Liquid Chromatography-tandem Mass Spectrometry Research Articles

Integrating metabolomics and bioinformatics to reveal the mechanism of Epimedium-induced liver injury.

Epimedium is a traditional Chinese medicine with a wide range of clinical applications; however, there have been numerous reports of adverse reactions in recent years. The most common side effect of Epimedium is liver injury. In this study, the liquid chromatography-mass spectrometry (LC-MS) method has been established to study the components of Epimedium and to identify the components absorbed into the blood of rats. Bioinformatics was used to screen out potential toxic components, and the integrating metabolomics method was used to explore the molecular mechanism of Epimedium-induced liver injury. The chemical constituents of Epimedium were identified by LC-MS, and 62 compounds were obtained, including 57 flavonoids, four organic acids and one alkaloid. The toxicity network of "Epimedium-component-target-liver injury" was constructed using bioinformatics research methods, and then the key hepatotoxic component icaritin was identified. Integrating metabolomics was used to investigate the changes in the metabolic profile of L-02 cells with different durations of icaritin administration compared with the control group, and 106 different metabolites were obtained. A total of 14 potential biomarkers significantly associated with cell survival were screened by Pearson correlation analysis combined with the L-02 cell survival rate. Our study preliminarily revealed the mechanism of hepatotoxicity induced by Epimedium.

Integrating metabolomics and bioinformatics to reveal the mechanism of Epimedium-induced liver injury.

Epimedium is a traditional Chinese medicine with a wide range of clinical applications; however, there have been numerous reports of adverse reactions in recent years. The most common side effect of Epimedium is liver injury. In this study, the liquid chromatography-mass spectrometry (LC-MS) method has been established to study the components of Epimedium and to identify the components absorbed into the blood of rats. Bioinformatics was used to screen out potential toxic components, and the integrating metabolomics method was used to explore the molecular mechanism of Epimedium-induced liver injury. The chemical constituents of Epimedium were identified by LC-MS, and 62 compounds were obtained, including 57 flavonoids, four organic acids and one alkaloid. The toxicity network of "Epimedium-component-target-liver injury" was constructed using bioinformatics research methods, and then the key hepatotoxic component icaritin was identified. Integrating metabolomics was used to investigate the changes in the metabolic profile of L-02 cells with different durations of icaritin administration compared with the control group, and 106 different metabolites were obtained. A total of 14 potential biomarkers significantly associated with cell survival were screened by Pearson correlation analysis combined with the L-02 cell survival rate. Our study preliminarily revealed the mechanism of hepatotoxicity induced by Epimedium.

Associations of endogenous estrogens, plasma Alzheimer’s disease biomarkers, and APOE4 carrier status on regional brain volumes in postmenopausal women

BackgroundWomen carrying the APOE4 allele are at greater risk of developing Alzheimer’s disease (AD) from ages 65–75 years compared to men. To better understand the elevated risk conferred by APOE4 carrier status among midlife women, we investigated the separate and interactive associations of endogenous estrogens, plasma AD biomarkers, and APOE4 carrier status on regional brain volumes in a sample of late midlife postmenopausal women.MethodsParticipants were enrolled in MsBrain, a cohort study of postmenopausal women (n = 171, mean age = 59.4 years, mean MoCA score = 26.9; race = 83.2% white, APOE4 carriers = 40). Serum estrone (E1) and estradiol (E2) levels were assessed using liquid chromatography–tandem mass spectrometry. APOE genotype was determined using TaqMan SNP genotyping assays. Plasma AD biomarkers were measured using single molecule array technology. Cortical volume was measured and segmented by FreeSurfer software using individual T1w MPRAGE images. Multiple linear regression models were conducted to determine whether separate and interactive associations between endogenous estrogen levels, plasma AD biomarkers (Aβ42/Aβ40, Aβ42/p-tau181), and APOE4 carrier status predict regional brain volume (21 regions per hemisphere, selected a priori); and, whether significant interactive associations between estrogens and AD biomarkers on brain volume differed by APOE4 carrier status.ResultsThere was no main effect of APOE4 carrier status on regional brain volumes, endogenous estrogen levels, or plasma AD biomarkers. Estrogens did not associate with regional brain volumes, except for positive associations with left caudal middle frontal gyrus and fusiform volumes. The interactive association of estrogens and APOE4 carrier status on brain volume was not significant for any region. The interactive association of estrogens and plasma AD biomarkers predicted brain volume of several regions. Higher E1 and E2 were more strongly associated with greater regional brain volumes among women with a poorer AD biomarker profile (lower Aβ42/40, lower Aβ42/p-tau181 ratios). In APOE4-stratified analyses, these interactions were driven by non-APOE4 carriers.ConclusionWe demonstrate that the brain volumes of postmenopausal women with poorer AD biomarker profiles benefit most from higher endogenous estrogen levels. These findings are driven by non-APOE4 carriers, suggesting that APOE4 carriers may be insensitive to the favorable effects of estrogens on brain volume in the postmenopause.

Detection of the selective androgen receptor modulator S-23 and its metabolites in equine urine and plasma following oral administration.

S-23 is an arylpropionamide selective androgen receptor modulator that has been investigated in animal models for use as a male hormonal contraceptive but is not yet available therapeutically. S-23 is available alongside other selective androgen receptor modulators (SARMs) to purchase online via uncontrolled sites, sold as supplement products. It has been detected in several human doping cases, highlighting the importance of identifying the best analytical targets for equine doping control. The purpose of this study was to investigate the detection of S-23 and its phase I metabolites in equine urine and plasma following a multiple dose oral administration to two Thoroughbred racehorses. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification, and liquid chromatography-tandem mass spectrometry was used for full sample analysis and generation of urine and plasma profiles. S-23 and seven phase I metabolites were observed in urine following enzyme hydrolysis and solvolysis. The most abundant analyte detected was the hydroxylated 4-amino-2-(trifluoromethyl)benzonitrile metabolite, which also allowed the longest duration of detection in urine from both horses, for up to 360 h following administration. The data suggest that this metabolite was likely to be highly conjugated with both sulphate and glucuronide moieties. In plasma, S-23 and two phase I metabolites were observed. S-23 was the most abundant analyte detected for both horses, allowing detection for up to 143 h post-administration. To the best of the authors' knowledge, this is the first report of S-23 and metabolites in equine urine and plasma samples.

Associations of endogenous estrogens, plasma Alzheimer's disease biomarkers, and APOE4 carrier status on regional brain volumes in postmenopausal women.

Women carrying the APOE4 allele are at greater risk of developing Alzheimer's disease (AD) from ages 65-75 years compared to men. To better understand the elevated risk conferred by APOE4 carrier status among midlife women, we investigated the separate and interactive associations of endogenous estrogens, plasma AD biomarkers, and APOE4 carrier status on regional brain volumes in a sample of late midlife postmenopausal women. Participants were enrolled in MsBrain, a cohort study of postmenopausal women (n = 171, mean age = 59.4 years, mean MoCA score = 26.9; race = 83.2% white, APOE4 carriers = 40). Serum estrone (E1) and estradiol (E2) levels were assessed using liquid chromatography-tandem mass spectrometry. APOE genotype was determined using TaqMan SNP genotyping assays. Plasma AD biomarkers were measured using single molecule array technology. Cortical volume was measured and segmented by FreeSurfer software using individual T1w MPRAGE images. Multiple linear regression models were conducted to determine whether separate and interactive associations between endogenous estrogen levels, plasma AD biomarkers (Aβ42/Aβ40, Aβ42/p-tau181), and APOE4 carrier status predict regional brain volume (21 regions per hemisphere, selected a priori); and, whether significant interactive associations between estrogens and AD biomarkers on brain volume differed by APOE4 carrier status. There was no main effect of APOE4 carrier status on regional brain volumes, endogenous estrogen levels, or plasma AD biomarkers. Estrogens did not associate with regional brain volumes, except for positive associations with left caudal middle frontal gyrus and fusiform volumes. The interactive association of estrogens and APOE4 carrier status on brain volume was not significant for any region. The interactive association of estrogens and plasma AD biomarkers predicted brain volume of several regions. Higher E1 and E2 were more strongly associated with greater regional brain volumes among women with a poorer AD biomarker profile (lower Aβ42/40, lower Aβ42/p-tau181 ratios). In APOE4-stratified analyses, these interactions were driven by non-APOE4 carriers. We demonstrate that the brain volumes of postmenopausal women with poorer AD biomarker profiles benefit most from higher endogenous estrogen levels. These findings are driven by non-APOE4 carriers, suggesting that APOE4 carriers may be insensitive to the favorable effects of estrogens on brain volume in the postmenopause.

Interaction of Serum and Plasma Proteins with Polyelectrolyte Microparticles with Core/Shell and Shell-Only Structures.

Polyelectrolyte microparticles (MPs) synthesized on calcium carbonate cores are considered a promising basis for new drug delivery systems. It is known that microparticles entering a physiological environment absorb proteins on their surface, which can change the properties of the microparticles and alter their functional activity. This study aimed to compare the compositions of the adsorbed protein layer formed on microparticles with the core/shell and shell structures obtained by layer-by-layer deposition. The difference in the microparticle structure was associated with changes in their surface topography and ζ-potential. These microparticles were incubated with human serum or plasma at 37°C for 24 h. The adsorbed proteins were eluted and analyzed by means of SDS-PAGE. The protein composition of the eluates was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS); a total of 357 proteins were identified, and 183 of them were detected in all samples. Our results demonstrate that the relative abundance of proteins of different functional groups (immunoglobulins, complement proteins, and apolipoproteins) varied depending on the structure and surface characteristics of the polyelectrolyte microparticles and the incubation medium. Our findings expand the understanding of the influence of the physicochemical properties of the microparticles on their interaction with proteins, which can help to improve the design of microparticles for drug delivery.

Exploring the Mechanism of Topical Application of Clematis Florida in the Treatment of Rheumatoid Arthritis through Network Pharmacology and Experimental Validation

Clematis Florida (CF) is a folk medicinal herb in the southeast of China, which is traditionally used for treating osteoarticular diseases. However, the mechanism of its action remains unclear. The present study used network pharmacology and experimental validation to explore the mechanism of CF in the treatment of rheumatoid arthritis (RA). Liquid chromatography–mass spectrometry (LC-MS/MS) identified 50 main compounds of CF; then, their targets were obtained from TCMSP, ETCM, ITCM, and SwissTargetPrediction databases. RA disease-related targets were obtained from DisGeNET, OMIM, and GeneCards databases, and 99 overlapped targets were obtained using a Venn diagram. The protein–protein interaction network (PPI), the compound–target network (CT), and the compound–potential target genes–signaling pathways network (CPS) were constructed and analyzed. The results showed that the core compounds were screened as oleanolic acid, oleic acid, ferulic acid, caffeic acid, and syringic acid. The core therapeutic targets were predicted via network pharmacology analysis as PTGS2 (COX-2), MAPK1, NF-κB1, TNF, and RELA, which belong to the MAPK signaling pathway and NF-κB signaling pathway. The animal experiments indicated that topical application of CF showed significant anti-inflammatory activity in a mouse model of xylene-induced ear edema and had strong analgesic effect on acetic acid-induced writhing. Furthermore, in the rat model of adjuvant arthritis (AA), topical administration of CF was able to alleviate toe swelling and ameliorate joint damage. The elevated serum content levels of IL-6, COX-2, TNF-α, IL-1β, and RF caused by adjuvant arthritis were reduced by CF treatment. Western blotting tests showed that CF may regulate the ERK and NF-κB pathway. The results provide a new perspective for the topical application of CF for treatment of RA.

Exploring the Mechanism of Topical Application of Clematis Florida in the Treatment of Rheumatoid Arthritis through Network Pharmacology and Experimental Validation.

Clematis Florida (CF) is a folk medicinal herb in the southeast of China, which is traditionally used for treating osteoarticular diseases. However, the mechanism of its action remains unclear. The present study used network pharmacology and experimental validation to explore the mechanism of CF in the treatment of rheumatoid arthritis (RA). Liquid chromatography-mass spectrometry (LC-MS/MS) identified 50 main compounds of CF; then, their targets were obtained from TCMSP, ETCM, ITCM, and SwissTargetPrediction databases. RA disease-related targets were obtained from DisGeNET, OMIM, and GeneCards databases, and 99 overlapped targets were obtained using a Venn diagram. The protein-protein interaction network (PPI), the compound-target network (CT), and the compound-potential target genes-signaling pathways network (CPS) were constructed and analyzed. The results showed that the core compounds were screened as oleanolic acid, oleic acid, ferulic acid, caffeic acid, and syringic acid. The core therapeutic targets were predicted via network pharmacology analysis as PTGS2 (COX-2), MAPK1, NF-κB1, TNF, and RELA, which belong to the MAPK signaling pathway and NF-κB signaling pathway. The animal experiments indicated that topical application of CF showed significant anti-inflammatory activity in a mouse model of xylene-induced ear edema and had strong analgesic effect on acetic acid-induced writhing. Furthermore, in the rat model of adjuvant arthritis (AA), topical administration of CF was able to alleviate toe swelling and ameliorate joint damage. The elevated serum content levels of IL-6, COX-2, TNF-α, IL-1β, and RF caused by adjuvant arthritis were reduced by CF treatment. Western blotting tests showed that CF may regulate the ERK and NF-κB pathway. The results provide a new perspective for the topical application of CF for treatment of RA.

Long-Term Biodegradation of Polyacrylamide Gel Residues in Mammary Glands: Physico-Chemical Analysis, Chromatographic Detection, and Implications for Chronic Inflammation.

In the past, polyacrylamide hydrogel was a popular choice for breast augmentation filler, and many women underwent mammoplasty with this gel. However, due to frequent complications, the use of polyacrylamide hydrogel in mammoplasty has been banned. Despite this ban, patients experiencing complications still seek medical treatment. The aim of this study was to investigate the fate of the polymer over a defined implantation period. Biopsies of breast implants were obtained from patients with 23 and 27 years of post-mammoplasty. These biopsies were meticulously purified from biological impurities and subjected to analysis using IR spectrometry, liquid chromatography-mass spectrometry, gas chromatography, and differential scanning calorimetry. The findings revealed the presence of polyacrylamide hydrogel residues, along with degradation products, within the infected material. Notably, the low-molecular-weight degradation products revealed via gas chromatography are aggressive and toxic substances capable of inducing chronic inflammation. This study sheds light on the long-term consequences of polyacrylamide hydrogel implantation, highlighting the persistence of harmful degradation products and their role in exacerbating patient complications.

Comparative proteomics in tall fescue to reveal underlying mechanisms for improving Photosystem II thermotolerance during heat stress memory

BackgroundThe escalating impacts of global warming intensify the detrimental effects of heat stress on crop growth and yield. Among the earliest and most vulnerable sites of damage is Photosystem II (PSII). Plants exposed to recurring high temperatures develop heat stress memory, a phenomenon that enables them to retain information from previous stress events to better cope with subsequent one. Understanding the components and regulatory networks associated with heat stress memory is crucial for the development of heat-resistant crops.ResultsPhysiological assays revealed that heat priming (HP) enabled tall fescue to possess higher Photosystem II photochemical activity when subjected to trigger stress. To investigate the underlying mechanisms of heat stress memory, we performed comparative proteomic analyses on tall fescue leaves at S0 (control), R4 (primed), and S5 (triggering), using an integrated approach of Tandem Mass Tag (TMT) labeling and Liquid Chromatography-Mass Spectrometry. A total of 3,851 proteins were detected, with quantitative information available for 3,835 proteins. Among these, we identified 1,423 differentially abundant proteins (DAPs), including 526 proteins that were classified as Heat Stress Memory Proteins (HSMPs). GO and KEGG enrichment analyses revealed that the HSMPs were primarily associated with the “autophagy” in R4 and with “PSII repair”, “HSP binding”, and “peptidase activity” in S5. Notably, we identified 7 chloroplast-localized HSMPs (HSP21, DJC77, EGY3, LHCA4, LQY1, PSBR and DEGP8, R4/S0 > 1.2, S5/S0 > 1.2), which were considered to be effectors linked to PSII heat stress memory, predominantly in cluster 4. Protein-protein interaction (PPI) analysis indicated that the ubiquitin-proteasome system, with key nodes at UPL3, RAD23b, and UCH3, might play a role in the selective retention of memory effectors in the R4 stage. Furthermore, we conducted RT-qPCR validation on 12 genes, and the results showed that in comparison to the S5 stage, the R4 stage exhibited reduced consistency between transcript and protein levels, providing additional evidence for post-transcriptional regulation in R4.ConclusionsThese findings provide valuable insights into the establishment of heat stress memory under recurring high-temperature episodes and offer a conceptual framework for breeding thermotolerant crops with improved PSII functionality.

Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling

BackgroundStem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection.MethodsEVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385.ResultsWe demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs.ConclusionsIn this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.

Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling

BackgroundStem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection.MethodsEVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385.ResultsWe demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs.ConclusionsIn this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titter of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titter of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

Effects of CYP3A4 genetic polymorphisms on the pharmacokinetics and efficacy of perampanel in Chinese pediatric patients with epilepsy

ObjectiveThis study was the first to evaluate the effect of CYP3A4 gene polymorphisms on the plasma concentration and effectiveness of perampanel (PER) in Chinese pediatric patients with epilepsy. MethodsWe enrolled 102 patients for this investigation. The steady-state concentration was determined after patients maintained a consistent PER dosing regimen for at least 21 days. Plasma PER concentrations were measured using liquid chromatography–tandem mass spectrometry. Leftover samples from standard therapeutic drug monitoring were allocated for genotyping analysis. The primary measure of efficacy was the rate of seizure reduction with PER treatment at the final check-up. ResultsThe CYP3A4×10 GC phenotype exhibited the highest average plasma concentration of PER at 491.1 ± 328.1 ng/mL, in contrast to the CC phenotype at 334.0 ± 161.1 ng/mL. The incidence of adverse events was most prominent in the CYP3A4×1 G TT and CYP3A4×10 GC groups, with rates of 77.8 % (7 of 9 patients) and 50.0 % (46 of 92 patients), respectively. Moreover, the percentage of patients for whom PER was deemed ineffective was least in the CYP3A4×1 G TT and CYP3A4×10 CC groups, recorded at 11.1 % (1 of 9 patients) and 10.0 % (1 of 10 patients), respectively. There was a significant correlation between PER plasma concentration and either exposure or toxicity (both with p < 0.05). We suggest a plasma concentration range of 625–900 ng/mL as a suitable reference for PER in Chinese patients with epilepsy. ConclusionThe CYP3A4×10 gene's genetic polymorphisms influence plasma concentrations of PER in Chinese pediatric patients with epilepsy. Given that both efficacy and potential toxicity are closely tied to plasma PER levels, the CYP3A4 genetic phenotype should be factored in when prescribing PER to patients with epilepsy.

Effect of concomitant use of yokukansan on steady-state blood concentrations of donepezil and risperidone in real-world clinical practice.

Yokukansan is one of the most frequently used herbal medicines that can improve the behavioral and psychological symptoms of dementia. In this exploratory study, we investigated whether yokukansan affects the steady-state blood concentrations of donepezil, risperidone, and the major metabolites of both drugs in a real-world clinical setting. A non-randomized, open-label, single-arm study examining drug-drug interactions was conducted. Fifteen dementia patients taking donepezil for at least 4 weeks and eight schizophrenia patients taking risperidone for at least 2 weeks were orally administered 2.5 g of yokukansan three times a day before or between meals, and blood samples were collected before and 8 weeks after starting co-treatment with yokukansan. Plasma concentrations of donepezil, risperidone, and each metabolite were measured using high-performance liquid chromatography-tandem mass spectrometry and compared before and after the 8-week administration of yokukansan. The plasma concentrations of donepezil and its metabolites (6-O-desmethyl-donepezil, 5-O-desmethyl-donepezil, and donepezil-N-oxide), risperidone, and its metabolite paliperidone did not differ before and after the 8-week treatment with yokukansan. The findings of this study show that the concomitant use of yokukansan may have little clinical impact on the steady-state blood levels of donepezil and risperidone in patients with dementia or schizophrenia.

Integrated proteomic and metabolomic profiling reveals novel insights on the inflammation and immune response in HFpEF

BackgroundThe precise mechanisms leading to the development of heart failure with preserved ejection fraction (HFpEF) remain incompletely defined. In this study, an integrative approach utilizing untargeted proteomics and metabolomics was employed to delineate the altered proteomic and metabolomic profiles in patients with HFpEF compared to healthy controls.Materials and methodsData were collected from a prospective cohort consisting of 30 HFpEF participants and 30 healthy controls, matched by gender and age. plasma samples were analyzed by multi-omics platforms. The quantification of plasma proteins and metabolites was performed using data-independent acquisition-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), respectively. Additionally, Proteomic and metabolomic results were analyzed separately and integrated using correlation and pathway analysis. This was followed by the execution of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment studies to elucidate the biological relevance of the observed results.ResultsA total of 46 significantly differentially expressed proteins (DEPs) and 102 differentially expressed metabolites (DEMs) were identified. Then, GO and KEGG pathway enrichment analyses were performed by DEPs and DEMs. Integrated analysis of proteomics and metabolomics has revealed Tuberculosis and African trypanosomiasis pathways that are significantly enriched and the DEPs and DEMs enriched within them, are associated with inflammation and immune response.ConclusionsIntegrated proteomic and metabolomic analyses revealed distinct inflammatory and immune response pathways in HFpEF, highlighting novel therapeutic avenues.

Methotrexate accumulation in target intestinal mucosa and white blood cells differs from non-target red blood cells of patients with Crohn's disease.

Intracellular methotrexate polyglutamates (MTX-PGs) concentrations are measurable in red blood cells (RBCs) during MTX treatment. MTX-PG3 concentrations correlate with efficacy in patients with Crohn's disease (CD). Since RBCs are not involved in pathogenesis of CD and lack extended MTX metabolism, we determined MTX-PGs accumulation in peripheral blood mononuclear cells (PBMCs: effector cells) and intestinal mucosa (target cells) and compared those with RBCs as a potential more precise biomarker. In a multicentre prospective cohort study, blood samples of patients with CD were collected during the first year of MTX therapy. Mucosal biopsies were obtained from non-inflamed rectum and/or inflamed intestine. MTX-PGs concentrations in mucosa, PBMCs and RBCs were measured by liquid chromatography-tandem mass spectrometry. From 80 patients with CD, a total of 27 mucosal biopsies, 9 PBMC and 212 RBC samples were collected. From 12 weeks of MTX therapy onwards, MTX-PG3 was the most predominant species (33%) in RBCs. In PBMCs, the distribution was skewed towards MTX-PG1 (48%), which accounted for an 18 times higher concentration than in RBCs. Long-chain MTX-PGs were highly present in mucosa: 21% of MTX-PGtotal was MTX-PG5. MTX-PG6 was measurable in all biopsies. MTX-PG patterns differ between mucosa, PBMCs and RBCs of patients with CD.

The detection of ACTH and insulin in equine plasma by solid-phase extraction and micro-flow LC/MSMS.

Previous liquid chromatography/mass spectrometry (LC/MS) methods for the detection of insulin and other similar peptide hormones in equine plasma relied on the use of antibody affinity extraction. As a result, these methods were not suitable for routine high-throughput analysis. A solid-phase extraction (SPE) method incorporating size exclusion as well as reversed-phase interactions allows the selective extraction of peptide hormones such as adrenocorticotropic hormone (ACTH), insulin and their synthetic analogues from equine plasma with approximately 80% extraction efficiencies. This extraction was combined with on-column derivatisation with acetic anhydride, followed by tryptic digestion and analysis by micro-LC/MSMS for high-sensitivity peptide hormone detection. The analysis of tryptic peptides provides greater sensitivity and more robust chromatography compared with the analysis of intact insulin and ACTH. For quantitative analysis, isotopically labelled internal standards of target peptides can be prepared in the laboratory through the use of deuterated acetic anhydride. The utility of the method was assessed for the analysis of ACTH and insulin in samples from horses suffering from pituitary pars intermedia dysfunction (PPID).

Plasma lipidomics profiling in predicting the chemo-immunotherapy response in advanced non-small cell lung cancer.

Advanced non-small cell lung cancer (NSCLC) presents significant treatment challenges, with chemo-immunotherapy emerging as a promising approach. This study explores the potential of lipidomic biomarkers to predict responses to chemo-immunotherapy in advanced non-small cell lung cancer (NSCLC) patients. A prospective analysis was conducted on 68 NSCLC patients undergoing chemo-immunotherapy, divided into disease control (DC) and progressive disease (PD) groups based on treatment response. Pre-treatment serum samples were subjected to lipidomic profiling using liquid chromatography-mass spectrometry (LC-MS). Key predictive lipids (biomarkers) were identified through projection to latent structures discriminant analysis. A biomarker combined model and a clinical combined model were developed to enhance the prediction accuracy. The predictive performances of the clinical combined model in different histological subtypes were also performed. Six lipids were identified as the key lipids. The expression levels of PC(16:0/18:2), PC(16:0/18:1), PC(16:0/18:0), CE(20:1), and PC(14:0/18:1) were significantly up-regulated. While the expression level of TAG56:7-FA18:2 was significantly down-regulated. The biomarker combined model demonstrated a receiver operating characteristic (ROC) curve of 0.85 (95% CI: 0.75-0.95) in differentiating the PD from the DC. The clinical combined model exhibited an AUC of 0.87 (95% CI: 0.79-0.96) in differentiating the PD from the DC. The clinical combined model demonstrated good discriminability in DC and PD patients in different histological subtypes with the AUC of 0.78 (95% CI: 0.62-0.96), 0.79 (95% CI: 0.64-0.94), and 0.86 (95% CI: 0.52-1.00) in squamous cell carcinoma, large cell carcinoma, and adenocarcinoma subtype, respectively. Pathway analysis revealed the metabolisms of linoleic acid, alpha-linolenic acid, glycerolipid, arachidonic acid, glycerophospholipid, and steroid were implicated in the chemo-immunotherapy response in advanced NSCLC. Lipidomic profiling presents a highly accurate method for predicting responses to chemo-immunotherapy in patients with advanced NSCLC, offering a potential avenue for personalized treatment strategies.

Plasma lipidomics profiling in predicting the chemo-immunotherapy response in advanced non-small cell lung cancer

BackgroundAdvanced non-small cell lung cancer (NSCLC) presents significant treatment challenges, with chemo-immunotherapy emerging as a promising approach. This study explores the potential of lipidomic biomarkers to predict responses to chemo-immunotherapy in advanced non-small cell lung cancer (NSCLC) patients.MethodsA prospective analysis was conducted on 68 NSCLC patients undergoing chemo-immunotherapy, divided into disease control (DC) and progressive disease (PD) groups based on treatment response. Pre-treatment serum samples were subjected to lipidomic profiling using liquid chromatography-mass spectrometry (LC-MS). Key predictive lipids (biomarkers) were identified through projection to latent structures discriminant analysis. A biomarker combined model and a clinical combined model were developed to enhance the prediction accuracy. The predictive performances of the clinical combined model in different histological subtypes were also performed.ResultsSix lipids were identified as the key lipids. The expression levels of PC(16:0/18:2), PC(16:0/18:1), PC(16:0/18:0), CE(20:1), and PC(14:0/18:1) were significantly up-regulated. While the expression level of TAG56:7-FA18:2 was significantly down-regulated. The biomarker combined model demonstrated a receiver operating characteristic (ROC) curve of 0.85 (95% CI: 0.75–0.95) in differentiating the PD from the DC. The clinical combined model exhibited an AUC of 0.87 (95% CI: 0.79–0.96) in differentiating the PD from the DC. The clinical combined model demonstrated good discriminability in DC and PD patients in different histological subtypes with the AUC of 0.78 (95% CI: 0.62–0.96), 0.79 (95% CI: 0.64–0.94), and 0.86 (95% CI: 0.52–1.00) in squamous cell carcinoma, large cell carcinoma, and adenocarcinoma subtype, respectively. Pathway analysis revealed the metabolisms of linoleic acid, alpha-linolenic acid, glycerolipid, arachidonic acid, glycerophospholipid, and steroid were implicated in the chemo-immunotherapy response in advanced NSCLC.ConclusionLipidomic profiling presents a highly accurate method for predicting responses to chemo-immunotherapy in patients with advanced NSCLC, offering a potential avenue for personalized treatment strategies.