Polar Metabolites Research Articles

Ion Mobility QTOF-MS Untargeted Lipidomics of Human Serum Reveals a Metabolic Fingerprint for GNE Myopathy

GNE myopathy, also known as hereditary inclusion body myopathy (HIBM), is a rare genetic muscle disorder marked by a gradual onset of muscle weakness in young adults. GNE myopathy (GNEM) is caused by bi-allelic variants in the UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase)/N-acetylmannosamine kinase (ManNAc kinase) gene (GNE), clinically resulting in the loss of ambulation within 10–20 years from the onset of the initial symptoms. The disease’s mechanism is poorly understood and non-invasive biomarkers are lacking, hindering effective therapy development. Based on the available evidence, we employed a lipidomic approach to study the serum lipid profile of GNE patients. The multivariate statistical analysis revealed a downregulation of carnitines, as well as of lysophosphatidylcholines, in sera samples derived from GNEM patients. Furthermore, we identified lower levels of sphingomyelins and, concomitantly, high levels of ceramides in serum samples from GNEM patients when compared to control samples derived from healthy donors. Moreover, the GNEM serum samples showed the upregulation of Krebs cycle intermediates, in addition to a decrease in oxaloacetic acid. The correlated data gathered in this study can offer a promising diagnostic panel of complex lipids and polar metabolites that can be used in clinic for GNEM in terms of a metabolic fingerprint measurable in a minimally invasive manner.

A systematic evaluation of quenching, extraction and analysis procedures for metabolomics study of the mechanism of QYSLD intervention in A549 cells.

The preparation of cellular metabolomics samples and how to achieve comprehensive coverage of different polar metabolites in cell samples in the analysis pose a challenge for cellular metabolomics. In this study, we optimized a metabolomics protocol based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) for theextraction and detection of metabolites in A549 cells and exploration of the intervention effect of Qi-Yu-San-Long decoction (QYSLD) on A549 cells. The results indicate that the lowest level of ATP leakage was observed when A549 cells were quenched under liquid nitrogen. MeOH/chloroform/H2O (1:2:1) extraction yielded more chromatographic peaks and excellent reproducibility, and the relative extraction efficiency of most target metabolites was also high. And we optimized thechromatographic separation conditions in both HILIC and RPLC modes, enabling comprehensive detection and analysis of metabolites with varying polarities. Then, we applied the optimized method to UPLC-Q-TOF/MS-based metabolomics of A549 cells to study the mechanism of QYSLD intervention in non-small cell lung cancer (NSCLC). The CCK-8, EdU staining, and cell cycle assay showed that QYSLD inhibited the proliferation of A549 cells by interfering with the cell cycle and blocking them in theG1 phase. A total of 36 differential metabolites associated with the antitumor effects of QYSLD on NSCLC were identified, mainly involving nicotinate and nicotinamide metabolism, sphingolipid metabolism, and glycerophospholipid metabolism. And western blotting confirmed that thechange in 1-methylnicotinamide levels after QYSLD intervention was associated with theinhibition of nicotinamide N-methyltransferase expression in A549 cells.

Altered Metabolism in Knockdown Lines of Two HXXXD/BAHD Acyltransferases During Wound Healing in Potato Tubers

Suberin biosynthesis involves the coordinated regulation of both phenolic and aliphatic metabolisms. HXXXD/BAHD acyltransferases occupy a unique place in suberization, as they function to crosslink phenolic and aliphatic monomers during suberin assembly. To date, only one suberin-associated HXXXD/BAHD acyltransferase, StFHT, has been described in potatoes, whereas, in Arabidopsis, at least two are implicated in suberin biosynthesis. RNAseq data from wound-induced potato tubers undergoing suberization indicate that transcripts for 28 HXXXD/BAHD acyltransferase genes accumulate in response to wounding. In the present study, we generated RNAi knockdown lines for StFHT and another highly wound-induced HXXXD/BAHD acyltransferase, designated StHCT, and characterized their wound-induced suberin phenotype. StFHT-RNAi and StHCT-RNAi knockdown lines share the same aliphatic suberin phenotype of reduced esterified ferulic acid and ferulates, which is similar to the previously described StFHT-RNAi knockdown suberin phenotype. However, the phenolic suberin phenotype differed between the two knockdown genotypes, with StHCT-RNAi knockdown lines having proportionately more p-hydroxyphenyl-derived moieties than either StFHT-RNAi knockdown or empty vector control lines. Analysis of soluble polar metabolites revealed that StHCT catalyzes a step upstream from StFHT. Overall, our data support the involvement of more than one HXXXD/BAHD acyltransferase in potato suberin biosynthesis.

Leveraging the potential of 1.0-mm i.d. columns in UHPLC-HRMS-based untargeted metabolomics.

Untargeted metabolomics UHPLC-HRMS workflows typically employ narrowbore 2.1-mm inner diameter (i.d.) columns. However, the wide concentration range of the metabolome and the need to often analyze small sample amounts poses challenges to these approaches. Reducing the column diameter could be a potential solution. Herein, we evaluated the performance of a microbore 1.0-mm i.d. setup compared to the 2.1-mm i.d. benchmark for untargeted metabolomics. The 1.0-mm i.d. setup was implemented on a micro-UHPLC system, while the 2.1-mm i.d. on a standard UHPLC, both coupled to quadrupole-orbitrap HRMS. On polar standard metabolites, a sensitivity gain with an average 3.8-fold increase over the 2.1-mm i.d., along with lower LOD (LODavg 1.48ng/mL vs. 6.18ng/mL) and LOQ (LOQavg 4.94ng/mL vs. 20.60ng/mL), was observed. The microbore method detected and quantified all metabolites at LLOQ with respect to 2.1, also demonstrating good repeatability with lower CV% for retention times (0.29% vs. 0.63%) and peak areas (4.65% vs. 7.27%). The analysis of various samples, in both RP and HILIC modes, including different plasma volumes, dried blood spots (DBS), and colorectal cancer (CRC) patient-derived organoids (PDOs), in full scan-data dependent mode (FS-DDA) reported a significant increase in MS1 and MS2 features, as well as MS/MS spectral matches by 38.95%, 39.26%, and 18.23%, respectively. These findings demonstrate that 1.0-mm i.d. columns in UHPLC-HRMS could be a potential strategy to enhance coverage for low-amount samples while maintaining the same analytical throughput and robustness of 2.1-mm i.d. formats, with reduced solvent consumption.

Detailed Profiling of 17-Hydroxygeranyllinalool Diterpene Glycosides from Nicotiana Species Reveals Complex Reaction Networks of Conjugation Isomers.

Specialised anti-herbivory metabolites are abundant in the solanaceous genus Nicotiana. These metabolites include the large family of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). Many HGL-DTGs occur exclusively within the Nicotiana genus, but information from the molecular model species N. tabacum, N. benthamiana, and the tree tobacco N. glauca is limited. We studied HGL-DTG occurrence and complexity in these species with the aim of providing in-depth reference annotations and comprehensive HGL-DTG inventories. We analysed polar metabolite extracts in comparison to the previously investigated wild reference species N. attenuata using positive ESI(+) and negative ESI(-) mode electrospray ionisation LC-MS and MS/MS. We provide annotations of 66 HGL-DTGs with in-source and MS/MS fragmentation spectra for selected HGL-DTGs with exemplary fragment interpretations of ESI(+) as well as less studied ESI(-) spectra. We assemble a potential biosynthesis pathway comparing the presence of HGL-DTGs in N. tabacum, N. glauca, and N. benthamiana to N. attenuata. Approximately one-third of HGL-DTGs are chromatographically resolved isomers of hexose, deoxyhexose, or malonate conjugates. The number of isomers is especially high for conjugates with low numbers of deoxyhexose moieties. We extend the number of known HGL-DTGs with a focus on Nicotiana model species and demonstrate that the HGL-DTG family of N. tabacum plants can be surprisingly complex. Our study provides an improved basis with detailed references to previous studies of wild Nicotiana species and enables inference of HGL-DTG pathways with required enzymes for the biosynthesis of this important family of specialised defence metabolites.

Multiomics Studies on Metabolism Changes in Alcohol-Associated Liver Disease.

Metabolic dysfunction in the liver represents a predominant feature in the early stages of alcohol-associated liver disease (ALD). However, the mechanisms underlying this are only partially understood. To investigate the metabolic characteristics of the liver in ALD, we did a relative quantification of polar metabolites and lipids in the liver of mice with experimental ALD using untargeted metabolomics and untargeted lipidomics. A total of 99 polar metabolites had significant abundance alterations in the livers of alcohol-fed mice. Pathway analysis revealed that amino acid metabolism was the most affected by alcohol in the mouse liver. Metabolites involved in glycolysis and the TCA cycle were decreased, while glycerol 3-phosphate (G3P) and long-chain fatty acids were increased. Relative quantification of lipids unveiled an upregulation of multiple lipid classes, suggesting that alcohol consumption drives metabolism toward lipid synthesis. Results from enzyme expression and activity detection indicated that the decreased activity of mitochondrial glycerol 3-phosphate dehydrogenase contributed to the disordered metabolism.

EMBL-MCF 2.0: an LC-MS/MS method and corresponding library for high-confidence targeted and untargeted metabolomics using low-adsorption HILIC chromatography

IntroductionOver the past two decades, liquid chromatography-mass spectrometry (LC-MS)-based metabolomics has experienced significant growth, playing a crucial role in various scientific disciplines. However, despite these advance-ments, metabolite identification (MetID) remains a significant challenge. To address this, stringent MetID requirements were established, emphasizing the necessity of aligning experimental data with authentic reference standards using multiple criteria. Establishing dependable methods and corresponding libraries is crucial for instilling confidence in MetID and driving further progress in metabolomics.ObjectiveThe EMBL-MCF 2.0 LC-MS/MS method and public library was designed to facilitate both targeted and untargeted metabolomics with exclusive focus on endogenous, polar metabolites, which are known to be challenging to analyze due to their hydrophilic nature. By accompanying spectral data with robust retention times obtained from authentic standards and low-adsorption chromatography, high confidence MetID is achieved and accessible to the metabolomics community.MethodsThe library is built on hydrophilic interaction liquid chromatography (HILIC) and state-of-the-art low adsorption LC hardware. Both high-resolution tandem mass spectra and manually optimized multiple reaction monitoring (MRM) transitions were acquired on an Orbitrap Exploris 240 and a QTRAP 6500+, respectively.ResultsImplementation of biocompatible HILIC has facilitated the separation of isomeric metabolites with significant enhancements in both selectivity and sensitivity. The resulting library comprises a diverse collection of more than 250 biologically relevant metabolites. The methodology was successfully applied to investigate a variety of biological matrices, with exemplary findings showcased using murine plasma samples.ConclusionsOur work has resulted in the development of the EMBL-MCF 2.0 library, a powerful resource for sensitive metabolomics analyses and high-confidence MetID. The library is freely accessible and available in the universal .msp file format under the CC-BY 4.0 license: mona.fiehnlab.ucdavis.edu https://mona.fiehnlab.ucdavis.edu/spectra/browse?query=exists(tags.text:%27EMBL-MCF_2.0_HRMS_Library%27), EMBL-MCF 2.0 HRMS https://www.embl.org/groups/metabolomics/instrumentation-and-software/#MCF-library.

8123 Identification Of Glutamine Metabolism As A Druggable Therapeutic Vulnerability For Adrenocortical Carcinoma

Abstract Disclosure: V. Chortis: None. C. Yao: None. C. Ribeiro: None. L. Kelley: None. L. Nagano: None. M. Berber: None. S. Raveenthiraraj: None. P. Vendramini: None. K. Kiseljak-Vassiliades: None. D.L. Carlone: None. M.C. Haigis: None. K.S. Borges: None. D.T. Breault: None. Dysregulation of cellular metabolism is a hallmark feature of cancer that can be targeted therapeutically but remains underexplored in adrenocortical carcinoma (ACC), despite the urgent need for improved treatments. We employed a recently developed transgenic mouse model of ACC (BPCre), driven by the two most common mutations in human ACC (β-catenin gain-of-function and p53 loss-of-function), aiming to characterize in vivo ACC metabolism at a tissue level and to identify metabolic vulnerabilities. We employed metabolomics (liquid chromatography-mass spectrometry) and transcriptomics (bulk RNA-seq) to compare tissue metabolism in spontaneous BPCre tumors with control adrenals. Polar metabolites were analyzed using a library that provides a representative cross-section of major carbon and nitrogen-handling pathways. We identified widespread dysregulation of tumor metabolism (64/175 metabolites significantly dysregulated, FDR<0.05), with the most significantly dysregulated pathways including purine and pyrimidine metabolism, proline and glutamate metabolism, glycolysis, and the pentose phosphate pathway (FDR<0.05). Orthogonal analysis by RNA-seq revealed congruent metabolic pathway alterations. Comparison of the most significantly dysregulated pathways between BPCre mouse tumors and human ACC using publicly accessible human transcriptomic datasets (TCGA and GTEx) revealed extensive overlap. Given the prominent dysregulation of several glutamine (Gln)-dependent metabolic pathways in mouse and human ACC, we hypothesized that Gln catabolism is likely to represent a targetable metabolic vulnerability in ACC. Pharmacological targeting of Gln metabolism using the Gln antagonist 6-Diazo-5-Oxo-L-Norleucine (L-DON) induced marked cytotoxicity in two cell lines derived from BPCre tumors and three human ACC cell lines (NCI-H295R, CU-ACC1-2), as assessed by cell viability and clonogenic assays. Remarkably, this effect was rescued only by nucleoside supplementation, suggesting that Gln’s contribution to de novo purine and pyrimidine biosynthesis is the critical metabolic dependency in ACC cells. Treatment with the L-DON pro-drug JHU-083, in vivo, led to marked growth inhibition of subcutaneous mouse ACC tumor implants in both immunocompetent and immunodeficient mice. As expected, tissue metabolomics of JHU-083-treated tumors confirmed extensive depletion of purine metabolites. Tumor immunophenotyping by flow cytometry revealed significantly increased infiltration with Natural Killer cells, likely potentiating the anti-tumor effect of glutamine antagonism in immunocompetent mice. JHU-083 was also effective against human NCI-H295R xenografts, in vivo. This work reveals important new insights into ACC metabolism and provides pre-clinical evidence supporting Gln antagonism as a new metabolic treatment approach for ACC. Presentation: 6/2/2024

HILIC-MS and CE-MS as complementary analytical approaches to assess the impact of exposure to polychlorinated biphenyls on the polar serum metabolome of pigs

BackgroundHydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE) coupled to mass spectrometry (MS) are two of the most widely used techniques to explore and characterize the polar metabolome. Both platforms have some peculiar characteristics that make them more suitable for studying different kind of metabolites and different parts of the metabolome. Consequently, their combined use is recommended to extend the coverage of the polar metabolome, particularly when multiple metabolic pathways are expected to be perturbed, as in toxicological studies related to exposure to environmental pollutants such as polychlorinated biphenyls (PCBs). It is unclear whether chronic exposure to low doses leads to effects on the most polar metabolites. ResultsIn this work, both HILIC-MS and CE-MS platforms have been used to characterize the polar metabolome of blood serum from pigs exposed to low-doses (20 ng/kg body weight (b.w.)/day) of Aroclor 1260 (a technical mixture of PCBs). The use of both platforms aims to cover large parts of the polar metabolome to better characterize the effects of PCB exposure. The results showed that the combined use of HILIC-MS and CE-MS effectively extended the coverage of the metabolome and the interpretability of the data, highlighting their complementarity in metabolic pathways analysis. The analysis of the polar metabolome evidenced a strong effect of exposure on amino acid metabolism, affecting 10 of the 20 proteic amino acids, and a consistent overflow in the urea cycle. SignificanceThis work highlights the need to implement analytical multi-platforms to better address metabolomics studies, showing the complementarity of two widely used analytical techniques (i.e. HILIC-MS and CE-MS) to study the polar metabolome in a risk assessment framework.

Interactions between prosulfocarb and trifluralin metabolism in annual ryegrass (Lolium rigidum).

Cross-resistance between pre-emergence herbicides is developing in Australian populations of annual ryegrass (Lolium rigidum Gaud.). A previous study has reported that selection with prosulfocarb (a pro-herbicide requiring bioactivation to its phytotoxic sulfoxide) can decrease metabolic resistance to trifluralin. Metabolism of prosulfocarb and trifluralin was investigated in L. rigidum populations with different levels of resistance to prosulfocarb, trifluralin and also pyroxasulfone, which is detoxified by glutathione (GSH) conjugation. Coleoptiles and radicles of herbicide-treated seedlings responded differently to the same herbicide. Radicles had a lower capacity for bioactivation of prosulfocarb, and this was correlated with a lower ability to metabolise trifluralin within and among populations. Coleoptile resistance to prosulfocarb sulfoxide was negatively correlated with abundance of a major polar metabolite. There was no evidence of GSH conjugation with the sulfoxide, making any potential links between prosulfocarb and pyroxasulfone resistance less obvious. Activation and metabolism of prosulfocarb in L. rigidum is complex and differentially regulated in different tissues. Selection with prosulfocarb may ameliorate trifluralin metabolism in the radicles, but the relationship between prosulfocarb and pyroxasulfone resistance is not GSH-mediated. When applying pre-emergence herbicides, care should be taken with the composition of mixtures and rotations to avoid selection of cross-resistance between pyroxasulfone and prosulfocarb. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Exploring electromembrane extraction coupled to fast LC-MS/MS as a high-throughput platform for determination of 12 polar endogenous metabolites in human plasma

High efficiency in the analytical workflow, including fast sample preparation and LC-MS/MS analysis, is an advantage when analyzing a high number of samples. It can however be a challenge when determining polar analytes in complex, biological samples, and one must expect to make a compromise between a simple sample preparation followed by a long chromatographic separation, or vice versa, to limit matrix effects. In this proof-of-concept work, a one-step 96-well (parallel extraction) electromembrane extraction (EME) method was coupled to flow injection-MS/MS of 0.7 min per sample, allowing a very high-throughput analysis of 12 polar, endogenous metabolites from unprecipitated plasma of limited dilution. The throughput of the EME method matched the subsequent analysis. Recoveries ranged from 6-93%, and repeatability and linearity were 2-15% and R2≥0.9949, respectively, for all but two compounds. Matrix effects were approximately 50% after EME and varied less than 11% between 6 plasma donors, which represented a major improvement relative to a simple protein precipitation where signals were entirely suppressed. The work demonstrates a potential for EME coupled to flow injection-MS/MS to serve as a high-throughput platform for bioanalysis, not just of polar analytes, but also hydrophobic drugs both basic and acidic.

Effects of thermal and UV stress on the polar and non-polar metabolome of photosymbiotic jellyfish and sea anemones

Recently, the impacts of climate change, notably ocean warming and solar ultraviolet radiation, have led to significant stress and mortality in cnidarians. The objective of this study is to decode the metabolic responses of sea anemones Entacmaea quadricolor and upside-down jellyfish Cassiopea andromeda upon exposure to thermal and ultraviolet stress. Gas chromatography-mass spectrometry and ultraperformance liquid chromatography coupled with high-resolution mass spectrometry targeting polar and non-polar metabolites were applied. In total, 72 polar and 242 lipophilic metabolites were detected in jellyfish and sea anemones, respectively. Amino acids are the major metabolite class in jellyfish, and triacylglycerides are the predominant lipids in jellyfish and anemones. Exposure to stressors led to metabolic alterations, marked by elevated amino acids in jellyfish and increased amino acids and sugar alcohols in sea anemones at 34 °C and after four days of ultraviolet radiation. Non-polar metabolome analysis indicated distinct responses to ultraviolet radiation and thermal stress in both species.

Advances in the application of ion chromatography-mass spectrometry in the fields of life and health

Ion chromatography is a technique commonly used to separate strongly polar and ionizable substances; it can be used to separate, identify, and quantify ionizable compounds in complex samples when coupled with mass spectrometry, and is currently being used in the application of food analysis, drug analysis, metabolomics and clinical poisoning analysis. Herein, we review the development of ion chromatography-mass spectrometry (IC-MS), its progress over the past 20 years, and future trends in the abovementioned areas. The IC-MS research progress and applications for the determination of inorganic anions, organic acids, polar pesticides, biogenic amines, and sugars in the food field are discussed. Drug analysis applications are discussed mainly in relation to the analysis of drug impurities, identifying drug degradation products, and determination of plasma concentration, while the separation and analysis of strongly polar metabolites, such as organic acids, sugar phosphates, and nucleotides in biological matrices are discussed in relation to metabolomics. Advances in the analysis of strongly polar or ionizable toxic compounds, such as alkyl methylphosphonic acid, methylphosphonic acid, glyphosate, 3-nitropropionic acid, and indandione rodenticides, are mainly discussed in clinical poisoning analysis field. This paper is expected to become a useful reference for the further expansion and application of IC-MS in the life and health fields.

A Comprehensive Strategy for Metabolites Profiling of Flowers and Leaves from Camellia tienii, an Endemic Golden Tea of Vietnam.

Golden camellia is defined as a species of the Camellia genus with yellow flowers, which have long been used as a medicine, food, and cosmetic in many Asian countries. To date, more than 50 golden camellia species are considered endemic in Vietnam; however, more information is needed about its chemical constituents and biological activity. This work aims to unveil the potential of Camellia tienii Ninh, a golden camellia species, as an herbal beverage by examining the presence and abundance of chemical components in flowers and leaves. A comprehensive strategy has been developed using both liquid and gas chromatography coupled with mass spectrometry. Specifically, LC-MS-based widely targeted analyses were opted to characterize 158 polar metabolites belonging mainly to flavonoids, catechins, and amino acids classes, and an untargeted approach using GC-MS annotated 42 major volatile compounds such as terpenes and fatty acids. The extensive profile revealed by these techniques could help understand the significant discrimination between two organs. C. tienii flowers accumulated more flavonoids, amino acids, and fatty acids, while leaves contain more terpenes, suggesting different pharmacological properties of these materials. Overall, this pipeline can be applied for other Camellia species and valorization of these valuable resources for health benefits purposes.

A key factor in monitoring cannabis consumption trends through wastewater analysis: Partitioning of THC[sbnd]COOH between the liquid and solid phase of influent wastewater

Current wastewater-based epidemiology (WBE) studies are predominantly focused on the analysis of urinary biomarkers present in the liquid phase of influent wastewater (IWW). This approach systematically underestimates less polar metabolites, such as cannabis biomarkers. These biomarkers can potentially sorb to and desorb from suspended particulate matter (SPM) present in IWW. This study investigates the bidirectional partitioning of THCCOOH between the liquid phase and SPM of IWW by performing multiple sorption experiments using THCCOOH-D9 as a surrogate due to the unavailability of blank SPM and blank IWW. In addition, this study involves the analysis of IWW collected from eight wastewater treatment plants (WWTP) (n = 56) across four European countries, where raw IWW, the liquid phase and SPM were separately analysed to identify potential trends in the fraction of THCCOOH in the SPM between and withing the examined locations. Based on the performed sorption experiments, bidirectional partitioning of THCCOOH between the liquid phase and SPM was noted, showing partition between both phases when only one of the two phases was spiked. It was illustrated that the concentration of SPM had a notable influence on the THCCOOH partitioning between both phases. In addition to the inter location variability (average THCCOOH present in the SPM ranged 31–59 %), a substantial intra location variation was also observed, e.g., in one location ranged 17–58 %. While the determination of a correction factor for the amount of THCCOOH present in SPM would be ideal, this is challenging, since the amount of SPM is not fixed in all IWW samples. Although SPM has influence on the THCCOOH partition, no correlation (p value Spearman correlation = 0.3160) was observed between the SPM concentration and the fraction of THCCOOH in the solid phase. Moreover, the collection of homogenized samples is difficult, and the time required to reach an equilibration in partitioning of THCCOOH between both IWW phases remains unclear. Due to i) the large inter-and intra-location variation of THCCOOH present in the SPM, ii) the variability in SPM concentration in IWW samples, and iii) the time required to reach a partitioning equilibration, an analytical procedure based on liquid-liquid extraction (LLE) that considers both the liquid phase and SPM of IWW is recommended to reduce the overall uncertainty for THCCOOH measurement in IWW. It was illustrated that this extraction method is capable to recover the total concentration of THCCOOH in both phases.

Gut microbiome shifts in adolescents after sleeve gastrectomy with increased oral-associated taxa and pro-inflammatory potential.

Bariatric surgery is highly effective in achieving weight loss in children and adolescents with severe obesity, however the underlying mechanisms are incompletely understood, and gut microbiome changes are unknown. 1) To comprehensively examine gut microbiome and metabolome changes after laparoscopic vertical sleeve gastrectomy (VSG) in adolescents and 2) to assess whether the microbiome/metabolome changes observed with VSG influence phenotype using germ-free murine models. 1) A longitudinal observational study in adolescents undergoing VSG with serial stool samples undergoing shotgun metagenomic microbiome sequencing and metabolomics (polar metabolites, bile acids and short chain fatty acids) and 2) a human-to-mouse fecal transplant study. We show adolescents exhibit significant gut microbiome and metabolome shifts several months after VSG, with increased alpha diversity and notably with enrichment of oral-associated taxa. To assess causality of the microbiome/metabolome changes in phenotype, pre-VSG and post-VSG stool was transplanted into germ-free mice. Post-VSG stool was not associated with any beneficial outcomes such as adiposity reduction compared pre-VSG stool. However, post-VSG stool exhibited an inflammatory phenotype with increased intestinal Th17 and decreased regulatory T cells. Concomitantly, we found elevated fecal calprotectin and an enrichment of proinflammatory pathways in a subset of adolescents post-VSG. We show that in some adolescents, microbiome changes post-VSG may have inflammatory potential, which may be of importance considering the increased incidence of inflammatory bowel disease post-VSG.

Abstract C044: PI5P4Kα regulates cell fitness through iron homeostasis in pancreatic cancer

Abstract PI5P4Ks (Phosphatidylinositol 5-phosphate 4-kinases) are a family of lipid kinases that phosphorylate phosphatidylinositol 5-monophosphate (PI-5-P) at the 4-position to generate distinct pools of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) largely on intracellular membranes. These lipid species are involved in cellular and metabolic stress responses, signaling and organelle communication. PI5P4Ks have been implicated in multiple cancers, with compelling evidence showing synthetic lethality when PI5P4Ks are inhibited in TP53-mutant cancer cells. Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive metabolic rewiring but despite the critical roles of PI5P4Ks in cellular metabolism, their role in PDAC remains completely unexplored. We have found that depletion of PI5P4Kα selectively induces apoptotic cell death in PDAC cells. To gain insights into the metabolic mechanisms underlying this apoptotic cell death, we quantified polar metabolites using gas chromatography- mass spectrometry (GC/MS) and found significant reductions in the ratio of monounsaturated to saturated fatty acids, lactate and TCA metabolites. Orthogonal rescue assays demonstrated that exogenous supplementation with fatty acids, pyruvate or lactate fail to rescue the observed apoptosis in PI5P4Kα-knockdown cells; however, TCA cycle metabolites did reverse the extent of apoptosis. Interestingly, we identify that these altered metabolic read-outs and the effects on cell fitness are dependent on iron. Consistent with these findings, intracellular iron levels are reduced upon PI5P4Kα inhibition, and this is directly linked to PI5P4Kα depletion disturbing the intracellular labile iron pools via iron import. Using a heterotopic xenograft mouse model, we demonstrate that PI5P4Kα knockdown leads to the abrogation of PDAC tumor growth due to increased intratumoral cell death. Validating the physiological relevance of PI5P4Kα in human PDAC, we find that PIP4K2A, the gene that encodes PI5P4Kα, is upregulated in human PDAC specimens relative to normal pancreas, and that PIP4K2A gene expression is strongly correlated with iron uptake related gene signatures. This study highlights the critical role of PI5P4Kα in PDAC and suggests that PI5P4Kα inhibition has therapeutic potential in pancreatic cancer. Citation Format: Gurpreet Kaur Arora, Ryan Loughran, Kyanh Ly, Cheska Marie Galapate, Alicia Llorente, Taylor R Anderson, Chantal Pauli, David A Scott, Yoav Altman, Cosimo Commisso, Brooke M Emerling. PI5P4Kα regulates cell fitness through iron homeostasis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C044.

Techniques, Databases and Software Used for Studying Polar Metabolites and Lipids of Gastrointestinal Parasites.

Gastrointestinal parasites (GIPs) are organisms known to have coevolved for millennia with their mammalian hosts. These parasites produce small molecules, peptides, and proteins to evade or fight their hosts' immune systems and also to protect their host for their own survival/coexistence. The small molecules include polar compounds, amino acids, lipids, and carbohydrates. Metabolomics and lipidomics are emerging fields of research that have recently been applied to study helminth infections, host-parasite interactions and biochemicals of GIPs. This review comprehensively discusses metabolomics and lipidomics studies of the small molecules of GIPs, providing insights into the available tools and techniques, databases, and analytical software. Most metabolomics and lipidomics investigations employed LC-MS, MS or MS/MS, NMR, or a combination thereof. Recent advancements in artificial intelligence (AI)-assisted software tools and databases have propelled parasitomics forward, offering new avenues to explore host-parasite interactions, immunomodulation, and the intricacies of parasitism. As our understanding of AI technologies and their utilisation continue to expand, it promises to unveil novel perspectives and enrich the knowledge of these complex host-parasite relationships.

Dual column chromatography combined with high-resolution mass spectrometry improves coverage of non-targeted analysis of plant root exudates

BackgroundWithin the plant kingdom, there is an exceptional amount of chemical diversity that has yet to be annotated. It is for this reason that non-targeted analysis is of interest for those working in novel natural products. To increase the number and diversity of compounds observable in root exudate extracts, several workflows which differ at three key stages were compared: 1) sample extraction, 2) chromatography, and 3) data preprocessing. ResultsPlants were grown in Hoagland's solution for two weeks, and exudates were initially extracted with water, followed by a 24-h regeneration period with subsequent extraction using methanol. Utilizing the second extraction showed improved results with less ion suppression and reduced retention time shifting compared to the first extraction. A single column method, utilizing a pentafluorophenyl column, paired with high-resolution mass spectrometry ionized and correctly identified 34 mock root exudate compounds, while the dual column method, incorporating a pentafluorophenyl column and a porous graphitic carbon column, retained and identified 43 compounds. In a pooled quality control sample of exudate extracts, the single column method detected 1,444 compounds. While the dual method detected fewer compounds overall (1,050), it revealed a larger number of small polar compounds. Three preprocessing methods (targeted, proprietary, and open source) successfully identified 43, 31, and 38 mock root exudate compounds to confidence level 1, respectively. SignificanceEnhancing signal strength and analytical method stability involves removing the high ionic strength nutrient solution before sampling root exudate extracts. Despite signal intensity loss, a dual column method enhances compound coverage, particularly for small polar metabolites. Open-source software proves a viable alternative for non-targeted analysis, even surpassing proprietary software in peak picking.

Biofluorescence imaging-guided spatial metabolic tracing: In vivo tracking of metabolic activity in circulating tumor cell-mediated multi-organ metastases

Circulating tumor cells (CTC) are considered metastatic precursors that are shed from the primary or metastatic deposits and navigate the bloodstream before undergoing extravasation to establish distant metastases. Metabolic reprogramming appears to be a hallmark of metastatic progression, yet current methods for evaluating metabolic heterogeneity within organ-specific metastases in vivo are limited. To overcome this challenge, we present Biofluorescence Imaging-Guided Spatial Metabolic Tracing (BIGSMT), a novel approach integrating in vivo biofluorescence imaging, stable isotope tracing, stain-free laser capture microdissection, and liquid chromatography-mass spectrometry. This innovative technology obviates the need for staining or intricate sample preparation, mitigating metabolite loss, and substantially enhances detection sensitivity and accuracy through chemical derivatization of polar metabolites in central carbon pathways. Application of BIGSMT to a preclinical CTC-mediated metastasis mouse model revealed significant heterogeneity in the in vivo carbon flux from glucose into glycolysis and the tricarboxylic acid (TCA) cycle across distinct metastatic sites. Our analysis indicates that carbon predominantly enters the TCA cycle through the enzymatic reaction catalyzed by pyruvate dehydrogenase. Thus, our spatially resolved BIGSMT technology provides fresh insights into the metabolic heterogeneity and evolution during melanoma CTC-mediated metastatic progression and points to novel therapeutic opportunities.