Dataset Information

Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

ABSTRACT: The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.

SUBMITTER: Behringer S

PROVIDER: S-EPMC6836102 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

Behringer Sidney S Wingert Victoria V Oria Victor V Schumann Anke A Grünert Sarah S Cieslar-Pobuda Artur A Kölker Stefan S Lederer Ann-Kathrin AK Jacobsen Donald W DW Staerk Judith J Schilling Oliver O Spiekerkoetter Ute U Hannibal Luciana L

Metabolites 20191018 10

The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cyste ...[more]

PMID: 31635306

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Project description:BackgroundHeart failure is a multifactorial disease associated with staggeringly high morbidity and motility. Recently, alterations of multiple metabolites have been implicated in heart failure; however, the lack of an effective technology platform to assess these metabolites has limited our understanding on how they contribute to this disease phenotype. We have successfully developed a new workflow combining specific sample preparation with tandem mass spectrometry that enables us to extract most of the targeted metabolites. 19 metabolites were chosen ascribing to their biological relevance to heart failure, including extracellular matrix remodeling, inflammation, insulin resistance, renal dysfunction, and cardioprotection against ischemic injury.ResultsIn this report, we systematically engineered, optimized and refined a protocol applicable to human plasma samples; this study contributes to the methodology development with respect to deproteinization, incubation, reconstitution, and detection with mass spectrometry. The deproteinization step was optimized with 20% methanol/ethanol at a plasma:solvent ratio of 1:3. Subsequently, an incubation step was implemented which remarkably enhanced the metabolite signals and the number of metabolite peaks detected by mass spectrometry in both positive and negative modes. With respect to the step of reconstitution, 0.1% formic acid was designated as the reconstitution solvent vs. 6.5 mM ammonium bicarbonate, based on the comparable number of metabolite peaks detected in both solvents, and yet the signal detected in the former was higher. By adapting this finalized protocol, we were able to retrieve 13 out of 19 targeted metabolites from human plasma.ConclusionsWe have successfully devised a simple albeit effective workflow for the targeted plasma metabolites relevant to human heart failure. This will be employed in tandem with high throughput liquid chromatography mass spectrometry platform to validate and characterize these potential metabolic biomarkers for diagnostic and therapeutic development of heart failure patients.

Dataset Information

Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

Publications

Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

Similar Datasets

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