Untargeted LC-MS Metabolomics for the Analysis of Micro-scaled Extracellular Metabolites from Hepatocytes

Abdalkader, Rodi; Chaleckis, Romanas; Meister, Isabel; Zhang, Pei; Wheelock, Craig E.; Kamei, Kazuhito

doi:10.2116/analsci.20n032

Cited by 8 publications

(9 citation statements)

References 24 publications

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“…However, since these columns showed a reduced lifetime at elevated pH values, they were not included in this study (Harrieder et al, 2022 ). Instead, a sulfobetaine (ZicHILIC) column was selected, since it was often used in other metabolomics studies and showed suitable separation by its zwitterionic stationary phase (Abdalkader et al, 2021 ; Trivedi et al, 2012 ; Steuer et al, 2020 ). The in-house HILIC reference column, an ammonium-sulfonic acid (Nucleodur), has also a zwitterionic functional group.…”

Section: Resultsmentioning

confidence: 99%

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

Hemmer,

Manier,

Wagmann

et al. 2024

Metabolomics

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Introduction Untargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the study purpose. Objectives The current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices. Methods Separation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C18, and Gold C18), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others. Results All three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column. Conclusion The results underline that the outcome of this untargeted toxicometabolomic study LC-HRMS metabolomic study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix. Graphical abstract

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Section: Resultsmentioning

confidence: 99%

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

Hemmer,

Manier,

Wagmann

et al. 2024

Metabolomics

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“…By using our previously developed untargeted LC-MS method for the measurement of extracellular metabolites at the microscale level, [21] we could annotate 104 metabolites at Metabolomics Standard Initiative annotation level 1. [32] Peak areas were used for metabolite semi quantification.…”

Section: Resultsmentioning

confidence: 99%

“…For the preparation of QC samples, 0 h cell culture medium sample was used from our previous study. [21] For each QC sample, 1 µL of cell culture medium was evaporated and processed together. After resuspension, the samples were centrifuged at 4°C for 15 min at 20000 g. Next, 40 µL of the supernatant was transferred to a 96-well 0.2 mL PCR plate (PCR-96-MJ; BMBio, Tokyo, Japan).…”

Section: Human Corneal Epithelial Cell Culture: Hce-t Cells Were Provmentioning

confidence: 99%

“…[19,20] Moreover, we have developed an LC-MS-based untargeted metabolomics workflow enabling non-invasive temporal quantification of micro-scaled extracellular metabolites. [21] Thus, the OoC platform in combination with LC-MS untargeted metabolomics might provide deeper insights into the metabolic and transport activities of the improved human corneal epithelial barrier than those provided by conventional cell culture and assay platforms.…”

Section: Introductionmentioning

confidence: 99%

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Spatiotemporal Determination of Metabolite Activities in Multiple Corneal Epithelium Barriers on a Chip

Abdalkader

Chaleckis

Wheelock

et al. 2021

Preprint

Self Cite

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The corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of corneal metabolism and determine the transportation of endogenous and exogenous molecules. This study introduces multiple corneal epithelium barriers on a chip, namely, Cornea-Chip, which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography–mass spectrometry metabolomics method, and 104 metabolites were annotated. The shifts in extracellular metabolites and quantitative analysis of the mRNA associated with membrane transporters could allow the investigation of the correlation between the expression of and the secretion and transportation of metabolites across the polarized corneal epithelial barrier. The Cornea-Chip might provide a non-invasive, simple, and effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as an alternative to animal experiments.

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“…The copyright holder for this preprint this version posted March 2, 2023. ; https://doi.org/10.1101/2023.03.02.530741 doi: bioRxiv preprint hundreds of metabolites in small volumes through a relatively straightforward sample preparation process 11,12,13 . In previous studies, we successfully used this technique to determine the metabolites in the cell culture medium (CCM) of liver cells (HepG2) and corneal epithelial cells (HCE-T) grown in microfluidic devices by collecting only a small volume at different time points 14,15 . Currently, there is no established method for evaluating the lineage deviation of hPSCs during differentiation by profiling extracellular metabolites.…”

Section: Introductionmentioning

confidence: 99%

Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis

Abdalkader

Chaleckis

Fujita

2023

Preprint

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Human induced pluripotent stem cells (hiPSCs) possess immense potential as a valuable source for the generation of a wide variety of human cells yet monitoring the early cell differentiation towards a specific lineage remains challenging. In this study, we employed a non-targeted metabolomic analysis technique to analyze the extracellular metabolites present in samples as small as one microliter. The hiPSCs were subjected to differentiation by initiating culture under the basal medium E6 in combination with chemical inhibitors that have been previously reported to direct differentiation towards the ectodermal lineage such as Wnt/β- catenin and TGF-β kinase/activin receptor alone or in combination with bFGF, and the inhibition of glycogen kinase 3 (GSK-3), which is commonly used for the diversion of hiPSCs towards mesodermal lineage. At 0 hr and 48 hrs 107 metabolites were identified, including biologically relevant metabolites such as lactic acid, pyruvic acid, and amino acids. By determining the expression of the pluripotency marker OCT3/4, we were able to correlate the differentiation status of cells with the shifted metabolites. The group of cells undergoing ectodermal differentiation showed a greater reduction in OCT3/4 expression. Moreover, metabolites such as pyruvic acid and kynurenine showed dramatic change under ectodermal differentiation conditions where pyruvic acid consumption increased 1-2-folds, while kynurenine secretion decreased 2-folds. Further metabolite analysis uncovered a group of metabolites specifically associated with ectodermal lineage, highlighting the potential of our findings to determine the characteristics of hiPSCs during cell differentiation, particularly under ectodermal lineage conditions.Graphical abstract

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Untargeted LC-MS Metabolomics for the Analysis of Micro-scaled Extracellular Metabolites from Hepatocytes

Cited by 8 publications

References 24 publications

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

Spatiotemporal Determination of Metabolite Activities in Multiple Corneal Epithelium Barriers on a Chip

Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis

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