Non-polar Metabolites Research Articles

Comprehensive metabolite analysis of wheat dough in a continuous heating process

Comprehensive metabolite analysis was carried out by gas chromatography combined with mass spectrometry to investigate the time-dependent metabolic changes during wheat dough heating. Thirty-five volatile metabolites comprising alcohols, ketones, aldehydes, aromatic compounds, furans, acids and esters were identified. Sixty-four non-volatile metabolites, which covered a broad spectrum of polar and non-polar constituents, were also identified and quantified. Results showed that the content of most volatile metabolites increased during heating. Meanwhile, the levels of non-volatile polar metabolites, such as sugar and amino acid, increased and the levels of non-volatile non-polar metabolites decreased or remained constant, including fatty acid methyl ester and free fatty acids. PCA results demonstrated that metabolic changes could be reflected by time-dependent shifts in the PCA loading scores during heating. Analysis of the loadings further showed that most volatile metabolites and non-volatile polar metabolites were the major contributors of the heating time-driven changes during heating. Furthermore, lipid oxidation mainly occurred in the residues of oleic acid and linoleic acid of triglycerides.

Unique Oxidative Metabolism of Bufalin Generates Two Reactive Metabolites That Strongly Inactivate Human Cytochrome P450 3A.

Identifying the alert groups of mechanism-based inactivators of human cytochrome P450s (hCYPs) is very helpful for early prediction of drug toxicity and for rational drug design to avoid idiosyncratic toxicity. Here, we report that a natural compound bufalin (BF) could time-dependently inactivate hCYP3A via complex CYP-catalyzed cascade oxidative metabolism. Metabolite profiling and time-dependent inhibition assays showed that 3-keto-bufalin (3-KBF), a unique nonpolar oxidative metabolite of BF, was the key substance responsible for hCYP3A inactivation. Further investigations demonstrated that 3-KBF was further metabolized by hCYPs to generate two mono-hydroxylated metabolites, which could be readily dehydrated and then covalently bound on glutathione or hCYP3A4. Collectively, this study uncovers unique CYP-catalyzed cascade oxidative pathways of BF in which two reactive intermediates bearing a Michael acceptor are finally formed as hCYP3A inactivators. These findings expand the current knowledge of CYP inactivators and suggest that some steroids bearing the 3-keto group may trigger time-dependent CYP3A inactivation.

P718 Characterization of microbiota and metabolomic profile in human colonic surgical resections of UC patients

Abstract Background Ulcerative colitis (UC) is characterized by a diffuse, continuous, and chronic inflammation of mucosa and submucosa layers in the colon whose etiology is still unknown. Intestinal microbiota dysbiosis and alterations in the metabolomic profile have been reported in mucosal biopsies from Inflammatory Bowel Disease (IBD) patients. We aim to characterize the microbiota composition and metabolomic profile in colonic resections of UC patients. Methods Colonic resections from UC (n=18) and non-IBD (n=20) patients were obtained. Microbiota identification, composition and classification was performed by 16S rRNA gene Illumina Miseq sequencing. The bioinformatic analysis of sequencing data was performed using constrained correspondence analysis (CCA) and non-parametric Wilcoxon test to compare species and genera proportions. Bacterial load was estimated by qPCR. Metabolomic analysis was performed using Nuclear Magnetic Resonance (NMR) to study polar metabolites and Gas-Chromatography or Liquid-Chromatography Mass-Spectrometry to study non-polar metabolites. Results Microbiota analysis revealed differences at genus and species level between UC and non-IBD patients. Cellulosimicrobium was found at 10-fold higher levels in UC samples while Escherichia genus was 3.88 times more abundant in controls. A reduction in bacterial load was seen in UC samples. Levels of short-chain fatty acids (FA), medium-chain FA, long-chain FA, carboxylic acids and amino acids were obtained from the metabolomics analysis and a positive correlation between propionic, aspartic and hydroxybutyric acids and Cellulosimicrobium and Pseudomonas was found (Fig1B). Gut commensals such as unclassified Agathobacter, Blautia faecis, Oscillospiraceae or Faecalibacterium prausnitzii positively correlated with the abundance of butyric acid, acetic acid and succinic acid; meanwhile Subdoligranulum, Lachnospireace or unclassified Blautia negatively correlated with some medium or long chain fatty acids (Fig1B). Fig1. Correlations between bacterial species and metabolites in (A) controls and (B) UC patients. Performed with mixOmics library in R. Conclusion Significant differences in microbiota composition and abundance are found in UC patients, which undergo a clear gut dysbiosis in colonic tissue. Correlation analysis suggests Cellulosimicrobium and Pseudomonas as sources of propionic, aspartic and hydroxybutyric acid. These results point at Cellulosimicrobium as a potential candidate in UC pathology.

P088 Metabolomic analysis reveals differences among UC and non-IBD human colonic resections: role of GPCRs

Abstract Background Metabolomics is a recent technique that has bounced into Inflammatory Bowel Diseases (IBD) due to its capacity to elucidate specific metabolites involved in the pathology and changes in the metabolomic profile have been detected in urine, blood or feces from UC patients. G-protein coupled receptors (GPCRs) have been recently identified as promising pharmacological targets. We aim to characterize the metabolomic profile and metabolite-sensing GPCRs expression in colonic resections from UC patients. Methods Colonic resections from UC (n=18) and non-IBD (n=20) patients were obtained. Metabolomic analysis was performed using Nuclear Magnetic Resonance (NMR) to study polar metabolites and Gas-Chromatography or Liquid-Chromatography Mass-Spectrometry to study non-polar metabolites. Results are expressed as μg of metabolite per gram of tissue. Gene expression of GPCRs was analyzed by qPCR. Data were expressed as fold induction vs control (mean±SEM) and compared by a t-test. A p-value<0.05 was considered statistically significant. Pearson correlation matrix were performed with R or mixOmics library in R. Results Metabolomic analysis revealed in colon of UC patients significant increased levels of propionic acid (7.7±1.1), undecanoic acid (0.3±0.02) decanoic acid (4.5±0.9), myristic acid (64.5±8.7), α-linoleic acid (23.1±4.0), aspartic acid (33.9±3.4), phenylalanine (8.8±1.6), glutamic acid, (136.3±10.8), b-hydroxybutyric (8.3±0.9) acid and lactic acid (297.5±34.7) vs non-IBD patients. Gene expression of GPR43 (22.7±9.5), GPR41 (4.7±1.7), GPR109a (19.1±5.3), GPR109b (13.6±4.6), GPR91 (13.2±5.7), GPR84 (6.7±2.0), GPR40 (6.6±1.8), GPR65 (3.0±0.5) and GPR68 (4.6±1.0) were significantly increased in UC patients compared with controls. In contrast, GPR120 (0.9±0.3), GPR119 (0.6±0.2) and GPR35 (0.5±0.1) were significantly reduced in UC patients. N-oleylethanolamide positively correlates with most of GPCRs, especially GPR68, GPR84 and GPR91 while acetic and succinic acids negatively correlate with GPR4 (Fig.1A). In UC patients, medium-chain fatty acids (FA) and most long-chain FA positively correlate with GPR84, GPR142, GPR43, GPR109a, GPR109b, GPR91 and GPR132 (Fig. 1C). Fig1. Graphs show the correlations between data relative to mRNA expression of GPCRs (expressed as ΔCt) vs metabolites. (A) Data from controls and UC. (B) Only controls. (C) Only UC. Conclusion Increased levels of several metabolites and GPCRs are detected in the colon of UC patients and their correlations suggest potential candidates to be involved in UC pathophysiology.

Chemical constituents and antibacterial activity of Bromelia laciniosa (Bromeliaceae): Identification and structural characterization

BackgroundBromelia laciniosa Mart. ex Schult. & Schult.f. is a Bromeliaceae plant species from the Brazilian Caatinga, popularly used for the treatment of childhood colic, diarrhea, fever, jaundice, dandruff and hepatitis. The objective of this work was to evaluate the chemical composition and antimicrobial activity of the species B. laciniosa. MethodIn this study, we evaluated the antimicrobial activity of ethanolic extract from the leaves of B. laciniosa by microdilution MIC method, and provided a comprehensive panel of B. laciniosa’s metabolite composition through mass spectrometry (MS) coupled to gas chromatography (GC–MS) and liquid chromatography (LC-MS). ResultsB. laciniosa ethanolic extract inhibited Staphylococcus aureus growth at the lowest concentration tested. The GC–MS analysis revealed 45 non-polar metabolites, including campesterol, palmitic acid, oleic acid and α-tocopherol. Isolation and structural elucidation by NMR experiments supported the identification of cirsilineol (1), and the mixture of steroids β-sitosterol (2) and stigmasterol (3). The LC-MS metabolite profiling was assisted by Global Natural Product Social Molecular Networking (GNPS) and led to the putative annotation of 22 metabolites, consisting of phenylpropanoids, C- and O-glycosyl-flavonoids and fatty acids. In total, 39 chemical constituents are being described for the first time in B. laciniosa. ConclusionsThe results show the diversity of secondary metabolites in the species B. laciniosa as well as antibacterial activity against S. aureus.

Wood profiling by non-targeted liquid chromatography high-resolution mass spectrometry: Part 2, Detection of the geographical origin of spruce wood (Picea abies) by determination of metabolite pattern

A non-targeted metabolomics-based approach using liquid chromatography high-resolution mass spectrometry was used to authenticate spruce wood (Picea abies) from two geographic source areas. The two sample sites were located in Germany and only 250 km apart. In order to achieve the highest possible metabolite coverage, the spruces samples were measured with four different methods using liquid chromatography high-resolution mass spectrometry. In this way, a total of approximately 4,100 features were detected, which included non-polar, polar, and intermediate-polar metabolites. Using supervised multivariate methods, a distinction between the two sample groups could be achieved on the basis of non-polar data sets. The major metabolites contributing to differentiation were identified by MS/MS experiments and were from the following classes of compounds: ceramides, fatty acids, glycerolipids, and phytosterols. Based on the soil descriptions of the two sites, it was concluded that there is probably a close relationship between nutrient availability and the differences in concentration of the marker compounds. The results show that a metabolomics-based approach is also suitable for differentiation of origin, even if the sample sites are close to each other.

Estrogenic mixtures induce alterations in lipidomic profiles in the gonads of female oysters

This study aimed to reveal possible alterations to lipidomic profiles in Sydney rock oysters, Saccostrea glomerata, exposed to estrogenic mixtures (i.e., estrone, E1; 17β-estradiol, E2; estriol, E3; 17α-ethinylestradiol, EE2; bisphenol A, BPA; 4-t-octylphenol, 4-t-OP; and 4-nonylphenol, 4-NP) at “low” and “high” concentrations, typical of those detected in Australian and global receiving waters. A seven-day acute exposure window exhibited significantly lower abundances of many non-polar metabolites in digestive gland, gills, and gonads. Overall, there was a strong effect of the carrier solvent ethanol (despite a low exposure of 0.0002%), with all solvent containing treatments exhibiting lower abundances of lipidic metabolites, especially in the gill and digestive gland. No significant changes of the lipidome were exhibited in the male gonad by estrogenic exposure. However, in the female gonad, significant reductions of phospholipids and phosphatidylcholine were associated with exposure to high estrogenic mixtures. We hypothesise that the decreases in these phospholipids in the female gonad may be attributable to 1) lower algal consumption and thus lower uptake of lipidic building blocks; 2) a reduction of available substrates for phospholipid and phosphatidylcholine synthesis; and/or 3) induction of reactive oxygen species via estrogen metabolism, which may cause lipid peroxidation and lower abundance of phospholipids.

Differentiating toxic and nontoxic congeneric harmful algae using the non-polar metabolome

Recognition and rejection of chemically defended prey is critical to maximizing fitness for predators. Paralytic shellfish toxins (PSTs) which strongly inhibit voltage-gated sodium channels in diverse animal taxa are produced by several species of the bloom-forming algal genus Alexandrium where they appear to function as chemical defenses against grazing copepods. Despite PSTs being produced and localized within phytoplankton cells, some copepods distinguish toxic from non-toxic prey, selectively ingesting less toxic cells, in ways that suggest cell surface recognition perhaps associated with non-polar metabolites. In this study LC/MS and NMR-based metabolomics revealed that the non-polar metabolomes of two toxic species (Alexandrium catenella and Alexandrium pacificum) vary considerably from their non-toxic congener Alexandrium tamarense despite all three being very closely related. Toxic and non-toxic Alexandrium spp. were distinguished from each other by metabolites belonging to seven lipid classes. Of these, 17 specific metabolites were significantly more abundant in both toxic A. catenella and A. pacificum compared to non-toxic A. tamarense suggesting that just a small portion of the observed metabolic variability is associated with toxicity. Future experiments aimed at deciphering chemoreception mechanisms of copepod perception of Alexandrium toxicity should consider these metabolites, and the broader lipid classes phosphatidylcholines and sterols, as potential candidate cues.

Assessment of Biphasic Extraction Methods of Mouse Fecal Metabolites for Liquid Chromatography-Mass Spectrometry-Based Metabolomic Studies.

With the increasing knowledge about the important roles of gut microbiota on the biological system, a systematic strategy to profile the fecal metabolome is urgently needed. Thus, an unbiased, efficient, and reproducible fecal metabolite extraction protocol needs to be established; however, the effect of biphasic extraction methods for the fecal samples remains unclear. In this study, five different methods were assessed in the extraction of polar and non-polar metabolites for the liquid chromatography-mass spectrometry (LC-MS)-based mouse fecal metabolomic study. First, the detection coverage of two extraction systems, the Bligh and Dyer extraction method (M1, chloroform/methanol/water, 2/2/1.8) and Matyash method (M2, methyl tert-butyl ether (MTBE)/methanol/water, 10/3/2.5), was compared; then, MTBE/methanol/water system with different solvent ratios (M3, 2.6/2.0/2.4; M4, 4.5/1/2.5; and M5, 3/2.5/2.5) were further evaluated. The results showed that M2 showed higher detection coverage than M1. For the MTBE/methanol/water system with different solvent ratios, M3 showed the largest detection coverage based on peak numbers and numbers of putatively annotated metabolites, while M4 presented the least overlap between two phases, higher peak intensities of metabolites, and superior reproducibility. Based on the above evidence, M4 was recommended for the biphasic extraction of fecal metabolites in the LC-MS-based mouse fecal metabolomic study.

Evaluation of liver tissue extraction protocol for untargeted metabolomics analysis by ultra-high-performance liquid chromatography/tandem mass spectrometry.

The aim of the untargeted metabolomics study is to obtain a global metabolome coverage from biological samples. Therefore, a comprehensive and systematic protocol for tissue metabolite extraction is highly desirable. In this study, we evaluated a comprehensive liver pretreatment strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to obtain more metabolites using four different protocols. These protocols included (A) methanol protein precipitation, (B) two-step extraction of dichloromethane-methanol followed by methanol-water, (C) two-step extraction of methyl tert-butyl ether-methanol followed by methanol-water, and (D) two-step extraction of isopropanol-methanol followed by methanol-water. Our results showed that protocol D was superior to the others due to more extracted features, annotated metabolites, and better reproducibility. And then, the stability and extraction sequence of protocol D were evaluated. The results showed that extraction with isopropanol-methanol followed by methanol-water was the optimum preparation sequence, which offered higher extraction efficiency, satisfactory repeatability, and acceptable stability. Furthermore, the optimal protocol was successfully applied by liver samples of rats after high-fat intervention. In summary, our protocol enabled a comprehensive and systematic evaluation of liver pretreatment to obtain more medium-polar and nonpolar metabolites and was suitable for high-throughput metabolomics analysis.

Front Cover: Evaluation of liver tissue extraction protocol for untargeted metabolomics analysis by ultra‐high performance liquid chromatography/tandem mass spectrometry

DOI: 10.1002/jssc.202100051 The cover picture shows a comprehensive liver pretreatment strategy based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The extraction with isopropanol-methanol followed by methanol-water offers higher extraction efficiency, satisfactory repeatability and acceptable stability. The optimal protocol has been successfully applied to liver samples of rats to obtain more medium-polar and nonpolar metabolites and is suitable for high-throughput metabolomics analysis.

A chemical window into the impact of RNAi silencing of the StNAC103 gene in potato tuber periderms: Soluble metabolites, suberized cell walls, and antibacterial defense

The growth and survival of terrestrial plants require control of their interactions with the environment, e.g., to defend against desiccation and microbial invasion. For major food crops, the protection conferred by the outer skins (periderm in potato) is essential to cultivation, storage, and marketing of the edible tubers and fruits. Potatoes are particularly vulnerable to bacterial infections due to their high content of water and susceptibility to mechanical wounding. Recently, both specific and conserved gene silencing (StNAC103-RNAi and StNAC103-RNAi-c, respectively) were found to increase the load of wax and aliphatic suberin depolymerization products in tuber periderm, implicating this NAC gene as a repressor of the wax and suberin biosynthetic pathways. However, an important gap in our understanding of StNAC103 silencing concerns the metabolites produced in periderm cells as antimicrobial defense agents and potential building blocks of the deposited suberin biopolymer. In the current work, we have expanded prior studies on StNAC103 silenced lines by conducting comprehensive parallel analyses to profile changes in chemical constituents and antibacterial activity. Compositional analysis of the intact suberized cell walls using solid-state 13C NMR (ssNMR) showed that NAC silencing produced an increase in the long-chain aliphatic groups deposited within the periderm cell walls. LC-MS of polar extracts revealed up-regulation of glycoalkaloids in both StNAC103-RNAi and StNAC103-RNAi-c native periderms but down-regulation of a phenolic amine in StNAC103-RNAi-c and a phenolic acid in StNAC103-RNAi native periderms. The nonpolar soluble metabolites identified using GC-MS included notably abundant long-chain alkane metabolites in both silenced samples. By coordinating the differentially accumulated soluble metabolites and the suberin depolymerization products with the ssNMR-based profiles for the periderm polymers, it was possible to obtain a holistic view of the chemical changes that result from StNAC103 gene silencing. Correspondingly, the chemical composition trends served as a backdrop to interpret trends in the chemical barrier defense function of native tuber periderms, which was found to be more robust for the nonpolar extracts.

On the Trail of the German Purity Law: Distinguishing the Metabolic Signatures of Wheat, Corn and Rice in Beer.

Here, we report a non-targeted analytical approach to investigate the influence of different starch sources on the metabolic signature in the final beer product. An extensive sample set of commercial beers brewed with barley, wheat, corn and/or rice were analyzed by both direct infusion Fourier transform ion cyclotron mass spectrometry (DI-FTICR MS, 400 samples) and UPLC-ToF-MS (100 samples). By its unrivaled mass resolution and accuracy, DI-FTICR-MS was able to uncover the compositional space of both polar and non-polar metabolites that can be traced back to the use of different starch sources. Reversed phase UPLC-ToF-MS was used to access information about molecular structures (MS2-fragmentation spectra) and isomeric separation, with a focus on less polar compounds. Both analytical approaches were able to achieve a clear statistical differentiation (OPLS-DA) of beer samples and reveal metabolic profiles according to the starch source. A mass difference network analysis, applied to the exact marker masses resolved by FTICR, showed a network of potential secondary metabolites specific to wheat, corn and rice. By MS2-similarity networks, database and literature search, we were able to identify metabolites and compound classes significant for the use of the different starch sources. Those were also found in the corresponding brewing raw materials, confirming the potential of our approach for quality control and monitoring. Our results also include the identification of the aspartic acid-conjugate of N-β-D-glucopyranosyl-indole-3-acetic acid as a potential marker for the use of rice in the brewing industry regarding quality control and food inspection purposes.

Metabolite profile of two Allium cepa L. aggregatum group cultivars by Nuclear Magnetic Resonance

Abstract. Basundari FRA, Sulistyaningsih E, Murti RH, Nuringtyas TR. 2021. Metabolite profile of two Allium cepa L. aggregatum group cultivars by Nuclear Magnetic Resonance. Biodiversitas 22: 3127-3135. Shallot, an Allium species with high economic value, is widely cultivated in Indonesia. The species includes numerous cultivars with unique characteristics, such as harvesting time, yield, taste, odor, and bulb color. Tuk Tuk and Trisula, the cultivars used in this experiment, have different bulb colors. Tuk Tuk’s bulb color is light red, while that of Trisula is dark red. This color difference may be attributed to variations in the metabolite contents of the shallot bulbs. The present experiment aimed to reveal differences in the metabolite contents of the two shallot cultivars by using Nuclear Magnetic Resonance (NMR) and then determine the specific metabolites contributing to these differences. Bulbs of Tuk Tuk and Trisula were planted in the field under the same conditions and then collected. The bulb samples were powdered, freeze-dried, and subjected to a two-phase extraction method with CDCl3 and D2O as solvents to separate the nonpolar metabolites from polar ones. The extracts were analyzed by 1H-NMR, and the spectra collected were analyzed using MNOVA software and Metaboanalyst.ca. A total of 23 metabolites were successfully identified and characterized in this experiment. The contents of eight of these metabolites, namely, sucrose, glutamine, citric acid, choline, methiin, propiin, threonine, and formic acid, were significantly higher in Trisula than in Tuk Tuk. These differences may be correlated with variations in the color intensity, pungency, and other traits of the cultivars. The results demonstrate that NMR metabolite profiling could effectively differentiate metabolite profile variations among shallot cultivars.

Intraspecific variation in polar and nonpolar metabolite profiles of a threatened Caribbean coral.

Research aimed at understanding intraspecific variation among corals could substantially increase understanding of coral biology and improve outcomes of active restoration efforts. Metabolomics is useful for identifying physiological drivers leading to variation among genotypes and has the capacity to improve our selection of candidate corals that express phenotypes beneficial to restoration. Our study aims to compare metabolomic profiles among known, unique genotypes of the threatened coral Acropora cervicornis. In doing so, we seek information related to the physiological characteristics driving variation among genotypes, which could aid in identifying genets with desirable traits for restoration. We applied proton nuclear magnetic resonance (1H-NMR) and liquid chromatography-mass spectrometry (LC-MS) to identify and compare metabolomic profiles for seven unique genotypes of A. cervicornis that previously exhibited phenotypic variation in a common garden coral nursery. Significant variation in polar and nonpolar metabolite profiles was found among A. cervicornis genotypes. Despite difficulties identifying all significant metabolites driving separation among genotypes, our data support previous findings and further suggest metabolomic profiles differ among various genotypes of the threatened species A. cervicornis. The implementation of metabolomic analyses allowed identification of several key metabolites driving separation among genotypes and expanded our understanding of the A. cervicornis metabolome. Although our research is specific to A. cervicornis, these findings have broad relevance for coral biology and active restoration. Furthermore, this study provides specific information on the understudied A. cervicornis metabolome and further confirmation that differences in metabolome structure could drive phenotypic variation among genotypes.

P078 Proteomics and Lipidomics Analysis Revealed Alterations of Complement Activation and Lipid Metabolism in peripheral blood mononuclear cells of Inflammatory Bowel Disease Patients

Abstract Background The lack of interaction between immune system cells and microbiota is considered a key driver of the pathogenesis of inflammatory bowel disease (IBD). The association of age-associated B cells (ABC) from antigen-experienced B cells and autoimmunity is now well established. The increased demands in lipid metabolism during immune response and cell activation upon autoimmune signals leads to lipid tag modifications of proteins. In IBD patients, we observed an impairment of B cell development, identifying a transitional B cell subset expressing CD38Hi, CD24Hi and CD19+. We hypothesize that metabolic alterations of proteins and lipids during immune cell activation might be a relevant mechanism of autoimmune disease. Methods An IBD cohort of patients in clinical remission under anti-TNF treatment was included in order to test metabolic and proteomic changes in peripheral blood mononuclear cells (PBMCs). Proteomic analysis was performed by Mass Spectrometry using a label free quantitative method (SWATH-MS analysis) and lead to identify a number of proteins underregulated (< 0.6) and overexpressed (> 1.5) fold change respect to control. Lipidomics of serum samples analyzed by proton Nuclear Magnetic Resonance spectroscopy (NMR)2 and multivariant statistics. Results Serum samples were obtained in Crohn disease (CD) n = 18, ulcerative colitis (UC) n = 9, before biological infusion and healthy controls (CTRL) n = 10. Proteins related with lipid metabolism like APOC2 for CD and APOB, APOA1 for UC, were differentially modulated. Significant differences in non-polar metabolites and lipids that phenotypically define CD and UC groups respect to CTRL were observed (Fig. 1) Fig. 1 Orthogonal-Partial Least Square-Discriminant Analysis (OPLS-DA) of the global NMR data of the serum samples of groups CD and CTRL. Indeed, proteins associated with complement activation like Complement Factor 1 and Complement C1q subcomponent subunit B were differentially expressed in CD group. These results were confirmed by lipidomic analysis, in which palmitic (Fig. 2) as well as other candidate lipids were more abundant in IBD groups than CTRL. Fig 2. Differences of palmitic acid concentration in CD and UC patients vs CTRL (Bonferroni test showed statistical difference between CTRL vs. CD and CTRL vs. UC with p = 0.02 and p = 0.004 respectively). Conclusion Our study detected high levels of proteins regulating lipid metabolism and palmitic in IBD patients. This observation can be interpreted as part of deleterious alterations of proteins through palmitoylation during B-cell activation and might be a relevant mechanism of disease. Further exploration of palmitate catabolism might shed light to the mechanisms that result in immunometabolic disorders.

Multi-Solvent Extraction Procedure for the Pioneer Fecal Metabolomic Analysis-Identification of Potential Biomarkers in Stable Kidney Transplant Patients.

Metabolic alteration plays a functional role in kidney allograft complications. Metabolomics is a promising high-throughput approach in nephrology but is still limited by the lack of overlap in metabolite coverage. We performed an untargeted fecal metabolomic analysis of forty stable kidney allograft recipients and twenty non-transplant controls. First, we applied the ultra-high performance liquid chromatography (UHPLC) analysis coupled with the Diod Array detector. The potential biomarkers were then collected and identified by gas chromatography-mass spectrometry (GCMS). In order to allow for complete coverage of the fecal polar and non-polar metabolites, the performance of five organic solvents with increasing polarity was investigated successively. UHPLC analysis revealed that the fecal metabolite profiles following the five extractions were significantly different between controls and kidney allografts. GC-MS analysis showed that the best predictors’ metabolites belonged mainly to long-chain fatty acids, phenolic compounds, and amino acids. Collectively, our results showed the efficiency of our pioneer method to successfully discriminate stable kidney-transplant recipients from controls. These findings suggest that distinct metabolic profiles mainly affect fatty acid biosynthesis and amino acid metabolism. In such a context, the novel insights into metabolomic investigation may be a valuable tool that could provide useful new relevant biomarkers for preventing kidney transplant complications.

Natural deep eutectic solvent formulations for spirulina: Preparation, intensification, and skin impact

Microalgae are a sustainable source of bioactive compounds like carotenoids, free fatty acids and especially polyunsaturated fatty acids. The extraction of such nonpolar metabolites usually involved nonpolar or toxic solvent like methanol or hexane. We report the first screening of Natural Deep Eutectic Solvents for the extraction of pigments and free fatty acids from spirulina. Natural Deep Eutectic Solvent with a wide range of polarity was screened, and the resulting extracts were compared according to their pigment and free fatty acid contents. The extraction performance was increased, and various metabolite profiles were obtained. A Natural Deep Eutectic Solvent based on glycerol/glucose exhibited a wide range of metabolites, which ranged from polar phycobiliproteins to free fatty acids. Natural Deep Eutectic Solvent based on a fatty acid mixture demonstrated the highest selectivity towards free fatty acids. After intensification of the extraction process, 6 spirulina- Natural Deep Eutectic Solvent formulations and Natural Deep Eutectic Solvent alone were evaluated in terms of the effects on cutaneous inflammation induced by Staphylococcus aureus, as well on 4 bacterial species that are part of the skin microbiota. The glycerol/glucose formulation exhibited an anti-inflammatory effect on keratinocytes stimulated by S. aureus. Nonpolar Natural Deep Eutectic Solvent alone and formulations impacted bacterial viability, especially for S. aureus, thus providing a new approach for the regulation of skin microbiota or product preservation.

Post-Harvest Treatment with Methyl Jasmonate Impacts Lipid Metabolism in Tomato Pericarp (Solanum lycopersicum L. cv. Grape) at Different Ripening Stages.

The application of exogenous jasmonate can stimulate the production of ethylene, carotenoids, and aroma compounds and accelerate fruit ripening. These alterations improve fruit quality and make fruit desirable for human consumption. However, fruit over-ripening results in large losses of fruit crops. This problem is overcome by applying 1-methylcyclopropene to the fruits, due to its capacity to block the ethylene receptors, suppressing fruit ripening. In this study, treatments with only 1-methylcyclopropene and both 1-methylcyclopropene and methyl jasmonate were administered to observe whether exogenous methyl jasmonate can improve the metabolite levels in fruits with blocked ethylene receptors. Fruit pericarps were analyzed at 4, 10, and 21 days after harvest (DAH) and compared with untreated fruits. The post-harvest treatments affected primary metabolites (sugars, organic acids, amino acids, and fatty acids) and secondary metabolites (carotenoids, tocopherols, and phytosterols). However, the lipid metabolism of the tomatoes was most impacted by the exogenous jasmonate. Fatty acids, carotenoids, tocopherols, and phytosterols showed a delay in their production at 4 and 10 DAH. Conversely, at 21 DAH, these non-polar metabolites exhibited an important improvement in their accumulation.

Non-Polar Myxococcus fulvus KYC4048 Metabolites Exert Anti-Proliferative Effects via Inhibition of Wnt/β-Catenin Signaling in MCF-7 Breast Cancer Cells.

The Wnt/β-catenin signaling pathway is involved in breast cancer and Myxococcus fulvus KYC4048 is a myxobacterial strain that can produce a variety of bioactive secondary metabolites. Although a previous study revealed that KYC4048 metabolites exhibit anti-proliferative effects on breast cancer, the biochemical mechanism involved in their effects remains unclear. In the present study, KYC4048 metabolites were separated into polar and non-polar (ethyl acetate and n-hexane) fractions via liquid-liquid extraction. The effects of these polar and non-polar KYC4048 metabolites on the viability of breast cancer cells were then determined by MTT assay. Expression levels of Wnt/β-catenin pathway proteins were determined by Western blot analysis. Cell cycle and apoptosis were measured via fluorescence-activated cell sorting (FACS). The results revealed that non-polar KYC4048 metabolites induced cell death of breast cancer cells and decreased expression levels of WNT2B, β-catenin, and Wnt target genes (c-Myc and cyclin D1). Moreover, the n-hexane fraction of non-polar KYC4048 metabolites was found most effective in inducing apoptosis, necrosis, and cell cycle arrest, leading us to conclude that it can induce apoptosis of breast cancer cells through the Wnt/β-catenin pathway. These findings provide evidence that the n-hexane fraction of non-polar KYC4048 metabolites can be developed as a potential therapeutic agent for breast cancer via inhibition of the Wnt/β-catenin pathway.