Liquid Chromatography-tandem Mass Spectrometry Research Articles

Gut microbiota induced abnormal amino acids and their correlation with diabetic retinopathy

AIM: To explore the correlation of gut microbiota and the metabolites with the progression of diabetic retinopathy (DR) and provide a novel strategy to elucidate the pathological mechanism of DR. METHODS: The fecal samples from 32 type 2 diabetes patients with proliferative retinopathy (PDR), 23 with non-proliferative retinopathy (NPDR), 27 without retinopathy (DM), and 29 from the sex-, age- and BMI- matched healthy controls (29 HC) were analyzed by 16S rDNA gene sequencing. Sixty fecal samples from PDR, DM, and HC groups were assayed by untargeted metabolomics. Fecal metabolites were measured using liquid chromatography-mass spectrometry (LC-MS) analysis. Associations between gut microbiota and fecal metabolites were analyzed. RESULTS: A cluster of 2 microbiome and 12 metabolites accompanied with the severity of DR, and the close correlation of the disease progression with PDR-related microbiome and metabolites were found. To be specific, the structure of gut microbiota differed in four groups. Diversity and richness of gut microbiota were significantly lower in PDR and NPDR groups, than those in DM and HC groups. A cluster of microbiome enriched in PDR group, including Pseudomonas, Ruminococcaceae-UCG-002, Ruminococcaceae-UCG-005, Christensenellaceae-R-7, was observed. Functional analysis showed that the glucose and nicotinate degradations were significantly higher in PDR group than those in HC group. Arginine, serine, ornithine, and arachidonic acid were significantly enriched in PDR group, while proline was enriched in HC group. Functional analysis illustrated that arginine biosynthesis, lysine degradation, histidine catabolism, central carbon catabolism in cancer, D-arginine and D-ornithine catabolism were elevated in PDR group. Correlation analysis revealed that Ruminococcaceae-UCG-002 and Christensenellaceae-R-7 were positively associated with L-arginine, ornithine levels in fecal samples. CONCLUSION: This study elaborates the different microbiota structure in the gut from four groups. The relative abundance of Ruminococcaceae-UCG-002 and Parabacteroides are associated with the severity of DR. Amino acid and fatty acid catabolism is especially disordered in PDR group. This may help provide a novel diagnostic parameter for DR, especially PDR.

(+)-Borneol Protects Dopaminergic Neuronal Loss in Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Parkinson's Disease Mice: A Study of Dopamine Level using In Vivo Brain Microdialysis.

Considerable research efforts have been directed toward the symptom relief of Parkinson's disease (PD) by attenuating dopamine (DA) depletion. One common feature of these existing therapies is their unavailability of preventing the neurodegenerative process of dopaminergic neurons. (+)-Borneol, a natural highly lipid-soluble bicyclic monoterpene, has been reported to regulate the levels of monoamine neurotransmitters in the central nervous system and exhibit neuroprotective effects. However, the effect of (+)-borneol on the dopaminergic neuronal loss of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice is not defined. Herein, we first report that 30 mg/kg (+)-borneol significantly attenuated the motor deficits of PD mice, which benefits from markedly increasing the level of DA and decreasing the metabolic rate of DA in the striatum of conscious and freely moving mouse detected by ultraperformance liquid chromatography tandem mass spectrometry online combined with in vivo brain microdialysis sampling. It is worth noting that the enhanced level of DA by (+)-borneol was enabled by the reduction in loss of tyrosine hydroxylase-immunoreactive dopaminergic neurons in the substantia nigra and striatum and promotion of reserpine- or nomifensine-induced DA release in PD mice. Interestingly, (+)-borneol evidently inhibited the decreased expression levels of DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) on the MPTP mouse model of PD. Moreover, (+)-borneol suppressed the neuroinflammation by inhibiting the production of IL-1β, IL-6, and TNF-α and attenuated oxidative stress by decreasing the level of MDA and increasing the activities of SOD and GSH-px in PD mice. These findings demonstrate that (+)-borneol protects DA neurons by inhibiting neuroinflammation and oxidative stress. Further research work for the neuroprotection mechanism of (+)-borneol will focus on reactive oxygen species-mediated apoptosis. Therefore, (+)-borneol is a potential therapeutic candidate for retarding the neurodegenerative process of PD.

Pharmacokinetics and Bioequivalence of Fixed-Dose Combination of Simvastatin and Ezetimibe Tablets: A Randomized, Crossover, Open-Label Study in Healthy Volunteers.

The current study aimed to evaluate the bioequivalence of a new generic combination of simvastatin and ezetimibe with the reference formulation. An open-label, randomized, 3-period, 3-sequence, crossover study, including 60 healthy volunteers, was implemented. Participants received the test and reference formulation, each containing 20mg of simvastatin and 10mg of ezetimibe as a single-dose tablet, separated by a minimum of 2-week washout periods. Blood samples were collected for 20 time points from predose to 72 hours after the dose. The total ezetimibe assay was carried out using a validated liquid chromatography-tandem mass spectrometry, while unconjugated ezetimibe, simvastatin, and simvastatin β-hydroxy acid determination was done via a validated ultra-performance liquid chromatography-tandem mass spectrometry. Each assay was preceded by a liquid-liquid extraction step. The pharmacokinetic parameters were derived using noncompartmental analysis and then compared between the reference and test formulations via a multivariate analysis of variance. No statistical difference was found in under the concentration-time curve from time 0 to the last quantifiable concentration and maximum concentration of unconjugated ezetimibe, total ezetimibe, and simvastatin between the reference and test formulations. The 90% confidence intervals of unconjugated ezetimibe, total ezetimibe, and simvastatin natural log-transformed under the concentration-time curve from time 0 to the last quantifiable concentration, and maximum concentration were in the range of 80%-125% as per the bioequivalence acceptance criteria. Therefore, the test formulation was bioequivalent to the reference formulation.

METTL5 promotes gastric cancer progression via sphingomyelin metabolism

BACKGROUND The treatment of gastric cancer (GC) has caused an enormous social burden worldwide. Accumulating studies have reported that N6-methyladenosine (m6A) is closely related to tumor progression. METTL5 is a m6A methyltransferase that plays a pivotal role in maintaining the metabolic stability of cells. However, its aberrant regulation in GC has not been fully elucidated. AIM To excavate the role of METTL5 in the development of GC. METHODS METTL5 expression and clinicopathological characteristics were analyzed via The Cancer Genome Atlas dataset and further verified via immunohistochemistry, western blotting and real-time quantitative polymerase chain reaction in tissue microarrays and clinical samples. The tumor-promoting effect of METTL5 on HGC-27 and AGS cells was explored in vitro by Cell Counting Kit-8 assays, colony formation assays, scratch healing assays, transwell assays and flow cytometry. The tumor-promoting role of METTL5 in vivo was evaluated in a xenograft tumor model. The EpiQuik m6A RNA Methylation Quantification Kit was used for m6A quantification. Next, liquid chromatography-mass spectrometry was used to evaluate the association between METTL5 and sphingomyelin metabolism, which was confirmed by Enzyme-linked immunosorbent assay and rescue tests. In addition, we investigated whether METTL5 affects the sensitivity of GC cells to cisplatin via colony formation and transwell experiments. RESULTS Our research revealed substantial upregulation of METTL5, which suggested a poor prognosis of GC patients. Increased METTL5 expression indicated distant lymph node metastasis, advanced cancer stage and pathological grade. An increased level of METTL5 correlated with a high degree of m6A methylation. METTL5 markedly promotes the proliferation, migration, and invasion of GC cells in vitro . METTL5 also promotes the growth of GC in animal models. METTL5 knockdown resulted in significant changes in sphingomyelin metabolism, which implies that METTL5 may impact the development of GC via sphingomyelin metabolism. In addition, high METTL5 expression led to cisplatin resistance. CONCLUSION METTL5 was found to be an oncogenic driver of GC and may be a new target for therapy since it facilitates GC carcinogenesis through sphingomyelin metabolism and cisplatin resistance.

Trauma patients with type O blood exhibit unique multi-omics signature with decreased lectin pathway of complement levels.

Patients with type O blood may have an increased risk of hemorrhagic complications due to lower baseline levels of von Willebrand Factor (vWF) and factor VIII, but the transition to a mortality difference in trauma is less clear. We hypothesized that type O trauma patients will have differential proteomic and metabolomic signatures in response to trauma beyond vWF and FVIII alone. Patients meeting the highest level of trauma activation criteria were prospectively enrolled. Blood samples were collected upon arrival to the emergency department. Proteomic and metabolomic (multi-omics) analyses of these samples were performed using liquid chromatography-mass spectrometry. Demographic, clinical, and multi-omics data were compared between patients with type O blood versus all other patients. There were 288 patients with multi-omics data; 146 (51%) had type O blood. Demographics, injury patterns, and initial vital signs and laboratory measurements were not different between groups. Type O patients had increased lengths of stay (7 vs. 6 days, p = 0.041) and a trend towards decreased mortality secondary to traumatic brain injury compared to other causes (TBI, 44.4 % vs. 87.5%, p = 0.055). Type O patients had decreased levels of mannose-binding lectin (MBL) and MBL associated serine proteases 1 and 2 which are required for the initiation of the lectin pathway of complement activation. Type O patients also had metabolite differences signifying energy metabolism and mitochondrial dysfunction. Blood type O patients have a unique multi-omics signature, including decreased levels of proteins required to activate the lectin complement pathway. This may lead to overall decreased levels of complement activation and decreased systemic inflammation in the acute phase possibly leading to a survival advantage, especially in TBI. However, this may later impair healing. Future work will need to confirm these associations, and animal studies are needed to test therapeutic targets. Retrospective Comparative Study, Level IV.

Association between secondhand smoke exposure and serum sex hormone concentrations among US female adults: a cross-sectional analysis using data from the National Health and Nutrition Examination Survey, 2013–2016

ObjectiveTo estimate the association between secondhand smoke (SHS) exposure and serum sex hormone concentrations in female adults (never smokers and former smokers).DesignCross-sectional analysis.SettingUS National Health and Nutrition Examination Survey, 2013–2016.Outcome...

Overcoming the nutritional immunity by engineering iron-scavenging bacteria for cancer therapy.

Certain bacteria demonstrate the ability to target and colonize the tumor microenvironment, a characteristic that positions them as innovative carriers for delivering various therapeutic agents in cancer therapy. Nevertheless, our understanding of how bacteria adapt their physiological condition to the tumor microenvironment remains elusive. In this work, we employed liquid chromatography-tandem mass spectrometry to examine the proteome of E. coli colonized in murine tumors. Compared to E. coli cultivated in the rich medium, we found that E. coli colonized in tumors notably upregulated the processes related to ferric ions, including the enterobactin biosynthesis and iron homeostasis. This finding indicated that the tumor is an iron-deficient environment to E. coli. We also found that the colonization of E. coli in the tumor led to an increased expression of lipocalin 2 (LCN2), a host protein that can sequester the enterobactin. We therefore engineered E. coli in order to evade the nutritional immunity provided by LCN2. By introducing the IroA cluster, the E. coli synthesizes the glycosylated enterobactin, which creates steric hindrance to avoid the LCN2 sequestration. The IroA-E. coli showed enhanced resistance to LCN2 and significantly improved the anti-tumor activity in mice. Moreover, the mice cured by the IroA-E. coli treatment became resistant to the tumor re-challenge, indicating the establishment of immunological memory. Overall, our study underscores the crucial role of bacteria's ability to acquire ferric ions within the tumor microenvironment for effective cancer therapy.

Preclinical pharmacokinetic studies of villocarine A, an active Uncaria alkaloid.

Villocarine A is a bioactive indole alkaloid isolated from the Uncaria genus. It has demonstrated vasorelaxation activity and potential to protect the central nervous system. To identify the pharmacokinetic properties of villocarine A, a series of in vitro and in vivo studies have been performed. Villocarine A was found to be highly permeable (15.6 ± 1.6*10-6cm/s) across human colorectal adenocarcinoma cell monolayer with high protein binding (>91%) in both rat and human plasma. Hepatic extraction ratio of villocarine A was 0.1 in pooled rat liver and 0.2 in human liver microsomes and was found stable in rat plasma at 37°C. Due to the high permeability and low rate of metabolism properties, villocarine A was initially considered suitable for preclinical development and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification (linearity: 1-150 ng/ml) in rat plasma was developed and validated for in vivo studies. Essential pharmacokinetic parameters included the volume of distribution and clearance of villocarine A, which were found to be 100.3 ± 15.6L/kg and 8.2 ± 1.1L/h/kg, respectively, after intravenous administration in rats. Following oral dosing, villocarine A exhibited rapid absorption as the maximum plasma concentration (53.2 ± 10.4ng/ml) occurred at 0.3 ± 0.1h, post-dose. The absolute oral bioavailability of villocarine A was 16.8 ± 0.1%. To our knowledge, this was the first pharmacokinetic study of villocarine A, which demonstrated the essential pharmacokinetic properties of villocarine A: large volume distribution, high clearance, and low oral bioavailability in rats.

A simple and highly sensitive LC–MS workflow for characterization and quantification of ADC cleavable payloads

Antibody–drug conjugates (ADC) payloads are cleavable drugs that act as the warhead to exert an ADC’s cytotoxic effects on cancer cells intracellularly. A simple and highly sensitive workflow is developed and validated for the simultaneous quantification of six ADC payloads, namely SN-38, MTX, DXd, MMAE, MMAF and Calicheamicin (CM). The workflow consists of a short and simple sample extraction using a methanol-ethanol mixture, followed by a fast liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The results showed that well-validated linear response ranges of 0.4–100 nM for SN38, MTX and DXd, 0.04–100 nM for MMAE and MMAF, 0.4–1000 nM for CM were achieved in mouse serum. Recoveries for all six payloads at three different concentrations (low, medium and high) were more than 85%. An ultra-low sample volume of only 5 µL of serum is required due to the high sensitivity of the method. This validated method was successfully applied to a pharmacokinetic study to quantify MMAE in mouse serum samples.

Bioequivalence Study of Atenolol Tablets in Healthy Chinese Subjects Under Fasting and Fed Conditions.

Atenolol, a cardioselective β1-blocker, exhibits efficacy in treating cardiovascular diseases. We conducted a single-center, randomized, open, single-dose, 2-preparation, 2-cycle, 2-sequence, double-crossover trial with a 7-day washout period to investigate the pharmacokinetics, bioequivalence (BE), and safety of test and reference atenolol tablets (25mg) in healthy Chinese volunteers. Forty-eight healthy participants were randomized into the fasting and fed arms. After administering a single oral dose of the test or reference formulation (25mg), plasma atenolol concentrations were measured using liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were obtained from concentration-time profiles. In total, 23 and 24 individuals were included in the fasting and fed arms, respectively. The mean concentration-time profiles for both formulations were similar, and Cmax, AUC0-t, and AUC0-∞ were within the BE range of 80%-125%. Thirteen adverse events (AEs) were observed in 7 participants in the fasting arm; 1 withdrew from the trial early owing to an AE. In the fed arm, 20 AEs were observed in 8 participants, and none withdrew from the trial. All adverse reactions were grade I, with no serious AEs or deaths. Therefore, the 2 tablets are bioequivalent in healthy Chinese individuals under fasting and fed conditions, supporting their further clinical development.

Comprehensive Glycosylation Characterization of Recombinant Human Erythropoietin by Electron-Activated Dissociation Mass Spectrometry.

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy, which has three N-glycosylation sites and one O-glycosylation site. It contains a variety of different glycosylation modifications, such as sialyation, O-acetylation on sialic acids, etc., which causes a big challenge for the glycosylation analysis of rhEPO. In this study, a liquid chromatography-mass spectrometry (LC-MS) method combined with electron-activated dissociation (EAD) technology was used in qualitative and quantitative characterization of rhEPO N-glycosylation and O-glycosylation in just one injection. The usage of EAD not only generated abundant MS/MS fragment ions of glycopeptides and improved the MS/MS sequence coverage but also preserved the glycan structures in the MS/MS fragment ions and the integrity of the glycosidic bond between the glycans and peptides. Three N-glycosylation sites (N24, N38, and N83) and one O-glycosylation site (S126) of rhEPO samples were successfully identified. Among them, the glycosylation ratios of N24, N38, and N83 sites were 82.7%, 100%, and 100% respectively, and 15, 10, and 12 different N-glycans could be identified at the glycopeptide level. The total average number of sialic acids, N-hydroxyacetylneuraminoic acid, and O-acetylation on sialic acid were 7.28, 4.21, and 0.66 at the intact protein level, respectively. For O-glycosylation site S126, O-glycosylation ratios analyzed at the intact protein level and the glycopeptide level were 80.2% and 80.3%, respectively, and two O-glycans were identified, including Core1_S1 and Core1_S2. This study also compared the difference of the glycans and their relative contents in batch-to-batch rhEPO samples. The results proved that the workflow using EAD fragmentation in LC-MS method could be effectively applied for characterizing the glycosylation analysis of rhEPO samples and batch-to-batch consistency analysis, which would help to reasonably guide the optimization of rhEPO production process.

A Study on the Performance, Structure, Composition, and Release Behavior Changes of Polybutylene Adipate Terephthalic Acid (PBAT) Film during Food Contact

Polybutylene adipate terephthalic acid (PBAT) is an emerging biodegradable material in food packaging. However, concerns have been raised regarding the potential hazards it could pose to food safety. In this study, the changes of PBAT films during food contact and the release of small molecules were inestigated by a multiscale approach. On a macro-scale, the surface roughness of the films increased with the reduction in the concentration of food simulants and the increase in contact temperatures, especially after immersion in acidic food environments. On a micro-scale, the crystallinity (Xc) and degradation indexes (DI) of the films increased by 5.7% - 61.2% and 7.8% - 48.6%, respectively, which led to a decrease in thermal stability. On a scale approaching the molecular level, 2,4-di-tert-butylphenol (2,4-DTBP) was detected by gas chromatography-mass spectrometry (GC-MS/MS) with the highest migration content, and the release behavior of 2,4-DTBP was further investigated by migration kinetics. In addition, terephthalic acid (TPA), a hydrolysis product of PBAT, was detected in acidic food environments by liquid chromatography-mass spectrometry (LC-MS/MS). The results of this study could provide practical guidance and assistance to promote sustainable development in the field of food packaging. Environment ImplicationPBAT film is a new biodegradable material in food packaging that may release small molecular during its contact with food, and pose potential risks to human health. In this study, PBAT films were immersed in a variety of food simulants, and it was found there released a variety of small molecules including TPA, resulting from the hydrolysis of PBAT. The study also revealed that some of these small molecules inhibited cell growth. These findings could promote the safe application of PBAT films in food packaging.

Predictive analysis of breast cancer response to neoadjuvant chemotherapy through plasma metabolomics.

Preoperative chemotherapy is a critical component of breast cancer management, yet its effectiveness is not uniform. Moreover, the adverse effects associated with chemotherapy necessitate the identification of a patient subgroup that would derive the maximum benefit from this treatment. This study aimed to establish a method for predicting the response to neoadjuvant chemotherapy in breast cancer patients utilizing a metabolomic approach. Plasma samples were obtained from 87 breast cancer patients undergoing neoadjuvant chemotherapy at our facility, collected both before the commencement of the treatment and before the second treatment cycle. Metabolite analysis was conducted using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS). We performed comparative profiling of metabolite concentrations by assessing the metabolite profiles of patients who achieved a pathological complete response (pCR) against those who did not, both in initial and subsequent treatment cycles. Significant variances were observed in the metabolite profiles between pCR and non-pCR cases, both at the onset of preoperative chemotherapy and before the second cycle. Noteworthy distinctions were also evident between the metabolite profiles from the initial and the second neoadjuvant chemotherapy courses. Furthermore, metabolite profiles exhibited variations associated with intrinsic subtypes at all assessed time points. The application of plasma metabolomics, utilizing CE-MS and LC-MS, may serve as a tool for predicting the efficacy of neoadjuvant chemotherapy in breast cancer in the future after all necessary validations have been completed.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites

BackgroundGlycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.MethodsWe enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS.ResultsHuman albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.ConclusionsAlbumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Effects of CYP3A5*3 genetic polymorphisms on the pharmacokinetics of perampanel in Chinese pediatric patients with epilepsy.

This study was the first to evaluate the effect of CYP3A5*3 gene polymorphisms on plasma concentration of perampanel (PER) in Chinese pediatric patients with epilepsy. We enrolled 98 patients for this investigation. 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 checkup. The plasma concentration showed a linear correlation with the daily dose taken ( r = 0.17; P < 0.05). The ineffective group showed a significantly lower plasma concentration of PER (490.5 ± 297.1 vs. 633.8 ± 305.5 μg/ml; P = 0.019). For the mean concentration-to-dose (C/D) ratio, the ineffective group showed a significantly lower C/D ratio of PER (3.2 ± 1.7 vs. 3.8 ± 2.0; P = 0.040). The CYP3A5*3 CC genotype exhibited the highest average plasma concentration of PER at 562.8 ± 293.9 ng/ml, in contrast to the CT and TT genotypes at 421.1 ± 165.6 ng/ml and 260.0 ± 36.1 ng/ml. The mean plasma PER concentration was significantly higher in the adverse events group (540.8 ± 285.6 vs. 433.0 ± 227.2 ng/ml; P = 0.042). The CYP3A5*3 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 CYP3A5*3 genetic genotype should be factored in when prescribing PER to patients with epilepsy.

Risk factors and lipid metabolism characteristics of early-onset male androgenetic alopecia: A pilot study.

Male androgenetic alopecia (MAA) is a multifactorial disease, with patients presenting at a younger age, which is a risk factor for many metabolic diseases. To explore the risk factors associated with early-onset of MAA and its metabolic characteristics. Forty patients with MAA and 45 healthy controls were collected. The serum levels of fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total testosterone (TT), uric acid (UA), and 25-hydroxyvitamin D (25(OH)D) were measured. Meanwhile, lipid metabolites were detected by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). 37.50% MAA patients had metabolic syndrome, compared to 17.78% in control group (p < 0.05). The levels of HDL-C, UA, and 25(OH)D were decreased in patients with MAA compared to healthy controls (p < 0.05). However, there was no significant difference in the level of TT between the two groups. Additionally, there were no significant differences in the levels of HDL-C, UA, 25(OH)D, and TT among different grades of hair loss (p > 0.05). The lipid profile of early-onset MAA differed significantly from healthy controls. In early-onset MAA, the levels of ceramide (Cer) and sphingomyelin (SM) were significantly lower. Cer(d38:5) and TG(15:0/18:1/18:1) may be the biomarkers. Low HDL-C, UA, and 25(OH)D may be the independent risk factors for early-onset MAA. Abnormal lipid metabolism was observed in early-onset MAA, wherein Cer and SM may serve as protective factors.

High-throughput identification of gut microbiome-dependent metabolites.

A significant hurdle that has limited progress in microbiome science has been identifying and studying the diverse set of metabolites produced by gut microbes. Gut microbial metabolism produces thousands of difficult-to-identify metabolites, which present a challenge to study their roles in host biology. In recent years, mass spectrometry-based metabolomics has become one of the core technologies for identifying small metabolites. However, metabolomics expertise, ranging from sample preparation to instrument use and data analysis, is often lacking in academic labs. Most targeted metabolomics methods provide high levels of sensitivity and quantification, while they are limited to a panel of predefined molecules that may not be informative to microbiome-focused studies. Here we have developed a gut microbe-focused and wide-spectrum metabolomic protocol using liquid chromatography-mass spectrometry and bioinformatic analysis. This protocol enables users to carry out experiments from sample collection to data analysis, only requiring access to a liquid chromatography-mass spectrometry instrument, which is often available at local core facilities. By applying this protocol to samples containing human gut microbial metabolites, spanning from culture supernatant to human biospecimens, our approach enables high-confidence identification of >800 metabolites that can serve as candidate mediators of microbe-host interactions. We expect this protocol will lower the barrier to tracking gut bacterial metabolism in vitro and in mammalian hosts, propelling hypothesis-driven mechanistic studies and accelerating our understanding of the gut microbiome at the chemical level.

Targeted metabolomics-based understanding of the sleep disturbances in drug-naïve patients with schizophrenia

BackgroundSleep disturbances are a common occurrence in patients with schizophrenia, yet the underlying pathogenesis remain poorly understood. Here, we performed a targeted metabolomics-based approach to explore the potential biological mechanisms contributing to sleep disturbances in schizophrenia.MethodsPlasma samples from 59 drug-naïve patients with schizophrenia and 36 healthy controls were subjected to liquid chromatography-mass spectrometry (LC-MS) targeted metabolomics analysis, allowing for the quantification and profiling of 271 metabolites. Sleep quality and clinical symptoms were assessed using the Pittsburgh Sleep Quality Index (PSQI) and the Positive and Negative Symptom Scale (PANSS), respectively. Partial correlation analysis and orthogonal partial least squares discriminant analysis (OPLS-DA) model were used to identify metabolites specifically associated with sleep disturbances in drug-naïve schizophrenia.Results16 characteristic metabolites were observed significantly associated with sleep disturbances in drug-naïve patients with schizophrenia. Furthermore, the glycerophospholipid metabolism (Impact: 0.138, p<0.001), the butanoate metabolism (Impact: 0.032, p=0.008), and the sphingolipid metabolism (Impact: 0.270, p=0.104) were identified as metabolic pathways associated with sleep disturbances in drug-naïve patients with schizophrenia.ConclusionsOur study identified 16 characteristic metabolites (mainly lipids) and 3 metabolic pathways related to sleep disturbances in drug-naïve schizophrenia. The detection of these distinct metabolites provide valuable insights into the underlying biological mechanisms associated with sleep disturbances in schizophrenia.

Macrophage-Induced Carboxypeptidase A4 Promotes the Progression of Anaplastic Thyroid Cancer.

Background: The density of tumor-associated macrophages in the tumor microenvironment of anaplastic thyroid cancer (ATC) is associated with poor prognosis. However, the crosstalk between macrophages and ATC cells is poorly understood. This study aimed to examine the impact of macrophages on cancer cell phenotypes. We found a new mediator between M2 macrophages and ATC cells through proteomics analysis. Methods: The role of macrophages in proliferation, migration, and invasion of ATC cells was evaluated using coculture assay and conditioned medium (CM). Secretory factors in the CM from single or coculture were identified using liquid chromatography-tandem mass spectrometry proteomics analysis. We evaluated the role of the secretory factor in proliferation, migration, and invasion of cancer cells. In vivo xenograft model was used to evaluate the effect of the factor. Results: M2 macrophages significantly increased the proliferation, migration, and invasion of ATC cells, whereas M1 macrophages decreased the proliferation, migration, and invasion of ATC cells. Based on proteomic analysis of CM, we identify carboxypeptidase A4 (CPA4) as a mediator of the crosstalk between macrophages and ATC cells. CPA4 was only detected in the coculture media of M2 macrophage/8505C, and its expression in cancer cells increased by M2 macrophage. The expression of CPA4 protein was significantly higher in human thyroid cancers, particularly in ATCs, than normal and benign tissues. A bioinformatics analysis of public data revealed that CPA4 expression was associated with poor prognosis and dedifferentiation of thyroid cancer. Knockdown of CPA4 suppressed proliferation, colony formation, migration, and invasion of ATC cells, consistent with the decrease of STAT3, ERK, and AKT/mTOR phosphorylation and epithelial-mesenchymal transition (EMT) marker expression. In addition, the increased expression of CPA4 in cancer cells by M2 macrophage stimulation induced the polarization of macrophages to the M2 phenotype, which formed a positive feedback loop. Xenograft tumors did not develop after CPA4 knockdown. Conclusions: Our data suggest that CPA4 stimulates the progression of thyroid cancer by mediating between M2 macrophages and ATC cells. CPA4 can be a new therapeutic target for the treatment of patients with ATC.

Lipidomic studies revealing serological markers associated with the occurrence of retinopathy in type 2 diabetes

PurposeThe duration of type 2 diabetes mellitus (T2DM) and blood glucose levels have a significant impact on the development of T2DM complications. However, currently known risk factors are not good predictors of the onset or progression of diabetic retinopathy (DR). Therefore, we aimed to investigate the differences in the serum lipid composition in patients with T2DM, without and with DR, and search for potential serological indicators associated with the development of DR.MethodsA total of 622 patients with T2DM hospitalized in the Department of Endocrinology of the First Affiliated Hospital of Xi’an JiaoTong University were selected as the discovery set. One-to-one case–control matching was performed according to the traditional risk factors for DR (i.e., age, duration of diabetes, HbA1c level, and hypertension). All cases with comorbid chronic kidney disease were excluded to eliminate confounding factors. A total of 42 pairs were successfully matched. T2DM patients with DR (DR group) were the case group, and T2DM patients without DR (NDR group) served as control subjects. Ultra-performance liquid chromatography–mass spectrometry (LC–MS/MS) was used for untargeted lipidomics analysis on serum, and a partial least squares discriminant analysis (PLS-DA) model was established to screen differential lipid molecules based on variable importance in the projection (VIP) > 1. An additional 531 T2DM patients were selected as the validation set. Next, 1:1 propensity score matching (PSM) was performed for the traditional risk factors for DR, and a combined 95 pairings in the NDR and DR groups were successfully matched. The screened differential lipid molecules were validated by multiple reaction monitoring (MRM) quantification based on mass spectrometry.ResultsThe discovery set showed no differences in traditional risk factors associated with the development of DR (i.e., age, disease duration, HbA1c, blood pressure, and glomerular filtration rate). In the DR group compared with the NDR group, the levels of three ceramides (Cer) and seven sphingomyelins (SM) were significantly lower, and one phosphatidylcholine (PC), two lysophosphatidylcholines (LPC), and two SMs were significantly higher. Furthermore, evaluation of these 15 differential lipid molecules in the validation sample set showed that three Cer and SM(d18:1/24:1) molecules were substantially lower in the DR group. After excluding other confounding factors (e.g., sex, BMI, lipid-lowering drug therapy, and lipid levels), multifactorial logistic regression analysis revealed that a lower abundance of two ceramides, i.e., Cer(d18:0/22:0) and Cer(d18:0/24:0), was an independent risk factor for the occurrence of DR in T2DM patients.ConclusionDisturbances in lipid metabolism are closely associated with the occurrence of DR in patients with T2DM, especially in ceramides. Our study revealed for the first time that Cer(d18:0/22:0) and Cer(d18:0/24:0) might be potential serological markers for the diagnosis of DR occurrence in T2DM patients, providing new ideas for the early diagnosis of DR.