Seasonal variations and sensory profiles of oolong tea: Insights from metabolic analysis of Tieguanyin cultivar.

The impact of seasonal variations on the quality of oolong tea products remains a subject of ongoing exploration. This study delves into the intricate relationships between seasonality, metabolites, and sensory characteristics in finished oolong tea products. Metabolomic data from 266 Tieguanyin oolong tea products harvested in both spring and autumn, along with corresponding sensory evaluations, were acquired. Using OPLS-DA and PLS-DA models with UPLC-QToF/MS data, our findings showed that seasonal effects were notably more pronounced in light-scented Tieguanyin products (lightly-roasted) compared to strong-scented products (moderately-roasted). Furthermore, over half of the identified key seasonal discriminant metabolites happened to be crucial for determining the sensory grade. The study marks the first-time recognition of triterpene saponins as critical factors in determining both the harvest season and the sensory grade of oolong tea. These insights deepen our understanding of the interplays between seasonal variations, metabolites, and sensory attributes in oolong tea products.

Targeted/untargeted metabolomics and antioxidant properties distinguish Citrus reticulata 'Chachi' from Citrus reticulata Blanco.

Dried citrus peel (DCP), also called "Chen Pi", has edible and medicinal value. However, the specific differences among various sources remain unknown. Herein, we collected six DCP species, namely, one Citrus reticulata 'Chachi' (CZG) and five Citrus reticulata Blanco (CRB). Targeted high-performance liquid chromatography and untargeted ultra-high-performance liquid chromatography-tandem mass spectrometry were employed to comprehensively compare the phenolic compounds and metabolites in DCP. Interestingly, 13 different phenolic compounds were noted in DCP. The total phenolic compound content in all CRB samples (58.86-127.65 mg/g) was higher than that of CZG (39.47 mg/g). Untargeted metabolomic revealed 1495 compounds, with 115 differentially expressed metabolites for CRBs and CZG, particularly flavonoids (38), terpenoids (15), and phenolic acids and derivatives (9). Lastly, antioxidant assays revealed that all CRB samples exhibited higher antioxidant activities compared with CZG. Therefore, our study results provide a theoretical basis for the high-value utilization of citrus peels and their metabolites.

Modular, Scalable, and Customizable LC-HRMS for Exposomics.

In this chapter, we describe a multi-purpose, reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflow for acquiring high-quality, non-targeted exposomics data utilizing data-dependent acquisition (DDA) combined with the use of toxicant inclusion lists for semi-targeted analysis. In addition, we describe expected retention times for >160 highly diverse xenobiotics in human plasma and serum samples. The method described is intended to serve as a generic LC-HRMS exposomics workflow for research and educational purposes. Moreover, it may be employed as a primer, allowing for further adaptations according to specialized research needs, e.g., by including reference and/or internal standards, by expanding to data-independent acquisition (DIA), or by modifying the list of compounds prioritized in fragmentation experiments (MS2).

Global Metabolomics Using LC-MS for Clinical Applications.

Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.

Distinguishing Artifactual Fatty Acid Dimers from Fatty Acid Esters of Hydroxy Fatty Acids in Untargeted LC-MS Pipelines.

Untargeted metabolomics is a powerful profiling tool for the discovery of possible biomarkers of disease onset and progression. Analytical pipelines applying liquid chromatography (LC) and mass spectrometry (MS)-based methods are widely used to survey a broad range of metabolites within various metabolic pathways, including organic acids, amino acids, nucleosides, and lipids. Accurate and complete identification of putative metabolites is an ongoing challenge in untargeted metabolomics studies. Highly sensitive instrumentation can result in the detection of adduct and fragment ions that form reproducibly and contain identifiable ions that are difficult to distinguish from metabolic pathway intermediates, which may result in false-positive identification. At concentrations as low as 10 µM, free fatty acids have been found to form homo- and heterodimers in untargeted metabolomics pipelines that resemble the lipid class fatty acid esters of hydroxy fatty acids (FAHFAs), resulting in misidentification. This chapter details a protocol for LC-MS-based untargeted metabolomics using hydrophilic interaction chromatography (HILIC) that specifically aids in distinguishing artifactual fatty acid dimers from endogenous FAHFAs.

CE-MS-Based Clinical Metabolomics of Human Plasma.

Capillary electrophoresis coupled to mass spectrometry (CE-MS) has emerged as a powerful analytical technique with significant implications for clinical research and diagnostics. The integration of information from CE and MS strengthens confidence in the identification of compounds present in clinical samples. The ability of CE to separate molecules based on their electrophoretic mobility coupled to MS enables the accurate identification and quantification of analytes, even in complex biological matrices such as human plasma.Here, we present a detailed protocol for an untargeted metabolomics study using CE-MS and its application in a study on human plasma from patients suffering Long COVID syndrome. The protocol ranges from sample preparation to biological interpretation, detailing a workflow enabling the analysis of cationic and anionic compounds, metabolite identification, and data processing.

Application of a Computational Metabolomics Workflow for the Diagnosis of Inborn Errors of Metabolism in a Laboratory Setting.

Inborn errors of metabolism constitute a set of hereditary diseases that impose severe medical and physical challenges in the affected individual, in particular, for the pediatric patient population. Timely diagnosis is crucial for these patients, as any delay could result in irreversible health damage, underscoring the importance of early initiation of personalized treatment. Current routine diagnostic screening for inborn errors of metabolism relies on various targeted analyses of established biomarkers. However, this approach is time-consuming, focuses on a limited number of tests (based on clinical information) with a relatively small number of biomarkers, and does not facilitate the identification of new markers. In contrast, untargeted metabolomics-based screening offers a more efficient diagnostic solution, by assessing thousands of metabolites across multiple metabolic pathways in a single test. This not only saves time but also conserves resources for clinicians, the diagnostic laboratory, and for patients.This chapter describes the computational workflow of our "Next Generation Metabolic Screening" approach, which is a metabolomics-based method that is currently applied at the Translational Metabolic Laboratory of the Radboud University Medical Center (the Netherlands) for the diagnosis of inborn errors of metabolism.

Effects of supplemental medusa (Rhopilema esculentum) on intestinal microbiota and metabolites in silver pomfret (Pampus argenteus).

Pampus argenteus demonstrates a preference for Rhopilema esculentum as prey, yet the ramifications of consuming supplemental medusa on fish microbiota and metabolism remain elusive. To elucidate these effects, 300 juvenile fish were divided into two groups: control group (C, given commercial food only) and supplemental medusa (SM) group (given supplemental medusa + commercial feed). After 15 days, fish in the SM group exhibited a significant increase in fatness, the amylase activity in the intestine significantly increased, and the intestinal microvilli were arranged more neatly. The comprehensive approach involving 16S rRNA amplicon sequencing and metabolomics was employed, leading to the identification of five genera within the SM group, namely Lactococcus, Cohaesibacter, Maritalea, Sulfitobacter, and Carnobacterium. Functional prediction analysis of the microbiota indicated that the consumption of supplemental medusa facilitated processes such as glycolysis/gluconeogenesis and amino acid absorption. Metabolomics analysis revealed significant enrichment of 85 differential metabolites, most of them belonging to fatty acids and conjugates. These differential metabolites primarily participated in processes such as amino acid metabolism, fatty acid synthesis, and disease. Notably, the consumption of medusa resulted in a significant reduction in nine lysophospholipids associated with cardiovascular disease and inflammation. Pearson's correlation coefficient analysis revealed associations between specific microorganisms and metabolites, indicating that Cobetia, Weissella, and Macrococcus exhibited an increased abundance in the SM group, positively correlating with apocynin, 12-Hete, and delta 9-THC-d3. The indicator bacteria Psychrobacter reduced in the SM group, exhibiting a negative correlation with cystathionine (a compound involved in glutathione synthesis). Overall, the supplementation of medusa may confer a beneficial effect on the immunity of the fish. This study contributes to the theoretical framework for fish feed development.

Genus-specific secondary metabolome in Allokutzneria and Kibdelosporangium.

Rare actinomycete genera are highly recognized as a promising source of structurally diverse and bioactive natural products. Among these genera, Allokutzneria and Kibdelosporangium are two phylogenetically closely related and have been reported to encode some valuable biosynthetic enzymes and secondary metabolites. However, there is currently no relevant systematic research available to outline the linkage of genomic and metabolomics for specific secondary metabolites in these two promising genera. In this study, we first investigated the genus-specific secondary metabolic potential in Allokutzneria and Kibdelosporangium by comparing the diversity and novelty of their secondary metabolite biosynthetic gene clusters (BGCs). The specific secondary metabolites produced by two representative strains of these genera were comprehensively investigated using untargeted metabolomics techniques. The findings unveiled that the majority (95.4%) of the gene cluster families (GCFs) encoded by Allokutzneria and Kibdelosporangium were genus-specific, including NRPS GCFs encoding siderophores. The untargeted metabolomics analysis revealed that the metabolic profiles of two representative strains exhibit extensive specificity, with the culture medium having a big impact on the metabolic profiles. Besides, an MS-cluster featuring a series of hydroxamate-type siderophores was identified from Allokutzneria albata JCM 9917, with two of them, including a novel one (N-deoxy arthrobactin A), being experimentally validated. The present study offers valuable insights for the targeted discovery of genus-specific natural products from microorganisms.

Anti-bacterial effects, and metabolites derived from bifidobacterial fermentation of an exopolysaccharide of Cs-HK1 medicinal fungus.

This study was to investigate the antibacterial effects and metabolites derived from bifidobacterial fermentation of an exopolysaccharide EPS-LM produced by a medicinal fungus Cordyceps sinensis, Cs-HK1. EPS-LM was a partially purified polysaccharide fraction which was mainly composed of Man, Glc and Gal at 7.31:12.95:1.00 mol ratio with a maximum molecular weight of 360 kDa. After fermentation of EPS-LM in two bifidobacterial cultures, B. breve and B. longum, the culture digesta showed significant antibacterial activities, inhibiting the proliferation and biofilm formation of Escherichia coli. Based on untargeted metabolomic profiling of the digesta, the levels of short chain fatty acids, carboxylic acids, benzenoids and their derivatives were all increased significantly (p < 0.01), which probably contributed to the enhanced antibacterial activity by EPS-LM. Since EPS-LM was only slightly consumed for the bifidobacterial growth, it mainly stimulated the biosynthesis of bioactive metabolites in the bifidobacterial cells. The results also suggested that EPS-LM polysaccharide may have a regulatory function on the bifidobacterial metabolism leading to production of antibacterial metabolites, which may be of significance for further exploration.

New insight into primary hyperparathyroidism using untargeted metabolomics.

Primary Hyperparathyroidism (PHPT) is characterized by excessive parathormone (PTH) secretion and disrupted calcium homeostasis. Untargeted metabolomics offers a valuable approach to understanding the complex metabolic alterations associated with different diseases, including PHPT. Plasma untargeted metabolomics was applied to investigate the metabolic profiles of PHPT patients compared to a control group. Two complementary liquid-phase separation techniques were employed to comprehensively explore the metabolic landscape in this retrospective, single-center study. The study comprised 28 female patients diagnosed following the current guidelines of PHPT diagnosis and a group of 30 healthy females as a control group. To evaluate their association with PHPT, we identified changes in plasma metabolic profiles in patients with PHPT compared to the control group. The primary outcome measure included detecting plasma metabolites and discriminating PHPT patients from controls. The study unveiled specific metabolic imbalances that may link L-amino acids with peptic ulcer disease, gamma-glutamyls with oxidative stress, and asymmetric dimethylarginine (ADMA) with cardiovascular complications. Several metabolites, such as gamma-glutamyls, caffeine, sex hormones, carnitine, sphingosine-1-phosphate (S-1-P), and steroids, were connected with reduced bone mineral density (BMD). Metabolic profiling identified distinct metabolic patterns between patients with PHPT and healthy controls. These findings provided valuable insights into the pathophysiology of PHPT.

Matcha alleviates obesity by modulating gut microbiota and its metabolites.

Matcha shows promise for diabetes, obesity, and gut microbiota disorders. Studies suggest a significant link between gut microbiota, metabolites, and obesity. Thus, matcha may have a positive impact on obesity by modulating gut microbiota and metabolites. This study used 16S rDNA sequencing and untargeted metabolomics to examine the cecal contents in mice. By correlation analysis, we explored the potential mechanisms responsible for the positive effects of matcha on obesity. The results indicated that matcha had a mitigating effect on the detrimental impacts of a high-fat diet (HFD) on multiple physiological indicators in mice, including body weight, adipose tissue weight, serum total cholesterol (TC), and low-density lipoprotein (LDL) levels, as well as glucose tolerance. Moreover, it was observed that matcha had an impact on the structural composition of gut microbiota and gut metabolites. Specifically, matcha was able to reverse the alterations in the abundance of certain obesity-improving bacteria, such as Alloprevotella, Ileibacterium, and Rikenella, as well as the abundance of obesity-promoting bacteria Romboutsia, induced by a HFD. Furthermore, matcha can influence the levels of metabolites, including formononetin, glutamic acid, pyroglutamic acid, and taurochenodeoxycholate, within the gastrointestinal tract. Additionally, matcha enhances caffeine metabolism and the HIF-1 signaling pathway in the KEGG pathway. The results of the correlation analysis suggest that formononetin, theobromine, 1,3,7-trimethyluric acid, and Vitamin C displayed negative correlation with both the obesity phenotype and microbiota known to exacerbate obesity, while demonstrating positive correlations with microbiota that alleviated obesity. However, glutamic acid, pyroglutamic acid, and taurochenodeoxycholate had the opposite effect. In conclusion, the impact of matcha on gut metabolites may be attributed to its modulation of the abundance of Alloprevotella, Ileibacterium, Rikenella, and Romboutsia within the gastrointestinal tract, thereby potentially contributing to the amelioration of obesity.

Comparative study of the oligosaccharide profile in goat, bovine, sheep, and human milk whey.

Milk oligosaccharides are high added value compounds that could be obtained by exploiting cheese whey, a byproduct of dairy industry. The objective was to compare the abundance and diversity of oligosaccharides in whey samples from domestic animals and humans. During fresh cheese making, whey samples were collected and analyzed by untargeted and targeted small molecule analysis using high-resolution mass-spectrometry. A great similarity in the metabolite profile between goat and sheep was observed. Up to 11 oligosaccharides were observed in the sheep whey from those typically found in humans. The concentration of 2'-Fucosyllactose (0.136 ± 0.055 g/L) and 3-Fucosyllactose (0.079 ± 0.009 g/L) were significantly higher (p-value <0.01) in sheep whey, while the concentration of 3'-Sialyllactose (0.826 ± 0.638 g/L) was higher in goat whey. No significant differences were observed between goat and sheep whey for the other oligosaccharides (p-value >0.05). Therefore, sheep and goat whey could become an important source of oligosaccharides through their revalorization.

LC-HRMS analysis of phospholipids bearing oxylipins.

BACKGROUND: Several oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developed. RESULTS: Optimized reversed-phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. PL sn-1/sn-2 isomers were identified based on the neutral loss of the fatty acyl in the sn-2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC. SIGNIFICANCE: The developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.

Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured Trichodesmium morphotypes.

Trichodesmium, a globally significant N2-fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of Trichodesmium species that form distinct colony morphotypes and are inhabited by diverse microorganisms. Trichodesmium colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct Trichodesmium colonies collected from the Red Sea. We also compared these results with two morphotypes of the cultivable Trichodesmium strain IMS101. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) coupled with liquid extraction surface analysis (LESA) tandem mass spectrometry (MS2), we identified and localized a wide range of small metabolites associated with single-colony Trichodesmium morphotypes. Our untargeted MALDI-MSI approach revealed 80 unique features (metabolites) shared between Trichodesmium morphotypes. Discrimination analysis showed spatial variations in 57 shared metabolites, accounting for 62% of the observed variation between morphotypes. The greatest variations in metabolite abundance were observed between the cultured morphotypes compared to the natural colony morphotypes, suggesting substantial differences in metabolite production between the cultivable strain IMS101 and the naturally occurring colony morphotypes that the cultivable strain is meant to represent. This study highlights the variations in metabolite abundance between natural and cultured Trichodesmium morphotypes and provides valuable insights into metabolites common to morphologically distinct Trichodesmium colonies, offering a foundation for future targeted metabolomic investigations.IMPORTANCEThis work demonstrates that the application of spatial mass spectrometry imaging at single-colony resolution can successfully resolve metabolite differences between natural and cultured Trichodesmium morphotypes, shedding light on their distinct biochemical profiles. Understanding the morphological differences between Trichodesmium colonies is crucial because they impact nutrient uptake, organic molecule production, and carbon and nitrogen export, and subsequently influence ocean biogeochemical cycles. As such, our study serves as an important initial assessment of metabolite differences between distinct Trichodesmium colony types, identifying features that can serve as ideal candidates for future targeted metabolomic studies.

Enhancing mass spectrometry interpretability by ComDim-ICA multi-block analysis: Geographical and varietal traceability of Brazilian Coffea canephora.

Mass spectrometry can gain analytical interpretability by studying complementarity and synergy between the data obtained by the same technique. To explore its potential in an untargeted metabolomic application, the objective of this work was to obtain organic and aqueous coffee extracts of three coffee Canephora groups produced in Brazil with distinctive aspects: geographical origin and botanical variety. Aqueous and organic extracts of roasted coffee beans were analyzed by direct infusion electrospray ionization mass spectrometry. Due to the large number of samples, the injector of the liquid chromatography system was used to automate the analysis. The column was removed, and a peak tube was added to connect the system directly to the mass spectrometer to inject both polar and nonpolar fractions of the coffee extracts individually. The technique provided characteristic fingerprinting mass spectra that not only allowed for differentiation of geographical origins but also between robusta and conilon botanical varieties. The mass spectra of the organic and water extracts represented two separate data blocks to be analyzed by the ComDim-ICA multi-block data analysis method. While the classical ComDim is based on applying PCA to the iteratively reweighted concatenated matrices, in the ComDim-ICA, the factorization is done using independent components analysis, which promotes specific improvements since it is based on extracting components that are statistically independent of one another. The results highlighted by ComDim-ICA show that both water and organic extracts contributed with important ions to the characterization of the coffee composition. However, the results revealed a high variability of metabolomic composition within each botanical variety (Robusta Amazônico and Conilon Capixaba) and geographical provenance (Rondônia indigenous-1, Rondônia non-indigenous-2 and Espírito Santo-3). Even so, water mass spectra differentiated the botanical variety Conilon from Robusta based on significant ions related to trigonelline, caffeic acid, caffeoylquinic acid, and methylpyridinium; both water and organic mass spectra differentiated Rondônia indigenous from Rondônia non-indigenous and Espírito Santo Conilon based on significant ions related to benzoic acid, pentose, coumaric acid, caffeine in the organic extract and malonic acid, pentose, caffeoylquinic acid, methyl pyridinium, caffeine, and sucrose present in the water extract. With the proposed approach acquiring ion fingerprints of different coffee extracts and their subsequent analysis by ComDim-ICA, new complementary chemical aspects of Brazilian Coffea canephora were put in evidence.

[Application of chromatographic retention time correction in untargeted screening].

OBJECTIVE: Chromatographic retention time correction is one of the important steps to effectively improve the accuracy of identification. This article proposed a strategy for untargeted screening of biological samples based on retention time correction. METHODS: A pre-treatment method for biological samples was established. The conditions of liquid chromatography and mass spectrometry were optimized. Fourteen compounds were selected as calibration agents. The retention time correction of different samples, different injection time, different brands of instruments, changing chromatographic column and changing mobile phase were investigated. RESULTS: Calibration agents had a wide coverage, good stability and no interference with sample determination. They could be uniformly distributed in the chromatogram in both positive and negative ion modes. The chromatogram was divided into several time intervals. Calibration agents in each time period were used for retention time linear correction, and the correction effects were good. CONCLUSION: The retention time correction method could eliminate the retention time drift caused by experimental conditions, improve the accuracy of qualitative analysis, and help to solve the problem of high false positive result based on mass spectrum information.

Investigating the effects of thermal processing on bitter substances in atemoya (Annona cherimola × Annona squamosa) through sensory-guided separation.

Atemoya (Annona cherimola × Annona squamosa) is a specialty crop in Taiwan. Thermal treatment induces bitterness, complicating seasonal production adjustments and surplus reduction. In this research, sensory-guided separation, metabolomics, and orthogonal partial least squares discrimination analysis (OPLS-DA) are used for identifying the bitterness in atemoya which originates from catechins, epicatechin trimers, and proanthocyanidins. Different thermal treatments (65 °C, 75 °C, and 85 °C) revealed that the glucose and fructose contents in atemoya significantly decreased, while total phenols, flavonoids, and tannins significantly increased. The concentration of 5-hydroxymethylfurfural (5-HMF) increased from 23.16 ng/g in untreated samples to 400.71 ng/g (AP-65), 1208.59 ng/g (AP-75), and 2838.51 ng/g (AP-85). However, these levels are below the 5-HMF bitterness threshold of 3780 ng/g. Combining mass spectrometry analysis with sensory evaluation, OPLS-DA revealed that atemoya treated at 65 °C, 75 °C, and 85 °C exhibited significant bitterness, with the main bitter components being proanthocyanidin dimers and trimers.

The prognosis and metabolite changes of NSCLC patients receiving first-line immunotherapy combined chemotherapy in different M1c categories according to 9th edition of TNM classification.

BACKGROUND: The 9th edition of the TNM Classification for lung cancer delineates M1c into two subcategories: M1c1 (Multiple extrathoracic lesions within a single organ system) and M1c2 (Multiple extrathoracic lesions involving multiple organ systems). Existing research indicates that patients with lung cancer in stage M1c1 exhibit superior overall survival compared to those in stage M1c2. The primary frontline therapy for patients with advanced non-small cell lung cancer (NSCLC), lacking driver gene mutations, involves the use of immune checkpoint inhibitors (ICIs) combined with chemotherapy. Nevertheless, a dearth of evidence exists regarding potential survival disparities between NSCLC patients with M1c1 and M1c2 undergoing first-line immune-chemotherapy, and reliable biomarkers for predicting treatment outcomes are elusive. Serum metabolic profiles may elucidate distinct prognostic mechanisms, necessitating the identification of divergent metabolites in M1c1 and M1c2 undergoing combination therapy. This study seeks to scrutinize survival discrepancies between various metastatic patterns (M1c1 and M1c2) and pinpoint metabolites associated with treatment outcomes in NSCLC patients undergoing first-line ICIs combined with chemotherapy. METHOD: In this study, 33 NSCLC patients lacking driver gene mutations diagnosed with M1c1, and 22 similarly diagnosed with M1c2 according to the 9th edition of TNM Classification, were enrolled. These patients received first-line PD-1 inhibitor plus chemotherapy. The relationship between metastatic patterns and progression-free survival (PFS) in patients undergoing combination therapy was analyzed using univariate and multivariate Cox regression models. Serum samples were obtained from all patients before treatment initiation for untargeted metabolomics analysis, aiming to identify differential metabolites. RESULTS: In the univariate analysis of PFS, NSCLC patients in M1c1 receiving first-line PD-1 inhibitor plus chemotherapy exhibited an extended PFS (HR = 0.49, 95% CI, 0.27-0.88, p = 0.017). In multivariate PFS analyses, these M1c1 patients receiving first-line PD-1 inhibitor plus chemotherapy also demonstrated prolonged PFS (HR = 0.45, 95% CI, 0.22-0.92, p = 0.028). The serum metabolic profiles of M1c1 and M1c2 undergoing first-line PD-1 inhibitors plus chemotherapy displayed notable distinctions. In comparison to M1c1 patients, M1c2 patients exhibited alterations in various pathways pretreatment, including platelet activation, linoleic acid metabolism, and the VEGF signaling pathway. Diminished levels of lipid-associated metabolites (diacylglycerol, sphingomyelin) were correlated with adverse outcomes. CONCLUSION: NSCLC patients in M1c1, devoid of driver gene mutations, receiving first-line PD-1 inhibitors combined with chemotherapy, experienced superior outcomes compared to M1c2 patients. Moreover, metabolomic profiles strongly correlated with the prognosis of these patients, and M1c2 patients with unfavorable outcomes manifested distinct changes in metabolic pathways before treatment. These changes predominantly involved alterations in lipid metabolism, such as decreased diacylglycerol and sphingomyelin, which may impact tumor migration and invasion.

HS-SPME-GC-MS untargeted metabolomics reveals key volatile compound changes during Liupao tea fermentation.

This study used headspace solid-phase microextraction-gas chromatography-mass spectrometry and multivariate statistical analysis to comprehensively analyze the volatile components in Liupao tea samples throughout fermentation. In total, 1009 volatile organic compounds were detected and identified, including terpenoids, heterocyclic compounds, esters, ketones, hydrocarbons, alcohols, aromatics, and acids. Principal component and hierarchical cluster analyses, characterize the volatile components of Liupao tea samples were characterized at various fermentation stages. Orthogonal partial least squares discriminant analysis identified 248 differentiating compounds (VIP ≥ 1, P < 0.05, and |Log2FC| ≥ 1.0) during fermentation. K-means clustering analysis showed that 11 metabolites increased significantly throughout the fermentation process, whereas 31 metabolites decreased continuously. Annotation of these differential compounds revealed significant changes in sensory flavor characteristics in "green, sweet, fruity, floral, and woody" flavors. The results demonstrated significant variations in the volatile components of Liupao tea fermentation, along with notable changes in flavor characteristics.