Liquid chromatography-tandem mass spectrometry based simultaneous quantification of tryptophan, serotonin and kynurenine pathway metabolites in tissues and cell culture systems
{"title":"Liquid chromatography-tandem mass spectrometry based simultaneous quantification of tryptophan, serotonin and kynurenine pathway metabolites in tissues and cell culture systems","authors":"Dennis Fröbel, Daniela Stanke, Mathias Langner, Gintare Žygienė, Nicole Bechmann, Mirko Peitzsch","doi":"10.1016/j.jchromb.2023.123870","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Kynurenine and respective metabolites exhibit bioactivity as well as tryptophan, an essential amino acid, and the neurotransmitter serotonin. Dysregulations in the kynurenine pathway are involved in neurodegenerative/neuropsychiatric disorders and diabetes mellitus type 2 but also in cancer. Therefore, measurements of kynurenine-related metabolites will improve the general understanding for kynurenine pathway relevance in disease pathogenesis.</p></div><div><h3>Methods</h3><p>Tryptophan, serotonin, picolinic acid, quinolinic acid, 3–OH-kynurenine, kynurenine, 3-OH-anthranilic acid, kynurenic acid, anthranilic acid as well as nicotinic acid and the redox cofactor NAD<sup>+</sup> were analyzed in heterogeneous matrices by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After validation, the described method was applied for measurements of native metabolite concentrations in murine tissues and cellular systems including pathway-shift monitoring after treatment with the tryptophan-2,3-dioxygenase-inhibitor 680C91. In addition, the method was evaluated for its ability for integration into multi-omics approaches using a single sample metabolite extraction procedure.</p></div><div><h3>Results</h3><p>A simple and sensitive UPLC-MS/MS method for simultaneous quantification of up to 10 kynurenine-related metabolites in four biological matrices was developed. Within a run time of 6.5 min, chromatographic separation of kynurenine-related metabolites, including the isomers nicotinic acid and picolinic acid, was achieved without derivatization. Validation parameters, including interday precision (<14.8%), mean accuracy (102.4% ± 12.9%) and linear detection ranges of more than three orders of magnitude, indicate method reliability. Depending the investigated sample matrix, the majority of metabolites were successfully detected and quantified in native murine and cell culture derived sample materials. Furthermore, the method allowed to monitor the impact of a tryptophan-2,3-dioxygenase-inhibitor on kynurenine pathway in a cellular system and is suitable for multi-assay analyses using aliquots from the same cell extract.</p></div><div><h3>Conclusion</h3><p>The described UPLC-MS/MS method provides a simple tool for the simultaneous quantification of kynurenine pathway metabolites. Due to its suitability for many physiological matrices, the method provides wide application for disease-related experimental settings.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1229 ","pages":"Article 123870"},"PeriodicalIF":2.8000,"publicationDate":"2023-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Chromatography B","FirstCategoryId":"1","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1570023223002805","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Background
Kynurenine and respective metabolites exhibit bioactivity as well as tryptophan, an essential amino acid, and the neurotransmitter serotonin. Dysregulations in the kynurenine pathway are involved in neurodegenerative/neuropsychiatric disorders and diabetes mellitus type 2 but also in cancer. Therefore, measurements of kynurenine-related metabolites will improve the general understanding for kynurenine pathway relevance in disease pathogenesis.
Methods
Tryptophan, serotonin, picolinic acid, quinolinic acid, 3–OH-kynurenine, kynurenine, 3-OH-anthranilic acid, kynurenic acid, anthranilic acid as well as nicotinic acid and the redox cofactor NAD+ were analyzed in heterogeneous matrices by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After validation, the described method was applied for measurements of native metabolite concentrations in murine tissues and cellular systems including pathway-shift monitoring after treatment with the tryptophan-2,3-dioxygenase-inhibitor 680C91. In addition, the method was evaluated for its ability for integration into multi-omics approaches using a single sample metabolite extraction procedure.
Results
A simple and sensitive UPLC-MS/MS method for simultaneous quantification of up to 10 kynurenine-related metabolites in four biological matrices was developed. Within a run time of 6.5 min, chromatographic separation of kynurenine-related metabolites, including the isomers nicotinic acid and picolinic acid, was achieved without derivatization. Validation parameters, including interday precision (<14.8%), mean accuracy (102.4% ± 12.9%) and linear detection ranges of more than three orders of magnitude, indicate method reliability. Depending the investigated sample matrix, the majority of metabolites were successfully detected and quantified in native murine and cell culture derived sample materials. Furthermore, the method allowed to monitor the impact of a tryptophan-2,3-dioxygenase-inhibitor on kynurenine pathway in a cellular system and is suitable for multi-assay analyses using aliquots from the same cell extract.
Conclusion
The described UPLC-MS/MS method provides a simple tool for the simultaneous quantification of kynurenine pathway metabolites. Due to its suitability for many physiological matrices, the method provides wide application for disease-related experimental settings.
期刊介绍：
The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis.
Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches.
Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
