Untargeted LC-MS Research Articles

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

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

Integrative LC-MS and GC-MS metabolic profiling unveils dynamic changes during barley malting

Malting involves complex biochemical transformations affecting sensory and quality attributes. Despite extensive research on storage carbohydrates and proteins in malting, the lack of a detailed metabolic understanding of this process limits our ability to assess and enhance malt quality. This study employed untargeted GC-MS and LC-MS metabolite profiling across six malting timepoints to identify 4980 known metabolites, 82 % of which exhibited significant changes during the malting process. Here we identified stage-dependent metabolic shifts and dynamic chemical classes and pathways between each studied stage. These results can guide the fine-tuning of malting conditions to improve malt quality for beer production and other malt-based applications. Additionally, metabolites with antimicrobial properties were identified, underscoring the interplay between barley and microbial metabolic processes during malting. Further research into these microbial metabolites and cognate microbes may lead to novel malting assessment traits for high-quality and safe malted barley.

Urinary Biomarkers of Strawberry and Blueberry Intake.

Introduction There is increasing interest in food biomarkers to address the shortcomings of self-reported dietary assessments. Berries are regarded as important fruits worldwide; however, there are no well-validated biomarkers of berry intake. Thus, the objective of this study is to identify urinary biomarkers of berry intake. Methods For the discovery study, participants consumed 192 g strawberries with 150 g blueberries, and urine samples were collected at 2, 4, 6, and 24 h post-consumption. A dose-response study was performed, whereby participants consumed three portions (78 g, 278 g, and 428 g) of mixed strawberries and blueberries. The urine samples were profiled by an untargeted LC-MS metabolomics approach in the positive and negative modes. Results Statistical analysis of the data revealed that 39 features in the negative mode and 15 in the positive mode significantly increased between fasting and 4 h following mixed berry intake. Following the analysis of the dose-response data, 21 biomarkers showed overall significance across the portions of berry intake. Identification of the biomarkers was performed using fragmentation matches in the METLIN, HMDB, and MoNA databases and in published papers, confirmed where possible with authentic standards. Conclusions The ability of the panel of biomarkers to assess intake was examined, and the predictability was good, laying the foundations for the development of biomarker panels.

Ningxiang Pig-Derived Microbiota Affects the Growth Performance, Gut Microbiota, and Serum Metabolome of Nursery Pigs.

The gut microbiota is crucial for maintaining the host's intestinal homeostasis and metabolism. This study investigated the effects of fecal microbiota transplantation (FMT) from Ningxiang pigs on the growth performance, fecal microbiota, and serum metabolites of the same-old DLY pigs. The results indicated that the average daily gain of FMT pigs was significantly greater than that of the control (CON) group. Compared to the CON group, the FMT group significantly improved the apparent digestibility of crude fiber, crude ash, gross energy, and calcium of the pigs. The analysis of serum antioxidant status revealed that the activities of total superoxide dismutase and catalase in the serum of pigs in the FMT group were significantly elevated, whereas the level of malondialdehyde was significantly reduced. Furthermore, 16S rRNA sequencing analysis revealed that the Ningxiang pig-derived microbiota altered the fecal microbiota structure and modulated the diversity of the gut microbiota in the DLY pigs. Untargeted LC-MS metabolomics demonstrated that pigs in the FMT group exhibited distinct metabolomic profiles compared to those in the CON group. Significant changes were observed in key metabolites involved in amino acid, lipid, and carbohydrate metabolism. Additionally, a correlation analysis between serum differential metabolites and the gut microbiota revealed that the relative abundance of Lachnospiraceae_NK4A136_group and Corynebacterium was highly correlated with lipid compounds. In conclusion, Ningxiang pig-derived microbiota can alleviate oxidative stress and enhance growth performance in DLY pigs by modulating their gut microbiota and metabolic features.

Ontogeny and organ-specific steroidal glycoside diversity is associated with differential expression of steroidal glycoside pathway genes in two Solanum dulcamara leaf chemotypes.

Solanaceous plants, such as Solanum dulcamara, produce steroidal glycosides (SGs). Leaf SG profiles vary among S. dulcamara individuals, leading to distinct phytochemical phenotypes ('chemotypes') and intraspecific phytochemical diversity ('chemodiversity'). However, if and how SG chemodiversity varies among organs and across ontogeny, and how this relates to SG metabolism gene expression is unknown. Among organs and across ontogeny, S. dulcamara plants with saturated (S) and unsaturated (U) SG leaf chemotypes were selected and clonally propagated. Roots, stems and leaves were harvested from vegetative and flowering plants. Extracts were analysed using untargeted LC-MS. Expression of candidate genes in SG metabolism (SdGAME9, SdGAME4, SdGAME25, SdS5αR2 and SdDPS) was analysed using RT-qPCRs. Our analyses showed that SG chemodiversity varies among organs and across ontogeny in S. dulcamara; SG richness (Dmg) was higher in flowering than vegetative plants. In vegetative plants, Dmg was higher for leaves than for roots. Lack of SdGAME25 expression in U-chemotype leaves, while readily expressed in roots and stems, suggests a pivotal role for SdGAME25 in differentiation of leaf chemotypes in vegetative and flowering plants. By acting as an ontogeny-dependent chemotypic switch, differential regulation of SdGAME25 enables adaptive allocation of SGs, thereby increasing SG chemodiversity in leaves. This indicates that differential expression and/or regulation of glycoalkaloid metabolism genes, rather than their presence or absence, explains observed chemotypic variation in SG chemodiversity among organs and across ontogeny.

The salivary metabolome of children and parental caregivers in a large-scale family environment study

Human metabolism is complex, and is impacted by genetics, cohabitation, diet, health, and environmental inputs. As such, we applied untargeted LC-MS metabolomics to 1425 saliva samples from a diverse group of elementary school-aged children and their caregivers collected during the Family Life Project, of which 1344 were paired into caregiver/child dyads. We compared metabolomes within and between homes, performed population-wide “metabotype” analyses, and measured associations between metabolites and salivary biomeasures of inflammation, antioxidant potential, environmental tobacco smoke (ETS) exposure, metabolic regulation, and heavy metals. Children and caregivers had similar salivary metabolomes, and dyad explained most metabolomic variation. Our data clustered into two groups, indicating that “metabotypes” exist across large populations. Lastly, several metabolites—putative oxidative damage-associated or pathological markers—were correlated with the above-mentioned salivary biomeasures and heavy metals. Implications of the family environment’s effects on metabolomic variation at population, dyadic, and individual levels for human health are discussed.

Wheat domestication alters root metabolic functions to drive the assembly of endophytic bacteria.

The domestication process progressively differentiated wild relatives from modern cultivars, thus impacting plant-associated microorganisms. Endophytic bacterial communities play vital roles in plant growth, development, and health, which contribute to the crop's sustainable development. However, how plant domestication impacts endophytic bacterial communities and relevant root exudates in wheat remains unclear. First, we have observed that the domestication process increased the root endophytic microbial community diversity of wheat while decreasing functional diversity. Second, domestication decreased the endophytic bacterial co-occurrence network stability, and it did significantly alter the abundances of core microorganisms or potential probiotics. Third, untargeted LC-MS metabolomics revealed that domestication significantly altered the metabolite profiles, and the abundances of various root exudates released were significantly correlated with keystone taxa including the Chryseobacterium, Massilia, and Lechevalieria. Moreover, we found that root exudates, especially L-tyrosine promote the growth of plant-beneficial bacteria, such as Chryseobacterium. Additionally, with L-tyrosine and Chryseobacterium colonized in the roots, the growth of wild wheat's roots was significantly promoted, while no notable effect could be found in the domesticated cultivars. Overall, this study suggested that wild wheat as a key germplasm material, and its native endophytic microbes may serve as a resource for engineering crop microbiomes to improve the morphological and physiological traits of crops in widely distributed poor soils.

Detection of pharmacolipidodynamic effects following the intravenous and oral administration of gefitinib to C57Bl/6JRj mice by rapid UHPLC-MS analysis of plasma

Omics-based biomarker technologies, including metabolic profiling (metabolomics/metabonomics) and lipidomics, are making a significant impact on disease understanding, drug development, and translational research. A wide range of patho-physiological processes involve lipids and monitoring changes in lipid abundance can give valuable insights into mechanisms of drug action, off target pharmacology and toxicity. Here we report changes, detected by untargeted LC–MS, in the plasma lipid profiles of male C57Bl/6JRj mice following the PO and IV administration of the epidermal growth factor receptor (EGFR) inhibitor gefitinib. Statistical analysis of the data obtained for both the IV and PO samples showed time-related changes in the amounts of lipids from several different classes. The largest effects were associated with a rapid onset of these changes following gefitinib administration followed by a gradual return by 24 h post dose to the type of lipid profile seen in predose samples. Investigation of the lipids responsible for the variance observed in the data showed that the PI, PC, LPC, PE and TG were subject to the largest disruption with both transient increases and decreases in relative amounts seen in response to administration of the drug. The pattern of the changes in the relative abundances of those lipids subject to variation appeared to be correlated to the pharmacokinetics of gefitinib (and its major metabolites). These observations support the concept of a distinct pharmacolipidodynamic relationship between drug exposure and plasma lipid abundance.

Metabolomics analysis of human spermatozoa reveals impaired metabolic pathways in asthenozoospermia.

Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia (AS), or low sperm motility, is a common cause of male infertility with complex aetiology, involving genetic and metabolic alterations, inflammation and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility. We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and AS (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate. We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa's metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered inAS. Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy and lipid metabolism in AS. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the aetiology of decreased motility in AS.

Comprehensive metabolome characterization of leaves, internodes, and aerial roots of Vanilla planifolia by untargeted LC-MS and GC × GC-MS.

Untargeted metabolomics is a powerful tool that provides strategies for gaining a systematic understanding of quantitative changes in the levels of metabolites, especially when combining different metabolomic platforms. Vanilla is one of the world's most popular flavors originating from cured pods of the orchid Vanilla planifolia. However, only a few studies have investigated the metabolome of V. planifolia, and no LC-MS or GC-MS metabolomics studies with respect to leaves have been performed. The aim of the study was to comprehensively characterize the metabolome of different organs (leaves, internodes, and aerial roots) of V. planifolia. Characterization of the metabolome was achieved using two complementary platforms (GC × GC-MS, LC-QToF-MS), and metabolite identification was based on a comparison with in-house databases or curated external spectral libraries. In total, 127 metabolites could be identified with high certainty (confidence level 1 or 2) including sugars, amino acids, fatty acids, organic acids, and amines/amides but also secondary metabolites such as vanillin-related metabolites, flavonoids, and terpenoids. Ninty-eight metabolites showed significantly different intensities between the plant organs. Most strikingly, aglycons of flavonoids and vanillin-related metabolites were elevated in aerial roots, whereas its O-glycoside forms tended to be higher in leaves and/or internodes. This suggests that the more bioactive aglycones may accumulate where preferably needed, e.g. for defense against pathogens. The results derived from the study substantially expand the knowledge regarding the vanilla metabolome forming a valuable basis for more targeted investigations in future studies, e.g. towards an optimization of vanilla plant cultivation.

New insights into the antibiofilm activity and mechanism of Mannosylerythritol Lipid-A against Listeria monocytogenes EGD-e

Listeria monocytogenes is one of the leading causative agents of foodborne disease outbreaks worldwide. Herein, the antibiofilm effect and mechanism of Mannosylerythritol Lipid-A against L. monocytogenes EGD-e is reported for the first time. MEL-A effectively attenuated biofilm formation while reducing the viability and motility of bacteria within the biofilm in the early stage, and influenced bacterial adhesion by affecting the secretion of extracellular polysaccharides and eDNA. RT-qPCR revealed that MEL-A significantly suppressed the expression of genes involved in flagellar movement and virulence. Untargeted LC-MS metabolomics indicated that MEL-A affected the fluidity and permeability of cell membranes by significantly upregulating unsaturated fatty acids, lipids and glycoside metabolites, and affected protein biosynthesis, nucleotide metabolism and DNA synthesis and repair by significantly downregulating amino acid metabolism and nucleic acid metabolism. These pathways may constitute the key targets of biofilm formation inhibition by MEL-A. Furthermore, MEL-A showed good removal effects on mature biofilms under different temperatures, different materials and milk. Our data indicated that MEL-A could be used as a novel antibiofilm agent to improve food safety. Our study provides new insights into the possible inhibitory mechanism of MEL-A and the response of L. monocytogenes EGD-e to MEL-A.

Gut Microbiota-Derived Butyrate Induces Epigenetic and Metabolic Reprogramming in Myeloid-Derived Suppressor Cells to Alleviate Primary Biliary Cholangitis

Background & AimsGut dysbiosis and myeloid-derived suppressor cells (MDSCs) are implicated in primary biliary cholangitis (PBC) pathogenesis. However, it remains unknown whether gut microbiota or their metabolites can modulate MDSCs homeostasis to rectify immune dysregulation in PBC. MethodsWe measured fecal short-chain fatty acids (SCFAs) levels by targeted GC-MS and analyzed circulating MDSCs by flow cytometry in two independent PBC cohorts. Human and murine MDSCs were differentiated in vitro in the presence of butyrate, followed by transcriptomic, epigenetic (CUT&Tag-seq and ChIP-qPCR) and metabolic (untargeted LC-MS, mitochondrial stress test, and isotope tracing) analyses. The in vivo role of butyrate-MDSCs was evaluated in 2-octynoic acid-BSA-induced cholangitis murine model. ResultsDecreased butyrate levels and defective MDSCs function were found in patients with incomplete response to ursodeoxycholic acid (UDCA), compared to those with adequate response. Butyrate induced expansion and suppressive activity of MDSCs in a manner dependent on PPARD-driven fatty acid β-oxidation (FAO). Pharmaceutical inhibition or genetic knockdown of the FAO rate-limiting gene CPT1A abolished the effect of butyrate. Furthermore, butyrate inhibited HDAC3 function, leading to enhanced H3K27ac modifications at promoter regions of PPARD and FAO genes in MDSCs. Therapeutically, butyrate administration alleviated immune-mediated cholangitis in mice via MDSCs, and adoptive transfer of butyrate-treated MDSCs also displayed protective efficacy. Importantly, reduced expression of FAO genes and impaired mitochondrial physiology were detected in MDSCs from UDCA non-responders, and their impaired suppressive function was restored by butyrate. ConclusionWe identify a critical role for butyrate in modulation of MDSCs homeostasis by orchestrating epigenetic and metabolic crosstalk, proposing a novel therapeutic strategy for treating PBC.

Untargeted LC–MS metabolomics reveals the metabolic responses in the Eriocheir sinensis gills exposed to salinity and alkalinity stress

In recent years, saline–alkaline aquaculture development has become an important measure for China to expand its fishery development space to ensure food safety. Previous studies have verified that salinity and alkalinity positively influence the quality of Chinese mitten crabs (Eriocheir sinensis). However, the regulatory mechanism of E. sinensis endures saline–alkaline stress which remains obscure. This study investigated the metabolic changes in puberty-molting E. sinensis gills exposed to freshwater (FW), sodium chloride salinity of 5 ppt (SW), and carbonate alkalinity 10.00 mmol/L (AW) for 50 days using untargeted liquid chromatography-mass spectrometry metabolomics (LC–MS). A total of 5802 (positive-ion mode) and 6520 (negative-ion mode) peaks were extracted by LC–MS, respectively. A total of 188 (50 upregulated and 138 downregulated), 141 (94 upregulated and 47 downregulated), and 130 (87 upregulated and 43 downregulated) significantly regulated metabolites (SRMs) were observed in the FW–SW, FW–AW, and SW–AW treatments, respectively, wherein 42 generic SRMs were also found by Venn diagram analysis. Seven of the top 10 SRMs with the highest (variable importance in projection) VIP values were similarly identified in FW–SW and SW–AW. Integrated analysis of key metabolic pathways revealed glycerophospholipid, choline in cancer, phenylalanine, and butanoate metabolism. Overall, significant differences were observed in the metabolites and key metabolic pathways of E. sinensis gill exposed to salinity and alkalinity stress. These results will be helpful in understanding the environmental adaptability of aquatic crustaceans to saline–alkaline water.

Comparative metabolomics reveals serum metabolites changes in goats during different developmental stages

Goats can provide meat, milk and skins for humans and are livestock with high economic benefits. However, despite their economic significance, the comprehensive analysis of goats’ serum metabolic profile and its intricate alterations throughout their developmental journey remains conspicuously absent. To investigate the stage-specificity and dynamic change characteristics of metabolites during the growth and development of goats, this study compared the alterations in serum hormone levels and serum biochemical markers across different developmental stages of female goats (1, 60, 120 and 180 days old; n = 5). Additionally, a serum untargeted LC–MS metabolomics analysis was conducted. A total of 504 DAMs were identified with age. The results indicated that PE, PC, Lyso-PE, Lyso-PC and FAFHA may play important roles in lipid metabolism in goats after birth. Weighted gene co-expression network analysis (WGCNA) identified two metabolite modules (Turquoise and Yellow) and key metabolites within these modules that were significantly associated with phenotypic features. l-carnitine may be a metabolite related to muscle development in goats. The findings of this study demonstrate notable variations in serum metabolites across distinct developmental phases in goats. Lipids and organic acids play important roles in different developmental stages of goats.

Abstract 1068: Plasma metabolite biomarkers for EGFR-mutated non-small cell lung cancer (NSCLC)

Abstract Background: Lung cancer is the leading cause of cancer death. More than half of the patients are diagnosed in the advanced stage for which the 5-year survival is below 10%. Lung cancer screening using low-dose CT scan reduced lung cancer mortality among heavy smokers. However, no standard screening method exists for non-smokers. Up to 35% and 90% of male and female lung cancer patients in Southeast Asia, respectively, do not smoke. Half of NSCLC patients in this region harbor EGFR mutations, which is common in women and non-smokers. Thus, we sought to explore novel biomarkers that help to identify lung cancer patients and those who have EGFR mutation. Metabolic reprogramming is one of the hallmarks of cancer. It leads to changes in metabolite levels in the blood. Hence, we conducted plasma metabolomic analysis to identify potential metabolite biomarkers in lung cancer patients. Methods: Plasma samples were collected from 69 lung cancer patients at Siriraj hospital and 10 healthy individuals during 2018-2022. Plasma metabolites from 29 treatment-naïve EGFR-mutated NSCLC patients, 30 treatment-naïve EGFR-wild-type NSCLC patients, 10 treatment-resistant EGFR T790M-mutated NSCLC patients, and 10 healthy individuals were profiled using GC-MS and untargeted LC-MS with Metlin database matching. EGFR mutation was detected from plasma ctDNA using real-time PCR. Negative plasma EGFR mutation results were confirmed with corresponding tumor tissues. Metabolomics data were processed by the metabox R package. Results: Among 114 candidate metabolites that passed the variable importance in projection (VIP) cutoff of 1.5, the top ten metabolites that differentiate NSCLC patients with EGFR mutation from those without were shown in Table 1. Many of them can also separate between NSCLC patients and healthy individuals. Table 1. ROC curve analysis Discriminant metabolites (Mutated EGFR vs Wild-type EGFR NSCLC) AUC Accuracy Sensitivity Specificity PG(a-13:0/i-12:0) 0.8000 0.7833 0.8333 0.7333 Nervonic ceramide 0.7736 0.6772 0.7259 0.6333 Valine 0.7678 0.7298 0.7259 0.7333 L-Histidinol 0.7644 0.7281 0.7222 0.7333 Leucine 0.7563 0.7632 0.7259 0.8000 Tryptophan 0.7425 0.6289 0.5593 0.7000 8-methylcaffeine 0.6793 0.6096 0.6222 0.6000 Formic acid 0.6759 0.6447 0.5889 0.7000 PC(20:0/24:0) 0.6506 0.6614 0.8259 0.5000 Sucrose 0.6471 0.6596 0.7593 0.5667 Discriminant metabolites (NSCLC patients vs Healthy individuals) AUC Accuracy Sensitivity Specificity Tryptophan 0.9986 0.9867 1.0000 0.9848 PC(20:0/24:0) 0.8609 0.9505 0.6111 1.0000 L-Histidinol 0.8572 0.9625 0.7778 0.9861 Formic acid 0.6696 0.9262 0.6667 0.9704 Conclusions: Plasma L-Histidinol, tryptophan, PC(20:0/24:0), and formic acid are potential metabolite biomarkers for NSCLC patients harboring EGFR mutation. Citation Format: Lucksamon Thamlikitkul, Siriphan Manocheewa, Suphitcha Limjiasahapong, Kwanjeera Wanichthanarak, Atikorn Panya, Naravat Poungvarin, Yotsawat Pomyen, Sakda Khoomrung. Plasma metabolite biomarkers for EGFR-mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1068.

Abstract 6379: Comparison of lipid profiles in children with solid tumors receiving chemotherapy and healthy children

Abstract Background: Lipid metabolism plays an important role in the regulation of synaptic function and plasticity.Cancer chemotherapy (chemo) can disturb the lipid profile, potentially linked with neuropsychological toxicities. This study aimed to compare the lipid profiles between children with cancer receiving chemo and healthy children. Methods: A prospective, observational study was conducted in 21 children with solid tumors enrolled from Children’s Healthcare of Atlanta (CHOA) and 16 controls. Children with solid tumors were consented precycle 2 chemo (T1) and followed at the end of chemo (T2). Controls completed one timepoint of data collection. At T1 (n=21) and T2 (n=13), fecal samples were collected for lipidomics analysis. Fecal lipids were processed using a high-resolution, untargeted lipidomics LC-MS approach. MetaboAnalyst and linear mixed models were used to analyze the data with false discovery rate correction (FDR 0.2). Results: There were no significant differences in age, sex, race, and body mass index between the groups. In total 241 lipid features were identified with high confidence and analyzed. A comparison between children with solid tumors at T2 (n=12) vs. controls (n=16) detected 8 differential lipids, which can be clustered into 4 classes, including 4 triglycerides (TG), 2 phosphatidylcholine (PC), 1 sphingomyelin (SM), and 1 lysophosphatidylcholine (LPC). Additionally, 24 differential lipids were detected for children with solid tumors from T1 to T2 (n=12), including 6 classes: 9 SM, 7 PC, 3 diacylglycerols (DG), 2 TG, 2 monoacylglycerols (MG), and 1 phosphatidylethanolamine (PE). Compared with controls, children with solid tumors at T2 had higher levels of SM (e.g., SM(d36:1)) and lower levels of TG (e.g., TG(18:0/18:0/20:0)), LPC (e.g., LPC(20:4)), and PC (e.g., PC(33:4). Compared with children at T1, those at T2 had higher levels of SM (e.g., SM(d36:1)), PE (e.g., PE(16:0p/20:4), PC (e.g., PC(30:0), and PC(36:1)), and lower levels of TG (e.g., TG(18:0/18:0/20:0) and TG(18:0/16:0/18:0)), LPC (e.g., LPC(20:4)), DG (e.g., DG(18:0/16:0) and DG(18:0/18:0)), and MG (e.g., MG(18:0)). Conclusion: Children with cancer chemo had alterations in fecal lipid profiles impacting the metabolism of PC, SM, TG, PE, MG, and DG. The increased PC and the dysregulation of LPC in comparison with controls suggest the decreased activity of phospholipase A2 (PLA2), primarily reported in inflammatory disease. Altered activity of lysophospholipid acyltransferase (LPCAT), diacylglyceride acyltransferase (DGAT), and phosphatidylethanolamine methyl transferase (PEMT) could also explain changes in PC and glycerolipids. Combined, these suggest dysregulation of the Kennedy or Lands pathways towards PC synthesis. Future work is needed to study whether the disturbed lipid metabolisms were associated with treatment toxicities and outcomes such as cognitive dysfunction based on literature. Citation Format: Jinbing Bai, Madelyn Houser, Melissa Martin, Christie Powell, Kristal Maner-Smith, Kathryn S. Sutton, Thomas Olson, Deborah W. Bruner. Comparison of lipid profiles in children with solid tumors receiving chemotherapy and healthy children [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6379.

Metabolomic Profiling Reveals the Quality Variations in Citri Reticulatae Pericarpium (Citrus reticulata Blanco cv. Chachiensis) with Different Storage Ages in Response to "Candidatus Liberibacter Asiaticus" Infection.

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, "Candidatus Liberibacter asiaticus" (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC-MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC.

Calotropis procera extract inhibits prostate cancer through regulation of autophagy.

Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen-independent PC-3 and androgen-sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound-healing assay and 2',7'-dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE-mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC-MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC-3 and 22Rv1, in a dose- and time-dependent manner. Also, CPE significantly inhibited the colony-forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF-κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE-treated PC-3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.

Development of an Untargeted LC-MS Metabolomics Method with Postcolumn Infusion for Matrix Effect Monitoring in Plasma and Feces.

Untargeted metabolomics based on reverse phase LC-MS (RPLC-MS) plays a crucial role in biomarker discovery across physiological and disease states. Standardizing the development process of untargeted methods requires paying attention to critical factors that are under discussed or easily overlooked, such as injection parameters, performance assessment, and matrix effect evaluation. In this study, we developed an untargeted metabolomics method for plasma and fecal samples with the optimization and evaluation of these factors. Our results showed that optimizing the reconstitution solvent and sample injection amount was critical for achieving the balance between metabolites coverage and signal linearity. Method validation with representative stable isotopically labeled standards (SILs) provided insights into the analytical performance evaluation of our method. To tackle the issue of the matrix effect, we implemented a postcolumn infusion (PCI) approach to monitor the overall absolute matrix effect (AME) and relative matrix effect (RME). The monitoring revealed distinct AME and RME profiles in plasma and feces. Comparing RME data obtained for SILs through postextraction spiking with those monitored using PCI compounds demonstrated the comparability of these two methods for RME assessment. Therefore, we applied the PCI approach to predict the RME of 305 target compounds covered in our in-house library and found that targets detected in the negative polarity were more vulnerable to the RME, regardless of the sample matrix. Given the value of this PCI approach in identifying the strengths and weaknesses of our method in terms of the matrix effect, we recommend implementing a PCI approach during method development and applying it routinely in untargeted metabolomics.

MetabCombiner 2.0: Disparate Multi-Dataset Feature Alignment for LC-MS Metabolomics.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS), as applied to untargeted metabolomics, enables the simultaneous detection of thousands of small molecules, generating complex datasets. Alignment is a crucial step in data processing pipelines, whereby LC-MS features derived from common ions are assembled into a unified matrix amenable to further analysis. Variability in the analytical factors that influence liquid chromatography separations complicates data alignment. This is prominent when aligning data acquired in different laboratories, generated using non-identical instruments, or between batches from large-scale studies. Previously, we developed metabCombiner for aligning disparately acquired LC-MS metabolomics datasets. Here, we report significant upgrades to metabCombiner that enable the stepwise alignment of multiple untargeted LC-MS metabolomics datasets, facilitating inter-laboratory reproducibility studies. To accomplish this, a "primary" feature list is used as a template for matching compounds in "target" feature lists. We demonstrate this workflow by aligning four lipidomics datasets from core laboratories generated using each institution's in-house LC-MS instrumentation and methods. We also introduce batchCombine, an application of the metabCombiner framework for aligning experiments composed of multiple batches. metabCombiner is available as an R package on Github and Bioconductor, along with a new online version implemented as an R Shiny App.