF THE STUDY: To explore the mechanism of action of BX in the treatment of ovariectomy-induced OP based function-oriented multi-omics analysis of gut microbiota (GM) and metabolites. MATERIALS AND METHODS: C57BL/6 mice were bilaterally ovariectomized to replicate the OP model. The therapeutic efficacy of BX was evaluated by bone parameters (BMD, BV/TV, Tb.N, Tb.Sp), hematoxylin and eosin (H&E) staining results, and determination of bone formation markers procollagen type â amino-terminal peptide (Pâ NP) and bone-specific alkaline phosphatase (BALP). Serum and fecal metabolomics and high-throughput 16S rDNA sequencing were performed to evaluate effects on endogenous metabolites and GM. In addition, an enzyme-based functional correlation algorithm (EBFC) algorithm was used to investigate functional correlations between GM and metabolites. RESULTS: BX improved OP in OVX mice by increasing BMD, BV/TV, serum Pâ NP, BALP, and improving Tb.N and Tb.Sp. A total of 59 differential metabolites were identified, and 9 metabolic pathways, including arachidonic acid metabolism, glycerophospholipid metabolism, purine metabolism, and tryptophan metabolism, were found to be involved in the progression of OP. EBFC analysis results revealed that the enzymes related to purine and tryptophan metabolism, which are from Lachnospiraceae_NK4A136_group, Blautia, Rs-E47_termite_group, UCG-009, and Clostridia_UCG-014, were identified as the intrinsic link between GM and metabolites. CONCLUSIONS: The regulation of GM and restoration of metabolic disorders may be the mechanisms of action of BX in alleviating OP. This research provides insights into the function-oriented mechanism discovery of traditional Chinese medicine in the treatment of OP.
Subject(s)
Coumarins , Gastrointestinal Microbiome , Mice, Inbred C57BL , Osteoporosis , Ovariectomy , Psoralea , Animals , Psoralea/chemistry , Female , Osteoporosis/drug therapy , Coumarins/pharmacology , Coumarins/isolation & purification , Coumarins/therapeutic use , Gastrointestinal Microbiome/drug effects , Mice , Bone Density/drug effects , Metabolomics , Disease Models, Animal , Fruit , MultiomicsABSTRACT
Herein, using an electrophoretic deposition strategy, a S-scheme CdS (cubic)/BiVO4 (monoclinic) heterostructured photocatalyst is fabricated. The as-synthesized photocatalysts exhibit high carrier separation efficiency, prominent hydrogen evolution ability and high stability. The results of the detailed density functional theory (DFT) prove that the photogenerated electrons and holes are located in BiVO4 and CdS components, respectively. Besides, an explicit solvent model based on the electron-enriched region in CdS/BiVO4 heterojunction is designed deliberately to investigate the solid/liquid interface issues. Intriguing findings demonstrate that the surface hydrogen diffusing rate in CdS/BiVO4/H2O is faster than that of BiVO4/H2O and is highly associated with the electron-enrich effect, which has a greater capacity to promote water decomposition, the possibility of proton collision and photocatalytic hydrogen evolution. Notably, the H p orbital can participate in the electron-enrich effect during solvation, thus reforming the orbital energy level and activating the HER of the BiVO4 component in the CdS/BiVO4 system.
ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is globally prevalent and seriously harms the economic efficiency of pig farming. Because of its immunosuppression and high incidence of mutant recombination, PRRSV poses a great challenge for disease prevention and control. Nonstructural protein 2 (NSP2) is the most variable functional protein in the PRRSV genome and can generate NSP2N and NSP2TF variants due to programmed ribosomal frameshifts. These variants are broad and complex in function and play key roles in numerous aspects of viral protein maturation, viral particle assembly, regulation of immunity, autophagy, apoptosis, cell cycle and cell morphology. In this paper, we review the structural composition, programmed ribosomal frameshift and biological properties of NSP2 to facilitate basic research on PRRSV and to provide theoretical support for disease prevention and control and therapeutic drug development.
Subject(s)
Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Animals , Swine , Porcine respiratory and reproductive syndrome virus/genetics , Porcine respiratory and reproductive syndrome virus/metabolism , Viral Nonstructural Proteins/metabolism , Cell Line , Gene Expression , Frameshifting, RibosomalABSTRACT
General anesthesia has been shown to induce significant changes in the functional connectivity of the cerebral cortex. However, traditional methods such as electroencephalography (EEG) or functional magnetic resonance imaging (fMRI) lack the spatial resolution to study the effects of general anesthesia on individual cortical neurons. This study aimed to use high-resolution two-photon imaging, which can provide single-neuron resolution, to investigate the characteristics of consciousness under general anesthesia. We used C57BL/6J and Thy1-GCamp6s mice and found that at similar levels of sedation, as measured by EEG, dexmedetomidine did not significantly inhibit the spontaneous activity of neuronal somata in the S1 cortex, but preserved the frequency of calcium events in neuronal spines. In contrast, propofol and ketamine dramatically inhibited the spontaneous activity of both neuronal somata and spines. The S1 cortex still responded to whisker stimulation under dexmedetomidine anesthesia, but not under propofol or ketamine anesthesia. Our results suggest that dexmedetomidine anesthesia has unique neuronal properties associated with its ability to facilitate easy awakening in the clinic. These findings provide insights into the development of more effective strategies for monitoring consciousness during general anesthesia.
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Simple, rapid and sensitive analysis of drug-derived pollutants is critically valuable for environmental monitoring. Here, taking acetaminophen, hydroquinone and catechol as a study example, a sensor based on an ITO/APTES/r-GO@Au electrode was developed for separate and simultaneous determination of phenolic pollutants. ITO electrodes that are modified with 3-aminopropyltriethoxysilane (APTES), graphene (GO) and Au nanoparticles (Au NPs) can significantly enhance the electronic transport of phenolic pollutants at the electrode surface. The redox mechanisms of phenolic pollutants include the electron transfer with the enhancement of r-GO@Au. The modified ITO electrode exhibits excellent electrical properties to phenolic pollutants and a good linear relationship between ECL intensity and the concentration of phenolic pollutants, with a limit of detection of 0.82, 1.41 and 1.95 µM, respectively. The separate and simultaneous determination of AP, CC and HQ is feasible with the ITO/APTES/r-GO@Au electrode. The sensor shows great promise as a low-lost, sensitive, and rapid method for simultaneous determination of drug-derived pollutants.
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Recording the highly diverse and dynamic activities in large populations of neurons in behaving animals is crucial for a better understanding of how the brain works. To meet this challenge, extensive efforts have been devoted to developing functional fluorescent indicators and optical imaging techniques to optically monitor neural activity. Indeed, optical imaging potentially has extremely high throughput due to its non-invasive access to large brain regions and capability to sample neurons at high density, but the readout speed, such as the scanning speed in two-photon scanning microscopy, is often limited by various practical considerations. Among different imaging methods, light field microscopy features a highly parallelized 3D fluorescence imaging scheme and therefore promises a novel and faster strategy for functional imaging of neural activity. Here, we briefly review the working principles of various types of light field microscopes and their recent developments and applications in neuroscience studies. We also discuss strategies and considerations of optimizing light field microscopy for different experimental purposes, with illustrative examples in imaging zebrafish and mouse brains.
Subject(s)
Microscopy , Neurosciences , Animals , Mice , Microscopy/methods , Zebrafish , Neurons/physiology , Brain/diagnostic imaging , Brain/physiologyABSTRACT
Although the use of the probiotic bacterium Lactobacillus for the treatment and prevention of diseases caused by various pathogenic bacteria has received increasing attention in recent years, its mechanism remains incompletely understood. Levilactobacillus brevis 23017 is a select probiotic strain that can regulate the immunity of host animals and resist pathogen infections. In this study, we analyzed the effect of L. brevis 23017 on Yersinia enterocolitica intestinal infection in a BALB/c mouse model and discussed its underlying mechanism. We found that in the mouse model, L. brevis 23017 prevented the damage of villi in the small intestine and decelerated weight loss after Y. enterocolitica infection. Moreover, we focused on the mechanism of the protective effect of L. brevis 23017 from the perspective of the damage and repair of the intestinal mucosal barrier. We observed that L. brevis 23017 maintained a normal mucosal barrier by altering the expression of tight junction proteins. Notably, our results indicated that L. brevis 23017 effectively promoted the secretion of the intestine-specific secretory immunoglobulin A (SIgA) by B cells via regulating cytokines and oxidative damage levels. This mechanism may be the reason for its protective role in Y. enterocolitica infection. In addition, our results demonstrated that the mechanism of L. brevis 23017 was related to antibacterial colonization and inflammation regulation and closely related to antioxidative stress and SIgA promotion. The protective effect of L. brevis 23017 on mice was related to the signaling pathway protein p38 MAPK and the phosphorylation levels of NF-κB. Our study provided novel insight into the mechanism of Lactobacillus against pathogenic bacterial infections. Such insight is of great importance for the prevention, diagnosis, and treatment of related diseases.
Subject(s)
Yersinia Infections , Yersinia enterocolitica , Animals , Disease Models, Animal , Immunoglobulin A, Secretory , Mice , Mice, Inbred BALB C , NF-kappa B/genetics , NF-kappa B/metabolism , Yersinia enterocolitica/metabolismABSTRACT
Ru(bpy)3Cl2/TPrA is a prominent and widely used ECL system in analytical science. However, the co-reactant TPrA restricts the variety of applications because of its toxicity, volatility, and high cost. Here, we use arginine (Arg) as an alternative co-reactant for Ru(bpy)3 2+ by taking advantage of its low cost, non-toxicity, and biocompatibility. The mechanism of the Ru(bpy)3 2+/Arg system is that the deprotonated Arg can react with Ru(bpy)3 2+ to release emission. The similarity between the Ru(bpy)3 2+/Arg, Ru(bpy)3 2+/TPrA, and Ru(bpy)3 2+/DBAE systems demonstrates that Arg can be used as an alternative co-reactant for Ru(bpy)3 2+ ECL. As a proof of concept, we achieve an excellent performance for acetaminophen (Ace) detection based on the specificity of Arg and Ace, with excellent linearity, low detection limits, and good recoveries. This work is promising to expand the scope of the Ru(bpy)3 2+/Arg system and move forward their applications in bioassays.
ABSTRACT
In present study, a comprehensive strategy integrating multiple chromatographic and chemometric methods to simultaneously characterize the volatile and non-volatile components was developed for the holistic quality evaluation of commercial Agastache rugosa (AR), a common edible and medicinal herb, collected in China. The volatile components and the non-volatile components were characterized by GC-MS and UPLC-QTOF-MS/MS, respectively. And the data were analyzed either independently or integratively by multivariate statistical analysis (MVS) for the quality assessment of commercial samples. The results revealed that the commercial AR samples were different in both the composition and the content of volatile components. However, the compositions of non-volatile components in commercial AR were generally similar, whereas the contents of some components were different. All the results indicated that the holistic quality of commercial AR was inconsistent, and the commercial samples collected could be classified into two main groups, the volatile components were majorly responsible for the classification. Whether or not the holistic quality variations affect the efficacy of AR deserves further investigation.
Subject(s)
Agastache , Plants, Medicinal , Chemometrics , Gas Chromatography-Mass Spectrometry , Tandem Mass SpectrometryABSTRACT
The difficulty of traditional Chinese medicine (TCM) researches lies in the complexity of components, metabolites, and bioactivities. For a long time, there has been a lack of connections among the three parts, which is not conducive to the systematic elucidation of TCM effectiveness. To overcome this problem, a classification-based methodology for simplifying TCM researches was refined from literature in the past 10 years (2011-2020). The theoretical basis of this methodology is set theory, and its core concept is classification. Its starting point is that "although TCM may contain hundreds of compounds, the vast majority of these compounds are structurally similar". The methodology is composed by research strategies for components, metabolites and bioactivities of TCM, which are the three main parts of the review. Technical route, key steps and difficulty are introduced in each part. Two perspectives are highlighted in this review: set theory is a theoretical basis for all strategies from a conceptual perspective, and liquid chromatography-mass spectrometry (LC-MS) is a common tool for all strategies from a technical perspective. The significance of these strategies is to simplify complex TCM researches, integrate isolated TCM researches, and build a bridge between traditional medicines and modern medicines. Potential research hotspots in the future, such as discovery of bioactive ingredients from TCM metabolites, are also discussed. The classification-based methodology is a summary of research experience in the past 10 years. We believe it will definitely provide support and reference for the following TCM researches.
Subject(s)
Chemistry Techniques, Analytical/methods , Chromatography, Liquid , Drugs, Chinese Herbal/chemistry , Mass Spectrometry , Medicine, Chinese Traditional/trends , Chemistry Techniques, Analytical/trends , Humans , Research DesignABSTRACT
Nitric oxide (NO) is a molecule of physiological importance, and the function of NO depends on its concentration in biological systems, particularly in cells. Concentration-based analysis of intracellular NO can provide insight into its precise role in health and disease. However, current methods for detecting intracellular NO are still inadequate for quantitative analysis. In this study, we report a quantitative mass spectrometry probe approach to measure NO levels in cells. The probe, Amlodipine (AML), comprises a Hantzsch ester group that reacts with NO to form a pyridine, Dehydro Amlodipine (DAM). Quantification of DAM by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) allows specific measurement of intracellular NO levels. Notably, the AML/NO reaction proceeds rapidly (within 1 s), which is favorable for NO detection considering its large diffusivity and short half-life. Meanwhile, studies under simulated physiological conditions revealed that the AML response to NO is proportional and selective. The presented UPLC-MS/MS method showed high sensitivity (LLOQ = 0.24 nM) and low matrix interference (less than 15%) in DAM quantification. Furthermore, the mass spectrometry probe approach was demonstrated by enabling the measurement of endogenous and exogenous NO in cells. Hence, the quantitative UPLC-MS/MS method developed using AML as a probe is expected to be a new method for intracellular NO analysis.
Subject(s)
Nitric Oxide , Tandem Mass Spectrometry , Chromatography, High Pressure Liquid , Chromatography, Liquid , Reproducibility of ResultsABSTRACT
Although Cynomorium songaricum Rupr. polysaccharide (CSP) has been examined for its effects on glucose regulation, its underlying mechanism is still unclear. To address this issue, a MS-based lipidomics strategy was developed to gain a system-level understanding of the mechanism of CSP on improving type 2 diabetes mellitus (T2DM). UPLC-QTOF/MS and multivariate statistical tools were used to identify the alteration of serum metabolites associated with T2DM and responses to CSP treatment. As a result, 35 potential biomarkers were found and identified in serum, amongst which 26 metabolites were regulated to normal like levels after the administration of CSP. By analyzing the metabolic pathways, glycerophospholipid metabolism was suggested to be closely involved. These results indicated that the intake of CSP exhibited promising anti-diabetic activity, largely due to the regulation of phospholipid metabolism, including phosphatidylcholines, lysophosphatydylcholines, phosphtatidylethanolamines and sphingomyelins.
Subject(s)
Cynomorium/chemistry , Diabetes Mellitus, Type 2/metabolism , Lipidomics , Polysaccharides/pharmacology , Animals , Humans , Lipid Metabolism/drug effects , Lipids/blood , Male , Mice , Mice, Inbred C57BL , Pancreas/drug effects , Pancreas/metabolism , Plant Extracts/pharmacologyABSTRACT
Neurotransmitter (NT) abnormalities in the enteric nervous system have been reported as crucial roles to regulate the intestinal inflammation and gut immune homeostasis. Capturing quantitative changes at the NT metabolome provides an opportunity to develop an understanding of neuroimmune-mediated inflammation. Given the wide diversity of chemical characterizations in the NTs, only partial coverage of the NT metabolome can be simultaneously quantified in a single-run analysis. Herein, we summarized the distribution of functional groups of compound entries in the NT metabolome. Based on this information, an orthogonal dansyl-labeling and label-free dual pretreatment approach was separately designed to target phenol and amine NTs and tertiary amine and choline NTs. By combining the dansyl-labeled and unlabeled NTs within a single vial, a comprehensive and practical approach was optimized for quantifying high coverage of NT metabolome in a single-run analysis on the reversed-phase C18 column. Method validation indicated good linearity with correlation coefficients (R2) > 0.99, intra- and interday accuracy with relative error < ±20%, and precision with relative standard deviations of ≤15%. With this method, we could simultaneously monitor the alterations of cholines, amines, amino acids, tryptophan and phenylalanine biological pathways in dextran sulphate sodium-induced colitis mice. The measured levels of NT metabolome ranged from 0.0007 to 3.540 µg/mg in intestinal contents and 0.013-154.54 µg/mL in serum samples. The NT metabolism was disrupted by colitis, characterized by the changed NT levels in serum and excessive amino acid NTs accumulation in the intestinal contents. We envisage that the orthogonal approach is of great significance for the comprehensive determination of targeted metabolomics. NTs have the potential to be biomarkers for clinical metabolomics.
Subject(s)
Enteric Nervous System , Metabolomics , Animals , Biomarkers/metabolism , Enteric Nervous System/metabolism , Metabolome , Mice , Neurotransmitter AgentsABSTRACT
[This corrects the article DOI: 10.3389/fphar.2017.00273.].
ABSTRACT
Long chain unsaturated fatty acids (LCUFAs) are emerging as critical contributors to inflammation and its resolution. Sensitive and accurate measurement of LCUFAs in biological samples is thus of great value in disease diagnosis and prognosis. In this work, a fluorous-derivatization approach for UPLC-MS/MS quantification of LCUFAs was developed by employing a pair of fluorous reagents, namely 3-(perfluorooctyl)-propylamine (PFPA) and 2-(perfluorooctyl)-ethylamine (PFEA). With this method, the LCUFAs in biological samples were perfluoroalkylated with PFPA and specifically retained on a fluorous-phase LC column, which largely reduced matrix interferences-induced quantitation deviation. Moreover, PFEA-labeled LCUFAs standards were introduced as one-to-one internal standards to farthest ensure unbiased results. Application of the proposed method enabled a reliable determination of eight typical LCUFAs with high sensitivity (LLOQ ranged from 30 amol to 6.25 fmol) and low matrix interferences (almost less than 10%). Such a high sensitivity could facilitate the determination of small-volume and low-concentration bio-samples. Further metabolic characterization of these targeted LCUFAs was monitored in OVA-induce asthma mice, requiring only 5 µL serum sample. Our results showed that asthmatic attack led to significant disturbances not only in the concentrations but also in the ratio among these LCUFAs. In view of the favorable advantages in sensitivity and accuracy, the present fluorous-paired derivatization approach will be expected to serve as a new avenue for dissecting the physiological and clinical implications of LCUFAs, thereby shedding light on the management of diseases related to their disturbances.
Subject(s)
Asthma , Tandem Mass Spectrometry , Animals , Chromatography, High Pressure Liquid , Chromatography, Liquid , Fatty Acids , Fatty Acids, Unsaturated , MiceABSTRACT
Recent research aimed at the design of mixed-matrix membrane (MMM) to be used for microextraction emphasized on membrane extraction phase with high surface area and porosity. This study explored the influence that surfactants have on MMM extraction efficiency for the first time. The zeolitic imidazolate framework 8-based MMM (ZIF-8-MMM) was synthesized by in situ self-assembly of ZIF-8 on the inner wall of a hollow fiber membrane with the aim of fabricating a microextraction device. By prompting the encapsulation of ionizable analytes in the polar core of reverse micelles, the presence of surfactants in extraction solvent assisted the dissolution of analytes in the fiber membrane lumen and enhanced their adsorption onto ZIF-8. Notably, hereby a microextraction method based on the novel ZIF-8-MMM-reverse micelle (ZIF-8-MMM-RM) system was developed and employed for the extraction and quantitation of two alkaloids (berberine and jatrorrhizine) and two flavonoids (wogonin and wogonoside) in biological samples. The main factors affecting microextraction performance, identity of the extraction solvent, surfactant concentration, sample solution pH and extraction time, were investigated in detail. The method showed good linearity (r2 > 0.99) and repeatability (RSD < 10%), low limits of detection (0.10-0.31 ng mL-1) and high relative recoveries (90.03-98.84%). The enrichment factor values ranged between 48.47 and 54.96. Reverse micelle formation prompted by surfactant addition was demonstrated to effectively assist the extraction of multiple ionizable analytes from biological samples, resulting in a marked improvement of ZIF-8-MMM extraction performance.
ABSTRACT
Mass spectrometry-based approaches enable us to capture changes in the metabolome in biological systems with high sensitivity and resolution. But global MS-based profiling of the bile acids (BAs) submetabolome is still a challenging task. Particularly for unconjugated BAs, the collision-induced dissociation (CID) fragment ions showed low ion intensities which were insufficient for analysis. This study is aimed at the development of an anion attachment MS-based approach for pseudotargeted profiling of the BAs submetabolome. We demonstrated that anion attachment MS with the combination use of ammonia fluoride (NH4F) and formate could provide stable anionic adduct ([M + HCOO]-) with good MS responses for unconjugated BAs. A mechanistic study revealed that the underlying rationale is due to the NH4F-induced approximate matching of attractions between BAs and anion for the 24-carboxyl hydrogen. This 24-carboxyl hydrogen regioselectivity is useful to screen for potential unconjugated BAs from the biological matrix. The stability and regioselectivity of anion attachment allowed the establishment of SRM transitions for unconjugated BAs for the first time. To profile conjugated BAs that come from the conjugation of glycine or taurine at 24-carboxyl hydrogen, specific precursor/fragment ion transitions were used for the detection. Finally, SRM-based UPLC-MS/MS method was developed for the pseudotargeted profiling of the BAs submetabolome with good linearity (r2â¯>â¯0.995) and high sensitivity (0.20-1.37â¯ngâ¯mL-1 for LLOQ). With this method, a total of 83 BAs, covering 45 unconjugated BAs and 38 conjugated BAs, were successfully determined in different biosamples from experimental colitis mice. The BAs metabolism homeostasis was disrupted by colitis, characterized by the decreased BAs levels in serum and excessive BAs accumuation in the gall bladder and colon. Overall, the present anion attachment MS-based approach is sufficiently sensitive and robust to comprehensively measure various BAs.
Subject(s)
Ammonium Compounds/chemistry , Bile Acids and Salts/analysis , Fluorides/chemistry , Metabolomics/methods , Animals , Bile/chemistry , Bile Acids and Salts/chemistry , Chromatography, High Pressure Liquid/methods , Colitis/chemically induced , Colitis/metabolism , Colon/metabolism , Gallbladder/metabolism , Male , Mice, Inbred C57BL , Sodium Dodecyl Sulfate , Spectrometry, Mass, Electrospray Ionization/methods , Sulfasalazine/pharmacologyABSTRACT
With the rapid development of immunometabolism, 2-hydroxyglutarate (2-HG) is being promoted as a key immunometabolite to regulate the immune system. Based on the well-established crosstalk between 2-HG and other immunometabolites, here we firstly constructed a 2-HG metabolic panel by mapping the related metabolic pathways. Quantitative methods to globally monitor 2-HG metabolic panel are of great importance for immunometabolism study. However, the existence of enantiomer hampers the accurate measurement of these immunometabolites. This study addressed an original isotopically-paired chiral derivatization approach for UPLC-MS/MS quantification of 2-HG metabolic panel. To achieve better chromatographic separation, N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was utilized as an optical resolving reagent to form diastereomers. For accurate quantitation, an 18O2-labeled-TSPC reagent was designed and readily synthesized to produce one-to-one internal standards. The developed approach enabled an accurate quantification of 13 immunometabolites in 2-HG metabolic panel with good linearity (R2â¯>â¯0.99) and high sensitivity (0.5-120â¯fmol for LLOQ). With this method, we were able to simultaneously monitor the specific alterations of 2-HG metabolic panel in collagen-induced rheumatoid arthritis (CIA) rats. The measured levels of this panel ranged from 0.02 to 85.14⯵gâ¯g-1 for synovium tissue and 0.012 to 87.75⯵molâ¯L-1 for serum samples. We envisage that the present isotopically-paired chiral derivatization approach will be practicable for different bio-samples to quantitatively profile the amino- and hydroxyl acids submetabolome, especially for the endogenous enantiomers. By virtue of the low cost of reagents and the simple procedure used in the assay, this method could be readily implemented.
Subject(s)
Glutarates/metabolism , Phenylalanine/analogs & derivatives , Animals , Chromatography, Liquid/methods , Female , Isotope Labeling , Limit of Detection , Metabolomics/methods , Oxygen/chemistry , Oxygen Isotopes/chemistry , Rats, Wistar , Reproducibility of Results , Stereoisomerism , Tandem Mass Spectrometry/methodsABSTRACT
Zebrafish is being increasingly used for metabolism and toxicity assessment. The drugs consumed in zebrafish metabolism studies are far less than those used in rat studies. In our study, zebrafish embryos were exposed to icariin, Baohuoside I (BI), Epimedin A (EA), Epimedin B (EB), Epimedin C (EC), Sagittatoside A (SA), Sagittatoside B (SB), and 2''-O-rhamnosylicariside II (SC), respectively, to examine the toxicity and metabolic profiles of these flavonoids. The order of toxicity was SC, SB > EC, SA > BI, icariin, EA, EB. After 24 h exposure to SB and SC, the mortality of zebrafish larvae reached 100% and yolk sac swollen was obvious. Both SC and SB caused severe hepatocellular vacuolization and liver cells degeneration in adult zebrafish after 15 consecutive days' treatment. The metabolic profiles of these flavonoids with trace amount were also monitored in larvae. BI was the common metabolite shared by icariin, EA, EB, SA, and SB, via deglycosylation. Both BI and SC remained as the prototype in the medium, suggesting that it is hard for BI and SC to cleave the rhamnose residue. EC was metabolized into SC and BI in zebrafish, inferring that SC might be responsible for the toxicity observed in EC group. The metabolites of icariin, EA, EB, EC, and BI in zebrafish larvae coincided with results from rats and intestinal flora. These data support the use of this system as a surrogate in predicting metabolites and hepatotoxicity risk, especially for TCM compound with trace amount.