Liquid chromatography-tandem mass spectrometry based simultaneous quantification of tryptophan, serotonin and kynurenine pathway metabolites in tissues and cell culture systems

by Selleckchem | Jan 05 2024

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.

Comments:

This research seems fascinating! The comprehensive method developed here for quantifying kynurenine-related metabolites using UPLC-MS/MS is impressive. Being able to simultaneously analyze multiple metabolites without derivatization in various biological matrices is a significant advancement.

The potential applications in understanding the role of the kynurenine pathway in diseases like neurodegenerative disorders, neuropsychiatric conditions, diabetes mellitus type 2, and cancer could be substantial. Being able to monitor the impact of inhibitors, such as the tryptophan-2,3-dioxygenase-inhibitor 680C91, on the pathway within cellular systems adds another layer of utility to this method.

Moreover, the adaptability of this method for integration into multi-omics approaches, utilizing a single sample metabolite extraction procedure, enhances its potential in broader experimental settings.

What's the next step for this research? Are there specific diseases or pathways this method will be applied to next?