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Abstract Introduction
In recent years, it has been revealed that tryptophan metabolism plays an important role in the metabolic control of immunological and neuropsychological processes [1]. Reliable analytical methods for the determination of tryptophan and its metabolites are required to understand the pathophysiological mechanisms of various diseases associated with tryptophan metabolism and to evaluate the therapeutic approaches [2]. Therefore, there is a need for a reliable analytical solution for comprehensive profiling of kynurenine pathway metabolites and for the quantitative determination of targeted analytes. In this context, many scientific findings demonstrate the relationship between kynurenine pathway metabolites and disease: The degradation of tryptophan associated with inflammation suggests that assessment of the metabolomic profile of patients with COVID-19 may assist in disease severity classification and even guide clinical decisions. Plasma levels of tryptophan and some of its metabolites via the kynurenine pathway vary according to the severity of COVID-19. It was determined that while tryptophan concentrations decreased significantly depending on the severity of the disease, kynurenine pathway metabolites such as 3-hydroxykynurenine increased [3,4,5,6]. The gut microbiota has also been shown to modulate the kynurenine pathway, thereby influencing the physiology of the digestive system and central nervous system. Indeed, the kynurenine pathway is thought to play a key role in the modulation of neurotransmission and immune functions. Fluctuations in the kynurenine pathway are associated with many psychiatric and gastrointestinal disorders [7]. Metabolic alterations in the kynurenine pathway may cause various biological responses in depression. Increasing evidence has been gathered regarding the relationship between the activation of the KYN pathway and the onset of depression [8].
Objectives
The main objective of this study was to achieve quantitative profiling of biomarkers related to the kynurenine pathway of tryptophan metabolism consisting of kynurenine (KYN), kynurenic acid (KA), 3-OH-kynurenine (3-HK), 3-OH-anthranilic acid (3-HA), picolinic acid (PA), quinolinic acid (QA), xanthurenic acid (XA), anthranilic acid (AA) and tryptophan (TRP) in human serum specimens by a simple and cost-effective LC-MS/MS-based methodology.
Methods
According to developed analysis method, serum samples were prepared by “protein crash and shoot” approach. Sample preparation procedure included three steps; 100 μL of calibrant/sample was pipetted into a centrifuge tube. Then, 50 μL of internal standard mixture was added and vortexed for 5 sec. Next, 250 μL of reagent (for deproteinization) was added to the tube, swirled for 5 sec. and centrifuge at 4500 rpm for 5 min. Finally decant the supernatant into a HPLC vial prior to LC-MS/MS injection. Analysis of samples were performed on Agilent HPLC system (Agilent Technologies, Santa Clara, CA, USA) consisting of flexible pump (G7104A), column compartment (G7116B) and autosampler (G7129C) coupled with Agilent Ultivo triple quadrupole LC/MS (6465B, Agilent Technologies, Santa Clara, CA, USA). The total run time from injection to injection was 12.0 min. Quantification of the metabolites was conducted by means of calibration curves established on the concentrations of the water-based calibrators with compensating for matrix effect according to yields of the assigned stable labelled isotope as internal standard. Data acquisition, qualification and quantification were carried out using Agilent MassHunter Acquisition (Version 1.2), Agilent MassHunter Qualitative Analysis (Version 10.0) and Agilent MassHunter Quantitative Analysis (Version 10.1) software programs, respectively.
Results
The methodological verification results showed that the method met the expected requirements and objectives in terms of sensitivity, accuracy, and precision. The calibration curves were linear over the concentration range of 3.0-600 µg/L for KA, QA, 3-HK, PA, XA (r² > 0.995), 2.0-400 µg/L for 3-HA, AA, 20.0-4000 µg/L for KYN (r² > 0.998) and 100.0-20000 µg/L for TRP (r² > 0.996), from three separately established quantitation batches on different days. The limits of detection (LODs) ranged in 0.14-1.16 µg/L and limits of quantitation (LOQs) ranged in 1.37-3.89 µg/L. Recovery % values for all analytes were found > 88 % and the intra-assay/inter-assay precision evaluated for low-and high-level spiked synthetic serum samples were less than 7.4 % and 7.8 %, respectively.
Conclusions
Tryptophan metabolism plays vital role in the regulation of immunity, neuronal function, and intestinal homeostasis. Establishing a reliable and accurate method for the determination of kynurenine pathway metabolites is useful for clinical diagnosis/monitoring and scientific research. In this work, a simple and rapid LC–MS/MS based bioanalytical method has been developed for the quantitation of serum KYN, KA, 3-HK, 3-HA, PA, QA, XA, AA and TRP in a single chromatographic run. A fit-for-purpose validation approach was adopted by employing water as surrogate matrix for calibration and synthetic serum-based QCs.
References
[1] Johanna Gostner et al., 2020, Neuropsychobiology, 79:89–99.
[2] Qing Tong et al., 2017, Biomedical Chromatography, 32, e4156.
[3] Judith Marín-Corral et al., 2021, International Journal of Molecular Sciences, 22, 4794.
[4] Nathan G. Lawler et al., 2021, Journal of Proteome Research, 20, 2796-2811.
[5] Xin Chen et al., 2021, Future Microbiology, 16(8), 577–588.
[6] Tiffany Thomas et al., 2020, JCI Insight.
[7] Luke Whiley et al., 2019, Analytical Chemistry, 91, 5207−5216.
[8] Masashi Sakurai et al., 2020, Nature Scientific Reports, 10:1961.
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= Discovery stage. (19.79%, 2022)
= Translation stage. (37.97%, 2022)
= Clinically available. (42.25%, 2022)
