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

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Laboratory of Clinical Biochemistry and Metabolism, Department of General Pediatrics, Adolescent Medicine and Neonatology, Faculty of Medicine, Medical Center, University of Freiburg, 79106 Freiburg, Germany
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Institute of Surgical Pathology, Faculty of Medicine, Medical Center, University of Freiburg, 79106 Freiburg, Germany
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Spemann Graduate School of Biology and Medicine, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
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Department of General Pediatrics, Adolescent Medicine and Neonatology, Faculty of Medicine, Medical Center, University of Freiburg, 79106 Freiburg, Germany
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Nordic European Molecular Laboratory (EMBL) Partnership, Centre for Molecular Medicine Norway, University of Oslo, 0318 Oslo, Norway
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Center for Pediatrics and Adolescent Medicine, Division of Pediatric Neurology and Metabolic Medicine, University Hospital Heidelberg, 69120 Heidelberg, Germany
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Center for Complementary Medicine, Institute for Infection Prevention and Hospital Epidemiology, Faculty of Medicine, Medical Center, University of Freiburg, 79106 Freiburg, Germany
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Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44106, USA
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Norwegian Center for Stem Cell Research, Department of Immunology, Oslo University Hospital, 0372 Oslo, Norway
*
Author to whom correspondence should be addressed.
Metabolites 2019, 9(10), 235; https://doi.org/10.3390/metabo9100235
Received: 12 September 2019 / Revised: 13 October 2019 / Accepted: 15 October 2019 / Published: 18 October 2019
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. View Full-Text
Keywords: methionine metabolism; homocysteine; glutathione; targeted metabolic profiling; mass spectrometry; thiol; biomarker methionine metabolism; homocysteine; glutathione; targeted metabolic profiling; mass spectrometry; thiol; biomarker
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Behringer, S.; Wingert, V.; Oria, V.; Schumann, A.; Grünert, S.; Cieslar-Pobuda, A.; Kölker, S.; Lederer, A.-K.; Jacobsen, D.W.; Staerk, J.; Schilling, O.; Spiekerkoetter, U.; Hannibal, L. Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine. Metabolites 2019, 9, 235.

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