Ganoderma lucidum Polysaccharide Supplementation Significantly Activates T-Cell-Mediated Antitumor Immunity and Enhances Anti-PD-1 Immunotherapy Efficacy in Colorectal Cancer.

Ganoderma lucidum polysaccharide (GLP) is a prebiotic with immunomodulatory effects. However, the therapeutic potential of GLP in tumor immunotherapy has not been fully explored, especially in T cell-mediated antitumor immunity. In this study, we found that GLP significantly inhibited tumor growth and activated antitumor immunity in colorectal cancer (CRC). In the spleens and tumor tissues, the proportion of cytotoxic CD8+T cells and Th1 helper cells increased, while immunosuppressive Tregs decreased. Additionally, microbiota dysbiosis was alleviated by GLP, and short-chain fatty acid production was increased. Meanwhile, GLP decreased the ratio of kynurenine and tryptophan (Kyn/Trp) in the serum, which contributed to antitumor immunity of T cells. More importantly, the combination of GLP and the immune checkpoint inhibitor anti-PD-1 monoclonal antibody further enhanced the efficacy of anti-PD-1 immunotherapy. Thus, GLP as a prebiotic has the potential to be used in tumor immunotherapy.

LPCAT2 inhibits colorectal cancer progression via the PRMT1/SLC7A11 axis.

Colorectal cancer (CRC) has a high degree of heterogeneity and identifying the genetic information of individual tumor cells could help enhance our understanding of tumor biology and uncover potential therapeutic targets for CRC. In this study, we identified LPCAT2+ tumor cell populations with less malignancy than LPCAT2- tumor cells in human and mouse CRC tissues using scRNA-seq. Combining in vitro and in vivo experiments, we found that LPCAT2 could inhibit the proliferation of CRC cells by inducing ferroptosis. Mechanistically, LPCAT2 arrested PRMT1 in cytoplasm of CRC cells via regulating acetylation of PRMT1 at the K145 site. In turn, PRMT1 enhanced SLC7A11 promoter activity. Thus, LPCAT2 attenuated the positive regulatory effect of PRMT1 on SLC7A11 promoter. Notably, SLC7A11 acts as a ferroptosis regulator. Furthermore, in LPCAT2 knockout mice (LPCAT2-/-) colon cancer model, we found that LPCAT2-/- mice exhibited more severe lesions, while PRMT1 or SLC7A11 inhibitors delayed the progression. Altogether, we elucidated that LPCAT2 suppresses SLC7A11 expression by inhibiting PRMT1 nuclear translocation, thereby inducing ferroptosis in CRC cells. Moreover, inhibitors of the PRMT1/SLC7A11 axis could delay tumor progression in CRC with low LPCAT2 expression, making it a potentially effective treatment for CRC.

Structural characterization, molecular dynamic simulation, and conformational visualization of a water-soluble glucan with high molecular weight from Gastrodia elata Blume.

Polysaccharides have been widely used in the development of natural drugs and health food. However, polysaccharide characterization lags due to inherently complicated features and the limitations of existing detection approaches. We aimed to provide new insight into the fine structure and conformational visualization of polysaccharides from Gastrodia elata Blume, a medicinal and edible plant. A water-soluble polysaccharide (GEP2-6) with the high molecular weight of 2.7 × 106 Da was first obtained, and its purity reached 99.2 %. Chemical and spectroscopic analyses jointly revealed that GEP2-6 was a glucan linked by α-(1 â†’ 4) and α-(1 â†’ 6) glycosidic bonds. After enzymolysis, the local structure of GEP2-6 included α-1,4-Glcp, α-1,6-Glcp, α-1,4,6-Glcp, and α-1-Glcp at a molar ratio of 31.27∶1.32∶1.08∶0.93. The glycosidic linkage pattern of repeating units was further simulated by a glycan database and spatial examination software. The good dissolution performance was interpreted by dynamics simulation and practical molecular characteristics. Spherical flexible chains and the porous stable conformation were corroborated using atomic force microscopy. In addition, GEP2-6 could effectively scavenge DPPH and hydroxyl radicals as a promising natural antioxidant. These efforts will contribute to the expansion of clinical applications of this G. elata polysaccharide and the structural elucidation for macromolecular polysaccharides combined with traditional and modern analysis techniques.

[LC-MS fingerprint and multi-indicator components analysis of classical formula Gualou Xiebai Banxia Decoction].

This study developed an optimal pre-processing technique for the reference substance of the classic formula Gualou Xiebai Banxia Decoction(GXBD) and established a comprehensive quality control method for GXBD reference substance to provide a reference for its overall quality evaluation. The authors prepared 15 batches of GXBD samples and innovatively used the extracted ion chromatogram under the base peak chromatogram mode to establish a liquid chromatography-mass spectrometry(LC-MS) fingerprint, identify characteristic peaks, and perform quantitative analysis of indicator components. The yield of the 15 batches of GXBD samples ranged from 50.28% to 76.20%. In the positive ion mode, 12 common characteristic peaks were detected in the LC-MS fingerprint, and the structures of five common peaks were identified by comparison with reference standards. The similarity between the fingerprint profiles of different batches of samples and the reference fingerprint profile ranged from 0.920 to 0.984. Finally, liquid chromatography-triple quadrupole mass spectrometry(LC-QQQ/MS) in multiple reaction monitoring(MRM) mode was used to determine the content of eight indicator components in GXBD, including loliolide, chrysoeriol, rutin, cucurbitacin D, macrostemonoside Ⅰ, 25S-timosaponin B Ⅱ, 25R-timosaponin B Ⅱ, and peptide proline-tryptophan-valine-proline-glycine(PWVPG). The method established in this study can reduce matrix interference in the compound, and it has good accuracy, stability, and practical value. It effectively reflects the quality attributes of GXBD samples and can be used for the comprehensive quality control of GXBD.

Identification of polyunsaturated fatty acids as potential biomarkers of osteoarthritis after sodium hyaluronate and mesenchymal stem cell treatment through metabolomics.

Introduction: Osteoarthritis (OA) is a prevalent joint disorder worldwide. Sodium hyaluronate (SH) and mesenchymal stem cells (MSCs) are promising therapeutic strategies for OA. Previous studies showed they could improve knee function and clinical symptoms of OA. However, the mechanism of the therapeutic effects on the improvement of OA has not been clearly explained. Methods: In our study, we used a technique called 5-(diisopropylamino)amylamine derivatization liquid chromatography coupled with mass spectrometry to find the metabolites in OA synovial fluid under different treatments. Results and Discussion: After looking into the metabolomics, we discovered that SH and MSC treatment led to the downregulation of ω-6 polyunsaturated fatty acids (PUFAs) and the upregulation of ω-3 PUFAs. Significantly, the contents of 5(S)-HETE, PGA2, PGB2, and PGJ2 were lower in the MSC group than in the SH group after quantification using 5-(diisopropylamino)amylamine derivatization-UHPLC-QQQ-MS. This is the first report on the relationship of 11(S)-HETE, PGA2, PGB2, PGF2ß, 11ß-PGF2α, and DK-PGE2 with OA. Moreover, the correlation analysis of metabolites and inflammation factors showed the positive association of ω-6 PUFAs with pro-inflammation cytokines, and of ω-3 PUFAs with anti-inflammation cytokines. Our results indicated the therapeutic effect of SH and MSCs in patients with OA. In addition, this reliable metabolic approach could uncover novel biomarkers to treat OA.

Ultrasensitive quantification of trace amines based on N-phosphorylation labeling chip 2D LC-QQQ/MS.

Trace amines (TAs) are metabolically related to catecholamine and associated with cancer and neurological disorders. Comprehensive measurement of TAs is essential for understanding pathological processes and providing proper drug intervention. However, the trace amounts and chemical instability of TAs challenge quantification. Here, diisopropyl phosphite coupled with chip two-dimensional (2D) liquid chromatography tandem triple-quadrupole mass spectrometry (LC-QQQ/MS) was developed to simultaneously determine TAs and associated metabolites. The results showed that the sensitivities of TAs increased up to 5520 times compared with those using nonderivatized LC-QQQ/MS. This sensitive method was utilized to investigate their alterations in hepatoma cells after treatment with sorafenib. The significantly altered TAs and associated metabolites suggested that phenylalanine and tyrosine metabolic pathways were related to sorafenib treatment in Hep3B cells. This sensitive method has great potential to elucidate the mechanism and diagnose diseases considering that an increasing number of physiological functions of TAs have been discovered in recent decades.

Polarity-extended composition profiling via LC-MS-based metabolomics approaches: A key to functional investigation of Citrus aurantium L.

Citrus is a genus containing diverse edible species, among them Citrus aurantium L. is widely utilized while short of composition research. Herein, utilizing multiple liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approaches, we comprehensively characterized its components. We first systematized both LC and MS characteristics of polymethoxyflavones (PMFs), by which 13 PMFs were identified in C. aurantium, and their biosynthesis pathway was further established. Using derivatization-LC-MS targeted metabolomics approaches, 28 carbohydrates and 18 carboxylic acids were firstly found in C. aurantium. Combined with untargeted metabolomics method, total 147 compositions were characterized, among which 92 were firstly reported in C. aurantium. We further obtained their geographical features and sought out principal discriminative compounds. Moreover, typical biofunctions of C. aurantium from diverse regions were speculated using pharmacological platform and partly verified by experiments. The present study provided systematic component information for C. aurantium, which laid the foundation for its further exploitation as functional food.

Identification of proline, 1-pyrroline-5-carboxylate and glutamic acid as biomarkers of depression reflecting brain metabolism using carboxylomics, a new metabolomics method.

AIM: Depression is a psychiatric disease which is accompanied by metabolic disorder. Though depression has been widely studied, its metabolism is yet to be illustrated. We aimed to manifest the underlying mechanisms to diagnose depression. METHODS: One hundred thirty serum samples, including 65 patients and 65 healthy controls from different hospitals (training and validation cohorts), were recruited into the research. Sensitive Profiling for ChemoSelective Derivatization Carboxylomics (SPCSDCarboxyl) was applied to deeply hunt for the differential metabolites. Then, the serum, CSF, and hippocampus from depression rat models (CUMS group) were used to further confirm the results. Additionally, the co-occurrence between enzymes and biomarkers, as well as the combinatorial marker panel and the correlation of biomarkers among serum, CSF, or hippocampus were elucidated. RESULTS: Two hundred eight metabolites were identified from the sera of patients. Proline, 1-pyrroline-5-carboxylate (P5C), and glutamic acid could discriminate patients from healthy humans and were confirmed to be the potential biomarkers. After further validation through CUMS rats, proline, and P5C were enriched, while glutamic acid was depleted in the CUMS group. The co-occurrence analysis of enzymes and biomarkers indicated that they could be used for the diagnosis of depression. Moreover, the combinatorial marker panel and the correlation analysis of biomarkers between serum and CSF or between serum and hippocampus revealed that serum could be an alternative approach to directly reflect the potential physiological mechanisms and diagnose depression instead of brain samples. CONCLUSION: These integrated methods may facilitate the identification of biomarkers and help manifest the underlying mechanisms of depression.

Spatiotemporal changes in snow depth and the influence factors in China from 1979 to 2019.

Snow depth is an important parameter to characterize the characteristics of snow cover, and it is also one of the most sensitive response factors to regional climate change. However, the extent of snow depth variability and its driving mechanisms are still unknown in China. Therefore, in this study, we used the regression analysis, root-mean-square error analysis, anomalous year analysis, and correlation analysis methods to explore the spatiotemporal variation characteristics of snow depth in China from 1979 to 2019 based on the reanalysis snow depth dataset. The results show that (1) the snow distribution in China is obviously spatially heterogeneous, and the southeastern, western, and southern regions of the Qinghai-Tibet Plateau, northern Xinjiang, and northeastern China have high values of snow depth; (2) the high-value regions are also the sensitive regions for anomalous variations in snow depth in China; (3) in the past 41 years, the interannual variability of snow depth in China has shown a significantly decreasing trend, and the linear tendency of snow depth is - 0.093 cm/10 a (p < 0.01) and the snow depth in four seasons showed a decreasing trend (p < 0.05); and (4) the driving factors of snow heterogeneity are dissimilar in different regions and seasons. In temperate zones, average air temperature is the main factor affecting snow depth in cold temperature, mid temperature, and warm temperature zones; the maximum air temperature is the main factor affecting snow depth in mid temperate and warm temperate zones. Both the minimum air temperature and the average land-surface temperature are important factors affecting the snow depth in the cold temperate, mid temperate and warm temperate zones, and all passed the significance test of 0.01.

Discovery of novel ascorbic acid derivatives and other metabolites in fruit of Rosa roxburghii Tratt through untargeted metabolomics and feature-based molecular networking.

Fruit of Rosa roxburghii Tratt (FRR) is widely used in functional food industry while short of metabolites research. Herein, we firstly identified 251 metabolites in FRR based on untargeted liquid chromatography-mass spectrometry (LC-MS) approach. Then, 42 differential compounds were sought out to avoid the confusing use of FRR and fruit of R. sterilis S. D. Shi (FRS), and FRR was evaluated exhibiting higher biofunction potential. Moreover, a quantitative LC-MS approach was established to determine the contents of 3 ascorbyl hexosides, and FRR with higher contents should be better source than FRS. Additionally, 17 ascorbic acid (AA) derivatives formed by conjugation of ascorbyl unit with organic acids, flavonoids, or glucuronic acid were also discovered in FRR through characteristic ions of AA and feature-based molecular networking (FBMN), enlightening that AA derivatives were not limited to ascorbyl glycosides. This study provided abundant metabolites information of FRR, laying the basis for exploitation of FRR.

Polarity-regulated derivatization-assisted LC-MS method for amino-containing metabolites profiling in gastric cancer.

Amino-containing compounds, including amino acids, aliphatic amines, aromatic amines, small peptides and catecholamines, are involved in various biological processes and play vital roles in multiple metabolic pathways. Previous studies indicated that some amino-containing metabolites are significant diagnostic and prognostic biomarkers of gastric cancer. However, the discovery of precise biomarkers for the preoperative diagnosis of gastric cancer is still in an urgent need. Herein, we established a polarity-regulated derivatization method coupled with liquid chromatography-mass spectrometry (LC-MS) for amino-containing metabolites profiling in the serum samples of patients with gastric cancer and healthy controls, based on our newly designed and synthesized derivatization reagent (S)-3-(1-(diisopropoxyphosphoryl) pyrrolidine-2-carboxamido)-N-hydroxysuccinimidyl ester (3-DP-NHS). Enhanced separation efficiency and detection sensitivity for amino-containing metabolites were achieved after derivatization. This method exhibited good linearity, recovery, intra- and inter-day precision and accuracy. Only 5 µL serum is needed for untargeted analysis, enabling 202 amino-containing metabolites to be detected. Statistical analysis revealed altered amino acid metabolisms in patients with gastric cancer. Furthermore, ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) analysis quantification revealed increased serum levels of tryptamine and decreased concentrations of arginine and tryptophan in patients with gastric cancer. Receiver operating characteristic (ROC) curves indicated that an increased tryptamine/tryptophan ratio could serve as a potential biomarker for gastric cancer diagnosis. This study demostrated the possibility of using serum amino acid biomarkers for gastric cancer diagnosis, providing new avenues for the treatment of gastric cancer.

The cholesterol uptake regulator PCSK9 promotes and is a therapeutic target in APC/KRAS-mutant colorectal cancer.

Therapeutic targeting of KRAS-mutant colorectal cancer (CRC) is an unmet need. Here, we show that Proprotein Convertase Subtilisin/Kexin type 9 (PSCK9) promotes APC/KRAS-mutant CRC and is a therapeutic target. Using CRC patient cohorts, isogenic cell lines and transgenic mice, we identify that de novo cholesterol biosynthesis is induced in APC/KRAS mutant CRC, accompanied by increased geranylgeranyl diphosphate (GGPP)─a metabolite necessary for KRAS activation. PCSK9 is the top up-regulated cholesterol-related gene. PCSK9 depletion represses APC/KRAS-mutant CRC cell growth in vitro and in vivo, whereas PCSK9 overexpression induces oncogenesis. Mechanistically, PCSK9 reduces cholesterol uptake but induces cholesterol de novo biosynthesis and GGPP accumulation. GGPP is a pivotal metabolite downstream of PCSK9 by activating KRAS/MEK/ERK signaling. PCSK9 inhibitors suppress growth of APC/KRAS-mutant CRC cells, organoids and xenografts, especially in combination with simvastatin. PCSK9 overexpression predicts poor survival of APC/KRAS-mutant CRC patients. Together, cholesterol homeostasis regulator PCSK9 promotes APC/KRAS-mutant CRC via GGPP-KRAS/MEK/ERK axis and is a therapeutic target.

Microbiota drive insoluble polysaccharides utilization via microbiome-metabolome interplay during Pu-erh tea fermentation.

Polysaccharides are abundant components in Pu-erh tea, yet the utilization of insoluble polysaccharides under the actions of microbiota has rarely been studied. The aim of this work was to study how insoluble polysaccharides were utilized during fermentation through the investigation of the variations and correlation of microbiota with polysaccharides degradation products. Genomics study revealed the significant changes of microbiota. Metabolomics analysis showed monosaccharides (types 1 and 3) were consumed during early and middle fermentation stages, while carboxylic acids and other monosaccharides (type 2) were accumulated at middle and late pile-fermentation stages. Correlation revealed that type 1 and 3 monosaccharides, which act as energy providers, were positively associated with Aspergillus, while type 2 monosaccharides possessing multiple bioactivities and carboxylic acids influencing tea taste were positively related to Rasamsonia, Thermomyces, Bacillaceae, and Lactobacillaceae. This study would be beneficial to improve production efficiency and provide basis for quality control of Pu-erh tea fermentation.

Umbilical cord blood metabolomics: association with intrauterine hyperglycemia.

BACKGROUND: Intrauterine hyperglycemia can harm a fetus's growth and development, and this can be seen in the umbilical cord blood metabolism disorder. However, the metabolites and metabolic mechanisms involved in the condition remain unknown. METHODS: Targeted metabolomics using liquid chromatography and MetaboAnalyst were conducted in this study to explore differences in metabolites and metabolic pathways between individuals with hyperglycemia or well-controlled gestational diabetes mellitus (GDM) and healthy controls. RESULTS: Univariate analysis found that the hyperglycemic and healthy control groups differed in 30 metabolites, while the well-controlled GDM and the healthy control groups differed only in three metabolites-ursodeoxycholic acid, docosahexaenoic acid, and 8,11,14-eicosatrienoic acid. Most of these metabolic variations were negatively associated with neonatal weights. Further research showed that the variations in the metabolites were primarily associated with the metabolic pathways of linoleic acid (LA) and alpha-linolenic acid (ALA). CONCLUSION: Gestational hyperglycemia and well-controlled GDM, which may play a major role by inhibiting the LA and ALA metabolic pathways, have detrimental effects on cord blood metabolism. IMPACT: The main point of this paper is that intrauterine hyperglycemia has a negative effect on cord blood metabolism mainly through the linoleic acid and alpha-linolenic acid metabolic pathways. This is a study to report a new association between well-controlled GDM and cord blood metabolism. This study provides a possible explanation for the association between intrauterine hyperglycemia and neonatal adverse birth outcomes.

Investigation and dynamic profiling of oligopeptides, free amino acids and derivatives during Pu-erh tea fermentation by ultra-high performance liquid chromatography tandem mass spectrometry.

Microbial fermentation is the critical step of Pu-erh tea manufacture, which will induce dramatic changes in the chemical composition and content of tea. In this research, we applied multi-methods based on UHPLC-Q-TOF/MS to profile the dynamic changes of oligopeptides, free amino acids, and derivatives (OPADs) during Pu-erh fermentation and predicted the potential bioactivities in silico. A total of 60 oligopeptides, 18 free amino acids, and 42 amino acid derivatives were identified, and the contents of most of them decreased after fermentation. But several N-acetyl amino acids increased 7-36 times after fermentation, and they might be the potential inhibitors of neurokinin-1 receptor. Moreover, the results of metamicrobiology showed Aspergillus niger and Aspergillus luchuensis were more prominent to metabolize protein, oligopeptides, and amino acids. Overall, these findings provide valuable insights about dynamic variations of OPADs during Pu-erh tea fermentation and are beneficial for guiding practical fermentation and quality control of Pu-erh tea.

Dynamic changes of phenolic acids and antioxidant activity of Citri Reticulatae Pericarpium during aging processes.

Citri reticulatae pericarpium (CRP) shows multiple bioactivities, including antioxidant, anti-tumor, and anti-inflammation. The folk proverb "CRP, the older, the better" means storing for longer time would improve its quality, which attributed to the influence of bioactive compounds. The aim of this work was to study which compounds are the factors that long storage would influence the quality of CRP. 161 compounds, including 65 flavonoids, 51 phenolic acids, 27 fatty acids, and 18 amino acids were identified through derivatization and non-derivatization liquid chromatography mass spectrometry approaches. Their dynamic changes indicated phenolic acids, which were reported to have various activities, were the main increased components. Furthermore, the representative phenolic acids were quantified and correlation analysis between their contents and antioxidant activity implicated they were the possible indicators that long storage would improve CRP quality. The results would provide basis for quality control of CRP during storage.

S-Propargyl-Cysteine Remodels the Gut Microbiota to Alleviate Rheumatoid Arthritis by Regulating Bile Acid Metabolism.

Background: Rheumatoid arthritis (RA) is a long-term autoimmune disorder characterized by chronic inflammation that results in swollen and painful joints and even cartilage and bone damage. The gut microbiota, a novel anti-inflammatory target, is considered an important environmental factor in the development of RA. S-propargyl-cysteine (SPRC), an amino acid analogue, exerts anti-inflammatory, cardioprotective effects, and neuroprotective effects on various diseases. In recent studies, an SPRC treatment exerted anti-inflammatory effects on RA. Meanwhile, gut microbiome dysbiosis in individuals with RA has also been reported by many researchers. However, the relationship between SPRC and gut microbiota in individuals with RA remains unclear. Methods: Thirty male Sprague-Dawley (SD) rats were randomly divided into three groups of 10 each, including the Control, Model, and SPRC groups. Adjuvant-induced arthritis (AIA) rats in SPRC group were treated with SPRC. Measurement of paw volume and serum tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels were applied to evaluate the inflammatory status. Fecal samples were collected on the 14th day and 28th day. Gut microbiota were analyzed using 16S ribosomal RNA (rRNA) gene amplicon sequencing. Untargeted metabolomics on plasma samples was applied to investigate the metabolic changes induced by the altered gut microbiota by using derivatization-UHPLC-Q-TOF/MS. Findings: Using 16S rRNA amplicon sequencing, we found that SPRC significantly altered the gut microbiota structure in AIA rats. In particular, Bifidobacterium, a genus of BSH (Bile Salt Hydrolase)-producing microbes, was overrepresented in SPRC-treated AIA rats. Additionally, a subsequent metabolomics analysis indicated that bile acid metabolism was also altered by SPRC treatment. Interestingly, glycochenodeoxycholic acid (GCDCA) and glycocholic acid (GCA), which are formed with the participation of BSH-producing microbes in the intestine, were identified as crucial biomarkers responding to SPRC treatment with significantly lowered levels. Interpretation: A mechanistic link between the gut microbiota and plasma metabolites was revealed in this study, which provides insights into the mechanism of SPRC treatment for RA from the perspective of the gut microbiota.

Identification and quantification of markers in Azedarach Fructus and Toosendan Fructus.

Toosendan Fructus with various pharmaceutical activities is a good source for the finding of new bioactive components, especially limonoids inside have been reported to have anticancer and antifeedant activities. To find more potential new bioactive compounds, the mass spectrometric characteristics of nimbolinin-type limonoids were first investigated. Utilizing these characteristics, totally 60 nimbolinins, including 33 new ones and at least 10 bioactive compounds, were identified by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). Furthermore, based on UHPLC-Q-TOF/MS and statistical analysis, 9 limonoids were identified to be the differential components between Toosendan Fructus and Azedarach Fructus. Particularly, nimbolinin A and toosendanin (TSN) with higher content in Azedarach Fructus and Toosendan Fructus respectively should be good markers. Finally, an UHPLC-triple quadrupole mass spectrometry (UHPLC-QQQ/MS) quantification approach for nimbolinin A and TSN was developed for their quality control. These results provided the basis for drug development and quality control of Toosendan Fructus and Azedarach Fructus.

Metabolomics reveals a correlation between hydroxyeicosatetraenoic acids and allergic asthma: Evidence from three years' immunotherapy.

BACKGROUND: Subcutaneous immunotherapy (SCIT) is an effective, safe, preventative treatment for allergic asthma; however, potential biomarkers for monitoring SCIT have rarely been reported. OBJECTIVE: Metabolomics was utilized for the discovery of new biomarkers and analyzing disease pathophysiology of allergic asthma, and it was also applied to determine the metabolomic profiles of serum samples from children with asthma undergoing SCIT and identify potential biomarkers for allergic asthma and its therapeutic monitoring. METHODS: Untargeted metabolomics using ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry was performed on 15 asthmatic and 15 healthy pediatric sera to profile carboxylic acids. Statistical analysis combined with pathway enrichment analysis was applied to identify potential biomarkers. Then, targeted metabolomics was performed to study longitudinal changes of eicosanoid profiles on sera from 20 participants with asthma who received SCIT at baseline, 6 months, one, two, and three years (ChiCTR-DDT-13003728). RESULTS: Metabolomic analysis revealed that levels of eicosanoids, particularly 12(S)-hydroxyeicosatetraenoic acid (HETE; AUC = 0.94, p < .0001) and 15(S)-HETE (AUC = 0.89, p = .0028), metabolized from arachidonic acid by lipoxygenase and glutathione peroxidase enzymes, were significantly higher in asthma group than in healthy individuals. Furthermore, levels of these important metabolites increased in the first year of SCIT treatment and then decreased from years one to three, being significantly lower after three years of treatment than baseline levels. CONCLUSION: 12(S)- and 15(S)-HETEs are potential biomarkers to participate in the pathogenesis and treatment of allergic asthma. Moreover, these metabolites may be a new target for biological indicators to monitor the therapeutic effect of SCIT, particularly in the setting of allergic asthma.

Stand out from matrix: Ultra-sensitive LC-MS/MS method for determination of histamine in complex biological samples using derivatization and solid phase extraction.

The determination of low abundant endogenous components is a challenge for the clinical samples. Histamine, a crucial endogenous component, fulfils various regulatory and mediatory functions in human, and the change of content is a critical index for the diagnosis of some diseases, especially allergy, asthma, and anaphylactic shock. However, it is challenging to detect histamine because of the low stability and concentration in complex biological samples. Here we developed an ultra-sensitive and accurate LC-MS/MS quantification method based on derivatization, isotope dilution, and solid phase extraction. The derivatization of histamine with diisopropyl phosphite (DIPP) not only enhanced the retention on the LC column but also improved the ionization efficiency. Next, solid phase extraction was applied to remove the interference, which finally resulted in standing out of the trace histamine from the high contents of the matrix. The lowest limit of quantification (LLOQ) was 0.1 pg/mL that is enough low to determine the histamine in one cell and low nano-liter of serum. This approach was successfully applied for the quantification of histamine in clinical serum samples of asthma patients and mast cell treated with chemicals modulating histamine release.