Mass Spectrometry-based Metabolomics Research Articles

Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry-Based Untargeted Metabolomics to Differentiate Guizhou Glutinous Sorghum and Hongyingzi Sorghum

Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry-Based Untargeted Metabolomics to Differentiate Guizhou Glutinous Sorghum and Hongyingzi Sorghum

NEK6 dampens FOXO3 nuclear translocation to stabilize C-MYC and promotes subsequent de novo purine synthesis to support ovarian cancer chemoresistance

De novo purine synthesis metabolism plays a crucial role in tumor cell survival and malignant progression. However, the specific impact of this metabolic pathway on chemoresistance in ovarian cancer remains unclear. This study aims to elucidate the influence of de novo purine synthesis on chemoresistance in ovarian cancer and its underlying regulatory mechanisms. We analyzed metabolic differences between chemosensitive and chemoresistant ovarian cancer tissues using mass spectrometry-based metabolomics. Cell growth, metabolism, chemoresistance, and DNA damage repair characteristics were assessed in vitro using cell line models. Tumor growth and chemoresistance were assessed in vivo using ovarian cancer xenograft tumors. Intervention of purines and NEK6-mediated purine metabolism on chemoresistance was investigated at multiple levels. Chemoresistant ovarian cancers exhibited higher purine abundance and NEK6 expression. Inhibiting NEK6 led to decreased de novo purine synthesis, resulting in diminished chemoresistance in ovarian cancer cells. Mechanistically, NEK6 directly interacted with FOXO3, contributing to the phosphorylation of FOXO3 at S7 through its kinase activity, thereby inhibiting its nuclear translocation. Nuclear FOXO3 promoted FBXW7 transcription, leading to c-MYC ubiquitination and suppression of de novo purine synthesis. Paeonol, by inhibiting NEK6, suppressed de novo purine synthesis and enhanced chemosensitivity. The NEK6-mediated reprogramming of de novo purine synthesis emerges as a critical pathway influencing chemoresistance in ovarian cancer. Paeonol exhibits the potential to interfere with NEK6, thereby inhibiting chemoresistance.

Comparative metabolites profiling of different solvent extracts of Asparagus species cladodes using liquid chromatography-mass spectrometry-based metabolomics and molecular networking.

Asparagus species are naturally distributed worldwide and are known for their pharmacological properties that offer cures for various ailments. However, the metabolic choreography of these Asparagus species is not well characterized, and the compounds contributing to their bioactivities remain unknown. This study aimed to profile and compare the metabolomes of three Asparagus species cladodes using different solvent extractions. An ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics and molecular networking approach was used to study the effects of different solvents (ethyl acetate, methanol, and chloroform) with varying polarity on metabolites extraction and identification of bioactive compounds from three Asparagus species cladodes (Asparagus falcatus, Asparagus plumosus, and Asparagus densiflorus 'Meyersii'). Multivariate statistical analyses (mainly principal component analysis) revealed a significant separation between the three solvents and the three species, indicating notable metabolic differences. A total of 118 metabolites were identified in the three species extracted with the different solvents, with methanolic and chloroform extracts containing more metabolites compared with ethyl acetate extracts. These metabolites were identified as belonging to the flavonoids, cinnamic acids, organooxygen compounds, steroids, fatty acids, benzenes, and glycerophospholipids compound classes. Furthermore, these compounds classes were differentially distributed among the three species, indicating chemical/chemotaxis differences between the compared species. Chloroform and methanol are recommended as the optimal solvents to obtain a high content of phytochemical compounds from Asparagus species cladodes.

Urine metabolomic profiles of autism and autistic traits-A twin study.

Currently, there are no reliable biomarkers for autism diagnosis. The heterogeneity of autism and several co-occurring conditions are key challenges to establishing these. Here, we used untargeted mass spectrometry-based urine metabolomics to investigate metabolic differences for autism diagnosis and autistic traits in a well-characterized twin cohort (N = 105). We identified 208 metabolites in the urine samples of the twins. No clear, significant metabolic drivers for autism diagnosis were detected when controlling for other neurodevelopmental conditions. However, we identified nominally significant changes for several metabolites. For instance, phenylpyruvate (p = 0.019) and taurine (p = 0.032) were elevated in the autism group, while carnitine (p = 0.047) was reduced. We furthermore accounted for the shared factors, such as genetics within the twin pairs, and report additional metabolite differences. Based on the nominally significant metabolites for autism diagnosis, the arginine and proline metabolism pathway (p = 0.024) was enriched. We also investigated the association between quantitative autistic traits, as measured by the Social Responsiveness Scale 2nd Edition, and metabolite differences, identifying a greater number of nominally significant metabolites and pathways. A significant positive association between indole-3-acetate and autistic traits was observed within the twin pairs (adjusted p = 0.031). The utility of urine biomarkers in autism, therefore, remains unclear, with mixed findings from different study populations.

Mass spectrometry-based metabolomics reveals metabolism of molnupiravir may lead to metabolic disorders and hepatotoxicity.

Molnupiravir (MO) is a pyrimidine nucleoside anti-SARS-CoV-2 drug. MO treatment could cause mild liver injury. However, the underlying mechanism of MO-induced liver injury and the metabolic pathway of MO in vivo are unclear. In this study, metabolomics analysis and molecular biology methods were used to explore these issues. Through metabolomics analysis, it was found that the homeostasis of pyrimidine, purine, lysophosphatidylcholine (LPC), and amino acids in mice was destroyed after MO treatment. A total of 80 changed metabolites were detected. Among these changed metabolites, 4-ethylphenyl sulfate, dihydrouracil, and LPC 20:0 was related to the elevation of alkaline phosphatase (ALP), interleukin-6 (IL6), and nuclear factor kappa-B (NF-κB). The levels of 4-ethylphenyl sulfate, dihydrouracil, and LPC 20:0 in plasma were positively correlated with their levels in the liver, suggesting that these metabolites were associated with MO-induced liver injury. MO treatment could increase NHC and cytidine levels, activate cytidine deaminase (CDA), and increase LPC levels. CDA and LPC could increase the mRNA expression level of toll-like receptor (TLR). The current study indicated that the elevation of hepatic TLR may be an important reason for MO leading to the liver injury.

Nanotopography Influences Host-Pathogen Quorum Sensing and Facilitates Selection of Bioactive Metabolites in Mesenchymal Stromal Cells and Pseudomonas aeruginosa Co-Cultures.

Orthopedic implant-related bacterial infections and resultant antibiotic-resistant biofilms hinder implant-tissue integration and failure. Biofilm quorum sensing (QS) communication determines the pathogen colonization success. However, it remains unclear how implant modifications and host cells are influenced by, or influence, QS. High aspect ratio nanotopographies have shown to reduce biofilm formation of Pseudomonas aeruginosa, a sepsis causing pathogen with well-defined QS molecules. Producing such nanotopographies in relevant orthopedic materials (i.e., titanium) allows for probing QS using mass spectrometry-based metabolomics. However, nanotopographies can reduce host cell adhesion and regeneration. Therefore, we developed a polymer (poly(ethyl acrylate), PEA) coating that organizes extracellular matrix proteins, promoting bioactivity to host cells such as human mesenchymal stromal cells (hMSCs), maintaining biofilm reduction. This allowed us to investigate how hMSCs, after winning the race for the surface against pathogenic cells, interact with the biofilm. Our approach revealed that nanotopographies reduced major virulence pathways, such as LasR. The enhanced hMSCs support provided by the coated nanotopographies was shown to suppress virulence pathways and biofilm formation. Finally, we selected bioactive metabolites and demonstrated that these could be used as adjuncts to the nanostructured surfaces to reduce biofilm formation and enhance hMSC activity. These surfaces make excellent models to study hMSC-pathogen interactions and could be envisaged for use in novel orthopedic implants.

Leech Poecilobdella manillensis protein extract ameliorated hyperuricemia by restoring gut microbiota dysregulation and affecting serum metabolites.

Hyperuricemia (HUA) is a public health concern that needs to be solved urgently. The lyophilized powder of Poecilobdella manillensis has been shown to significantly alleviate HUA; however, its underlying metabolic regulation remains unclear. To explore the underlying mechanisms of Poecilobdella manillensis in HUA based on modulation of the gut microbiota and host metabolism. A mouse model of rapid HUA was established using a high-purine diet and potassium oxonate injections. The mice received oral drugs or saline. Additionally, 16S rRNA sequencing and ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry-based untargeted metabolomics were performed to identify changes in the microbiome and host metabolome, respectively. The levels of uric acid transporters and epithelial tight junction proteins in the renal and intestinal tissues were analyzed using an enzyme-linked immunosorbent assay. The protein extract of Poecilobdella manillensis lyophilized powder (49 mg/kg) showed an enhanced anti-trioxypurine ability than that of allopurinol (5 mg/kg) (P < 0.05). A total of nine bacterial genera were identified to be closely related to the anti-trioxypurine activity of Poecilobdella manillensis powder, which included the genera of Prevotella, Delftia, Dialister, Akkermansia, Lactococcus, Escherichia_Shigella, Enterococcus, and Bacteroides. Furthermore, 22 metabolites in the serum were found to be closely related to the anti-trioxypurine activity of Poecilobdella manillensis powder, which correlated to the Kyoto Encyclopedia of Genes and Genomes pathways of cysteine and methionine metabolism, sphingolipid metabolism, galactose metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. Correlation analysis found that changes in the gut microbiota were significantly related to these metabolites. The proteins in Poecilobdella manillensis powder were effective for HUA. Mechanistically, they are associated with improvements in gut microbiota dysbiosis and the regulation of sphingolipid and galactose metabolism.

GC–MS-based untargeted plasma metabolomics identifies a 2-biomarker panel for possible diagnosis of precancerous cervical intraepithelial neoplasia stages from cervical cancer

Cervical cancer (CC) remains a major health concern globally, much of the brunt of which is experienced by the low- and middle-income countries where screening in terms of cytology and DNA genotyping for the high-risk oncogenic subtypes of the human papilloma virus (hr-HPV) is either inadequate or performed rather late. In this study, we aimed to determine biomarkers or panels of biomarkers that are capable of diagnosing the precancerous cervical intraepithelial neoplasia (CIN) stages from healthy and CC patients via untargeted gas chromatography–mass spectrometry-based metabolomics. Various cross-comparisons were conducted from which differential metabolites were identified. The underlying metabolic pathways based on the differential metabolites identified from the various cross-comparisons mainly related to amino acids biosynthesis and metabolism and steroid hormone biosynthesis. From all cross-comparisons, two common metabolites namely, 2-methyl-1-propylamine (also known as isobutylamine) and estrone were found to possess excellent to good diagnostic abilities, especially in distinguishing the early stages of CIN (CIN I, CIN II) from healthy women and CC patients. These findings have clinical significance in the sense that, once validated the 2-biomarker panel could be adopted in clinical practice for early diagnosis of CIN and invasive carcinoma. This would therefore inform the choice of treatment to be initiated by the clinician.

Integrated metabolomics and proteomics analyses to reveal anticancer mechanism of hemp oil extract in colorectal cancer

Cannabis sativa L., with a rich history in Chinese folk medicine, includes hemp strains that offer substantial economic and medical benefits due to their non-addictive properties. Hemp has demonstrated various pharmaceutical activities, including anti-inflammatory, antioxidant, and anti-tumor effects. This study explores the potential of hemp oil extract (HOE) in treating colorectal cancer (CRC). Despite its promise, the specific anticancer mechanisms of HOE have not been well understood. To elucidate these mechanisms, we employed mass spectrometry-based metabolomics and proteomics to investigate the global effects of HOE on CRC cells. Additionally, bioinformatics approaches, including bulk RNA-seq and single-cell RNA-seq, were used to identify gene expression differences and cellular heterogeneity. The results were validated using flow cytometry, western blotting, and immunohistochemistry. Our findings reveal that HOE induces significant alterations in purine metabolism pathways, down-regulates c-MYC, and inhibits the expression of cell cycle-related proteins such as CCND1, CDK4, and CDK6, leading to cell cycle arrest in the G1 phase. This comprehensive analysis demonstrates that HOE effectively blocks the cell cycle in the G1 phase, thereby inhibiting colorectal cancer cell proliferation. These findings provide experimental evidence supporting the potential therapeutic use of hemp in medicine.

Chronic Exposure to Petroleum-Derived Hydrocarbons Alters Human Skin Microbiome and Metabolome Profiles: A Pilot Study.

Petroleum-derived substances, like industrial oils and grease, are ubiquitous in our daily lives. Comprised of petroleum hydrocarbons (PH), these substances can come into contact with our skin, potentially causing molecular disruptions and contributing to the development of chronic disease. In this pilot study, we employed mass spectrometry-based untargeted metabolomics and 16S rRNA gene sequencing analyses to explore these effects. Superficial skin samples were collected from subjects with and without chronic dermal exposure to PH at two anatomical sites: the fingers (referred to as the hand) and arms (serving as an intersubject variability control). Exposed hands exhibited higher bacterial diversity (Shannon and Simpson indices) and an enrichment of oil-degrading bacteria (ODB), including Dietzia, Paracoccus, and Kocuria. Functional prediction suggested enriched pathways associated with PH degradation in exposed hands vs non-exposed hands, while no differences were observed when comparing the arms. Furthermore, carboxylic acids, glycerophospholipids, organooxygen compounds, phenol ethers, among others, were found to be more abundant in exposed hands. We observed positive correlations among multiple ODB and xenobiotics, suggesting a chemical remodeling of the skin favorable for ODB thriving. Overall, our study offers insights into the complex dysregulation of bacterial communities and the chemical milieu induced by chronic dermal exposure to PH.

Liquid chromatography-mass spectrometry quantification of phytochemicals in Withania somnifera using data-dependent acquisition, multiple-reaction-monitoring, and parallel-reaction-monitoring with an inclusion list.

Characterization of botanical extracts by mass spectrometry-based metabolomics analysis helps in determining the phytochemical composition that underlies their bioactivity and potential health benefits, while also supporting reproducibility of effects in clinical trials. The quantification of seven withanolides in Withania somnifera using three mass-spectrometry methods was evaluated using Deming regression. Two high-resolution time-of-flight mass spectrometry methods were used, one operating in data-dependent acquisition mode and the other in parallel-reaction-monitoring mode with an inclusion list. The two high-resolution time-of-flight mass spectrometry methods were compared to a multiple-reaction-monitoring method. We evaluated in-source fragmentation of steroidal glycosides and optimized the methods accordingly. A novel software approach to integrating parallel-reaction-monitoring data acquired with an inclusion list was developed. Combining and comparing quantitative results allowed for quantitative specificity, good precision, and adjustment of instrument source conditions for optimal quantification by multiple-reaction-monitoring mass spectrometry, an analytical method that is widely accessible in analytical and phytochemical laboratories.

Mass spectrometry-based metabolomics for the investigation of antibiotic-bacterial interactions.

With the development of analytical technologies especially mass spectrometry, metabolomics is becoming increasingly hot in the field of studying antibiotic-bacterial interactions. On the one hand, metabolomics can reveal metabolic perturbations in bacteria in the presence of antibiotics and expose metabolic mechanisms. On the other hand, through in-depth analysis of bacterial metabolic profiles, biomarkers and bioactive secondary metabolites with great potential as drug precursors can be discovered. This review focuses on the experimental workflow of bacterial metabolomics and its application to study the interaction between bacteria and antibiotics. Metabolomics improves the understanding of antibiotic lethality, reveals metabolic perturbations in antibiotic-resistant bacteria, guides the diagnosis and antibiotic treatment of infectious diseases, and aids in the exploration of antibacterial metabolites in nature. Furthermore, current limitations and directions for future developments in this area are discussed.

Metabolic Profiling Changes Induced by Fermented Blackberries in High-Fat-Diet-Fed Mice Utilizing Gas Chromatography-Mass Spectrometry Analysis.

The aim of this study was to investigate the metabolic changes associated with the anti-obesity effects of fermented blackberry extracts in the liver tissues of high-fat-diet-fed mice using mass spectrometry-based metabolomics analysis. C57BL/6J mice were divided into eight groups: normal-diet-fed mice, high-fat-diet-fed mice, high-fat diet treated with blackberry extract, high-fat-diet mice treated with blackberry fermented by L. plantarum, and high-fat diet with blackberry fermented by L. brevis. After 12 weeks, the high-fat-diet group exhibited a greater increase in liver weight compared to the control group, and among the groups, the group administered with blackberry fermented with L. plantarum showed the most pronounced reduction in liver weight. As the primary organ responsible for amino acid metabolism, the liver is crucial for maintaining amino acid homeostasis. In our study, we observed that the levels of several essential amino acids, including isoleucine and valine, were decreased by the high-fat diet, and were recovered by administration of blackberry extract fermented with L. plantarum. Our results demonstrated the potential of blackberry extract fermented with L. plantarum as a functional material for metabolic disorders by restoring some of the amino acid metabolism disturbances induced by a high-fat diet.

Urinary metabolomics signature of animal and plant protein intake and its association with 24-h blood pressure: the African-PREDICT study.

The contrasting relationships of plant and animal protein intake with blood pressure (BP) may be partially attributed to the differential non-protein (e.g., saturated fat and fibre) and amino acid (AA) compositions. This study determined whether animal and plant protein intake were related to differential metabolomic profiles associated with BP. This study included 1008 adults from the African-PREDICT study (aged 20-30 years). Protein intake was determined using 24-h dietary recalls. Twenty-four-hour ambulatory BP was measured. Amino acids and acylcarnitines were analysed in spot urine samples using liquid chromatography-tandem mass spectrometry-based metabolomics. Participants with a low plant, high animal protein intake had higher SBP (by 3 mmHg, p = 0.011) than those with high plant, low animal protein intake (low-risk group). We found that the relationships of plant and animal protein intake with 24-h SBP were partially mediated by BMI and saturated fat intake, which were independently associated with SBP. Protein intake was therefore not related to SBP in multiple regression analysis after adjusting for confounders. In the low-risk group, methionine (Std. β = -0.217; p = 0.034), glutamic acid (Std. β = -0.220; p = 0.031), glycine (Std. β = -0.234; p = 0.025), and proline (Std. β = -0.266; p = 0.010) were inversely related to SBP, and beta-alanine (Std. β = -0.277; p = 0.020) to DBP. Ultimately a diet high in animal and low in plant protein intake may contribute to higher BP by means of increased BMI and saturated fat intake. Conversely, higher levels of urinary AAs observed in adults consuming a plant rich diet may contribute to lower BP.

Multi-scale variational autoencoder for imputation of missing values in untargeted metabolomics using whole-genome sequencing data

BackgroundMissing data is a common challenge in mass spectrometry-based metabolomics, which can lead to biased and incomplete analyses. The integration of whole-genome sequencing (WGS) data with metabolomics data has emerged as a promising approach to enhance the accuracy of data imputation in metabolomics studies. MethodIn this study, we propose a novel method that leverages the information from WGS data and reference metabolites to impute unknown metabolites. Our approach utilizes a multi-scale variational autoencoder to jointly model the burden score, polygenetic risk score (PGS), and linkage disequilibrium (LD) pruned single nucleotide polymorphisms (SNPs) for feature extraction and missing metabolomics data imputation. By learning the latent representations of both omics data, our method can effectively impute missing metabolomics values based on genomic information. ResultsWe evaluate the performance of our method on empirical metabolomics datasets with missing values and demonstrate its superiority compared to conventional imputation techniques. Using 35 template metabolites derived burden scores, PGS and LD-pruned SNPs, the proposed methods achieved R2-scores > 0.01 for 71.55 % of metabolites. ConclusionThe integration of WGS data in metabolomics imputation not only improves data completeness but also enhances downstream analyses, paving the way for more comprehensive and accurate investigations of metabolic pathways and disease associations. Our findings offer valuable insights into the potential benefits of utilizing WGS data for metabolomics data imputation and underscore the importance of leveraging multi-modal data integration in precision medicine research.

Sex Differences in the Blood Metabolome During Acute Response to Ischemic Stroke.

Introduction: Females suffer greater lifetime risk of stroke and greater morbidity and mortality from stroke compared with males. This study's objective was to identify differences in metabolomic profiling of females and males with stroke and which differences were associated with neurological outcome. Methods: Females and males with acute ischemic stroke enrolled in the Emergency Medicine Specimen Bank at a comprehensive stroke center provided whole blood samples upon arrival for mass spectrometry-based metabolomics. We used descriptive statistics to characterize the cohort. A linear regression model was fit for individual metabolites to determine differences in relative abundance between males and females while controlling for covariates (age, race/ethnicity, postmenopausal status, cardiovascular risk factors, depression, time between sample collection and last known well, and initial National Institutes of Health Stroke Scale [NIHSS] score). For each differentially expressed metabolite, a linear regression model was fit to determine the association between the metabolite and NIHSS at 24 hours after admission while controlling for the covariates and acute treatments. Results: After adjusting for covariates, eight metabolites differed in females and males with a stroke. These included amino acids or their metabolites (proline and tryptophan), nucleotides (guanosine diphosphate [GDP], and inosine-3',5'-cyclic monophosphate), citrate, dehydroascorbate, choline, and acylcarnitine-(5-OH). GDP and dehydroascorbate were significantly associated with 24-hour NIHSS (p = 0.0991). Conclusions: Few metabolites were differentially abundant in blood after a stroke when comparing females with males and controlling for confounders, but the interactions between biological sex and GDP, as well as biological sex and dehydroascorbate, were associated with 24-hour neurological function. This has important implications for future studies that evaluate the therapeutic potential of these metabolites in ischemic stroke.

Mass spectrometry-based metabolomics study of nicotine exposure in THP-1 monocytes

The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and l-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and l-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.

Challenges and recent advances in quantitative mass spectrometry-based metabolomics.

The field of metabolomics has gained tremendous interest in recent years. Whether the goal is to discover biomarkers related to certain pathologies or to better understand the impact of a drug or contaminant, numerous studies have demonstrated how crucial it is to understand variations in metabolism. Detailed knowledge of metabolic variabilities can lead to more effective treatments, as well as faster or less invasive diagnostics. Exploratory approaches are often employed in metabolomics, using relative quantitation to look at perturbations between groups of samples. Most metabolomics studies have been based on metabolite profiling using relative quantitation, with very few studies using an approach for absolute quantitation. Using accurate quantitation facilitates the comparison between different studies, as well as enabling longitudinal studies. In this review, we discuss the most widely used techniques for quantitative metabolomics using mass spectrometry (MS). Various aspects will be addressed, such as the use of external and/or internal standards, derivatization techniques, in vivo isotopic labelling, or quantitative MS imaging. The principles, as well as the associated limitations and challenges, will be described for each approach.

Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation.

The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis.

ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart.

Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear. We utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate Acly in cardiomyocytes of MyH6-Cas9 mice. In vivo, positron emission tomography and ex vivo stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level. Here, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. Ex vivo isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate. This study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.